AU2014250730B2 - Human antigen binding proteins that bind beta-Klotho, FGF receptors and complexes thereof - Google Patents

Abstract

The present disclosure provides compositions and methods relating to or derived from antigen binding proteins activate FGF21-mediated signaling. In embodiments, the antigen binding proteins specifically bind to (i) j -Klotho; (ii) FGFRIc, FGFR2c, 5 FGFR3c or FGFR4; or (iii) a complex comprising j -Klotho and one of FGFRIc, FGFR2c, FGFR3c, and FGFR4. In some embodiments the antigen binding proteins induce FGF21-like signaling. In some embodiments, the antigen binding proteins are fully human, humanized, or chimeric antibodies, binding fragments and derivatives of such antibodies, and polypeptides that specifically bind to (i) j -Klotho; (ii) FGFRIc, 10 FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising j -Klotho and one of FGFRIc, FGFR2c, FGFR3c, and FGFR4. Other embodiments provide nucleic acids encoding such antigen binding proteins, and fragments and derivatives thereof, and polypeptides, cells comprising such polynucleotides, methods of making such antigen binding proteins, and fragments and derivatives thereof, and polypeptides, and methods 15 of using such antigen binding proteins, fragments and derivatives thereof, and polypeptides, including methods of treating or diagnosing subjects suffering from type 2 diabetes, obesity, NASH, metabolic syndrome and related disorders or conditions.

Description

HUMAN ANTIGEN BINDING PROTEINS THAT BIND β-KLOTHO, FGF RECEPTORS AND COMPLEXES THEREOF

The present application is a divisional application of Australian Application No. 2010328444, which is incorporated in its entirety herein by reference.

This application claims the benefit of U.S. Provisional Application No. 61/267,321 filed December 7, 2009 and U.S. Provisional Application No. 61/381,846 filed September 10, 2010, which are incorporated by reference herein.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on November 24, 2010, is named A-1519-WO-PCT.txt and is 645,894 bytes in size.

FIELD OF THE INVENTION

The present disclosure relates to nucleic acid molecules encoding antigen binding proteins that bind to (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4. The present disclosure also provides antigen binding proteins that bind to (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4, including antigen binding proteins that induce FGF21-like signaling, as well as pharmaceutical compositions comprising antigen binding proteins that bind to (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4, including antigen binding proteins that induce FGF21-like signaling, and methods for treating metabolic disorders using such nucleic acids, polypeptides, or pharmaceutical compositions. Diagnostic methods using the antigen binding proteins are also provided.

BACKGROUND

Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

Fibroblast Growth Factor 21 (FGF21) is a secreted polypeptide that belongs to a subfamily of Fibroblast Growth Factors (FGFs) that includes FGF19, FGF21, and FGF23 (Itoh et al., (2004) Trend Genet. 20:563-69). FGF21 is an atypical FGF in that it is heparin -7 independent and functions as a hormone in the regulation of glucose, lipid, and energy metabolism.

It is highly expressed in liver and pancreas and is the only member of the FGF family to be primarily expressed in liver. Transgenic mice overexpressing FGF21 exhibit metabolic phenotypes of slow growth rate, low plasma glucose and triglyceride levels, and an absence of age-associated type 2 diabetes, islet hyperplasia, and obesity. Pharmacological administration of recombinant FGF21 protein in rodent and primate models results in normalized levels of plasma glucose, reduced triglyceride and cholesterol levels, and improved glucose tolerance and insulin sensitivity. In addition, FGF21 reduces body weight and body fat by increasing energy expenditure, physical activity, and metabolic rate. Experimental research provides support for the pharmacological administration of FGF21 for the treatment of type 2 diabetes, obesity, dyslipidemia, and other metabolic conditions or disorders in humans. FGF21 is a liver derived endocrine hormone that stimulates glucose uptake in adipocytes and lipid homeostasis through the activation of its receptor. Interestingly, in addition to the canonical FGF receptor, the FGF21 receptor also comprises the membrane associated β-Klotho as an essential cofactor. Activation of the FGF21 receptor leads to multiple effects on a variety of metabolic parameters.

In mammals, FGFs mediate their action via a set of four FGF receptors. FGFRI - 4, that in turn are expressed in multiple spliced variants, e.g., FGFRIc, FGFR2c, FGFR3c and FGFR4. Each FGF receptor contains an intracellular tyrosine kinase domain that is activated upon ligand binding, leading to downstream signaling pathways involving MAPKs (Erkl/2), RAF1, AK.T1 and STATs. (Kharitoncnkov et ah, (2008) BioDrugs 22:37-44). Several reports suggested that the “c”-rcporter splice variants of FGFRI -3 exhibit specific affinity to β-Klotho and could act as endogenous receptor for FGF21 (Kurosu et al., (2007),/. Biol. Chem. 282:26687-26695): Ogawa et al., (2007) Proc. Natl. Acad. Sci. USA 104:7432-7437); Kharitonenkov et al., (2008) ,7. Cel! Physiol. 215:1-71. In the liver, which abundantly expresses both β-Klotho and FGFR4, FGF21 does not induce phosphorylation of MAPK albeit the strong binding of FGF2I to the β-Klotho-FGFR4 complex. In 3T3-LI cells and white adipose tissue, FGFRI is by far the most abundant receptor, and it is therefore most likely that FGF21 's main functional receptors in this tissue are the β-Klotho/FGFRlc complexes.

The present disclosure provides a human (or humanized) antigen binding protein, such as a monoclonal antibody, that induces FGF21-like signaling, e.g., an agonistic antibody that mimics the function of FGF21. Such an antibody is a molecule with FGF21-like activity and selectivity but with added therapeutically desirable characteristics typical for an antibody such as protein stability, lack of immunogenicity, ease of production and long half-life in vivo.

SUMMARY

According to a first aspect of the invention there is provided an isolated antibody or antibody fragment thereof that mimics FGF21-mediated signaling comprising: a heavy chain variable region CDR1 comprising the sequence set forth in SEQ ID NO: 122; a heavy chain variable region CDR2 comprising the sequence set forth in SEQ ID NO: 133; a heavy chain variable region CDR3 comprising the sequence set forth in SEQ ID NO: 148; a light chain variable region CDR1 comprising the sequence set forth in SEQ ID NO: 166; a light chain variable region CDR2 comprising the sequence set forth in SEQ ID NO: 176; and a light chain variable region CDR3 comprising the sequence set forth in SEQ ID NO: 188.

According to a second aspect of the invention there is provided an isolated antibody or antibody fragment thereof that mimics FGF21-mediated signalling, wherein said antibody or fragment thereof competes for binding to: b-Klotho; FGFRlc, FGFR2c, FGFR3c or FGFR4; or a complex comprising b-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4, with an antibody of the invention.

According to a third aspect of the invention there is provided a pharmaceutical composition comprising one or more antibodies or antibody fragments thereof of the invention in admixture with a pharmaceutically acceptable carrier.

According to a fourth aspect of the invention there is provided a pharmaceutical composition comprising an antibody or antibody fragment thereof of the invention in admixture with a pharmaceutically acceptable carrier.

According to a fifth aspect of the invention there is provided an isolated nucleic acid comprising a polynucleotide sequence encoding the antibody or antibody fragment thereof of the invention.

According to a sixth aspect of the invention there is provided an expression vector comprising the nucleic acid of the invention.

According to a seventh aspect of the invention there is provided an isolated cell comprising the nucleic acid of the invention.

According to an eighth aspect of the invention there is provided an isolated cell comprising the expression vector of the invention.

According to a ninth aspect of the invention there is provided a method of producing an antibody or antibody fragment thereof that specifically binds to (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4, comprising incubating the cell of the invention under conditions that allow it to express the antibody.

According to a tenth aspect of the invention there is provided an antibody or antibody fragment thereof when produced by the method of the invention.

According to an eleventh aspect of the invention there is provided a method of preventing or treating a condition in a subject in need of such treatment comprising administering the antibody or antibody fragment thereof of the invention, or a therapeutically effective amount of the composition of the invention to the subject, wherein the condition is treatable by lowering blood glucose, insulin or serum lipid levels.

According to a twelfth aspect of the invention there is provided use of the antibody or antibody fragment thereof of the invention in the manufacture of a medicament for preventing or treating a condition, wherein the condition is treatable by lowering blood glucose, insulin or serum lipid levels.

An isolated antigen binding protein that induces FGF21-mediated signaling is also provided.

Also provided is an isolated antigen binding protein that specifically binds to at least one of: (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; and (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c and FGFR4 wherein the antigen binding protein induces FGF21-mediated signaling.

In one embodiment, the provided antigen binding proteins comprise an amino acid sequence selected from the group consisting of: (a) a light chain CDR3 comprising a sequence selected from the group consisting of: (i) a light chain CDR3 sequence that differs by no more than a total of three amino acid additions, substitutions, and/or deletions from a CDR3 sequence selected from the group consisting of the light chain CDR3 sequences of L1-L18, SEQ ID NOs: 180-194; (ii) QVWDX1X2SDHVV (SEQ ID NO: 276); (iii) QQX3GX4X5X6X7T (SEQ ID NO: 283); (iv) LQHNSYPLT (SEQ ID NO: 267); (v) MQSLQTPFT (SEQ ID NO: 268); (vi) QQYNNWPPT (SEQ ID NO: 269); (vii) MQSIQLPRT (SEQ ID NO: 270); (viii) QQANDFPIT (SEQ ID NO: 271); (ix) MQALQTPCS (SEQ ID NO: 272); (b) a heavy chain CDR3 sequence comprising a sequence selected from the group consisting of: (i) a heavy chain CDR3 sequence that differs by no more than a total of four amino acid additions, substitutions, and/or deletions from a CDR3 sequence selected from the group consisting of the heavy chain CDR3 sequences of H1-H18, SEQ ID NOs: 145-157; (ii) X34X16X17X18GX19YYYX20GMDV (SEQ ID NO: 322); (iii) SUVVX21VY X22LDX23 (SEQ ID NO: 326); (iv) IVVVPAAIQSYYYYYGMGV (SEQ ID NO: 311); (v) DPDGDYYYYGMDV (SEQ ID NO: 312); (vi) TYSSGWYVWDYYGMDV (SEQ ID NO: 313); (vii) DRVLSYYAMAV (SEQ ID NO: 314); (viii) VRIAGDYY YYYGMDV (SEQ ID NO: 315); (ix) ENIVVIPAAIFAGWFDP (SEQ ID NO: 316); and (x) DRA AAGLH YYY GMD V (SEQ ID NO: 317); or (c) the light chain CDR3 sequence of (a) and the heavy chain CDR3 sequence of (b); wherein, Xi is G, S or N; X2 is N, S or T; X3 is C, Y or_ S; X4 is G or S; X$ is A or S; X<, is P or F; X? is L or absent: X?4 is I, V or S; Xp, is L or V; Xp is L, T or V; X^ is L, V, G or T; Xp is A, G or absent; X2o is Y, C or D; X2i is I or Μ: X22 is A or V: and X2? is H or Y; and wherein the antigen binding protein specifically binds (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-KJotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4.

In another embodiment the provided antigen binding proteins comprise either: (a) a light chain CDR1 sequence selected from the group consisting of: (i) a light chain CDR1 that differs by no more than three amino acids additions, substitutions, and/or deletions from a CDR1 sequence of L1-L18, SEQ ID NOs: 158-170; (ii) RASQ X9 XpXnXpXpXuLA (SEQ ID NO: 304); (iii) GGNNIGSXisSVH (SEQ ID NO: 307); (iv) RSSQSLLXMXioNGXiiX^Xi.LD (SEQ ID NO: 310); (v) RASQSVNSNLA (SEQ ID NO: 295); (vi) RASQDIRYDLG (SEQ ID NO: 296); (vii) RASQG1SIWLA (SEQ ID NO: 297); and (viii) KSSQSLLQSDGKTYLY (SEQ ID NO: 298); (b) a light chain CDR2 sequence selected from the group consisting of: (i) a fight chain CDR2 that differs by no more than two amino acid additions, substitutions, and/or deletions from a CDR2 sequence of LI-LI8, SEQ ID NOs:171-179: (it) LGSX27RAS (SEQ ID NO: 290); (iii) GXSSX2SRAT (SEQ ID NO: 294); (iv) AASSLQS (SEQ ID NO: 284); (v) G VST RAT (SEQ ID NO: 285); (vi) DDSDRPS (SEQ ID NO: 286); (vii) EVSNRFS (SEQ ID NO: 287); (c) a heavy chain CDR1 sequence selected from the group consisting of: (i) a heavy chain CDR1 that differs by no more than two amino acid additions, substitutions, and/or deletions from a CDR1 sequence of H1-H18, SEQ ID NOs: 121 -131; and (ii) NARMGVX.p (SEQ ID NO: 352); (iii) X4()YGIH (SEQ ID NO: 355); (iv) DLSMH (SEQ ID NO: 345); (v) DAWMS (SEQ ID NO: 346); (vi) TYAMS (SEQ ID NO: 347); (vii) SYFWS (SEQ ID NO: 348); (viii) SYYWS (SEQ ID NO: 131); (ix) SGGYNWS (SEQ ID NO: 349); (d) a heavy chain CDR2 selected from the group consisting of: (i) a heavy sequence that differs by no more than three amino acid additions, substitutions, and /or deletions from a CDR2 sequence of H1-H18, SEQ ID NOs: 132-144; (ii) HIFSNDEKSYSTSLKX24 (SEQ ID NO: 333); (iii) X25ISGSGVSTX2cYADSVKG (SEQ ID NO: 338); (iv) VIWYDGSX.^KYYX^DSVKG (SEQ ID NO: 341); (v) X^lYXjsSGSTX^YNPSLKS (SEQ ID NO: 344); (vi) GFDPEDGETIYAQKFQG (SEQ ID NO: 327); (vii) RIKSKTDGGTTDYAAPVKG (SEQ ID NO: 328); (viii) R1YTSGSTNYNPSLKS (SEQ ID NO: 329); (ix) R1KSKDGGTTDYAAPVKG (SEQ ID NO: 330); (x) R1KSKX42DGGTTDYAAPVKG (SEQ ID NO: 483); wherein X9 is N or S; Χκι is V or F; Xu is D or S; X12 is G or S; Xu is S, N or T; Xu is S or Y; Xu is E or Q; X29 is Y or H; X,0 is Y or S; Xu is F or Y; X32 is T or N; X33 is Y or F; X27 is N or D; Xs is A or T; X2S is S or F; XJ9 is S or N; XM is S or N; X2S is G or A; X20 is Η, Y or N; X?5 is D or I; XM is A or G; X37 is N or R; X3g is Y or T; Xji is Y or N; X42 is T or absent; (e) the light chain CDR1 of (a) and the light chain CDR2 of (b); (f) the light chain CDR1 of (a) and the heavy chain CDR1 of (c); (g) the light chain C'DRl of (a) and the heavy chain CDR2 of (d); (h) the light chain C'DRl (b) and the heavy chain CDR1 of (c); (i) the heavy chain CDR1 of (c) and the heavy chain CDR2 of (d); (]) the light chain CDR2 of (b) and the heavy chain CDR2 of (d); (k) the light chain CDRI of fa), the light chain CDR2 of (b), and the heavy chain CDRI of (c); (1) the light chain CDR2 of (b), the heavy CDRI of (c), and the heavy chain CDR2 of (d); (m) the light chain CDRI of (a), the heavy chain CDRI of fc), and the heavy chain CDR2 of (d); or (η) the light chain C’DRl of (a), the light chain CDR2 of (b), the heavy chain CDR2 of (c), and the heavy chain CDR2 of (d), wherein said antigen binding protein specifically binds (i) β-Klotho; (ii) FGFRIc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRIc, FGFR2c, FGFR3c, and FGFR4.

In yet another embodiment the provided antigen binding proteins comprise cither: (a) a light chain variable domain comprising; (i) a light chain CDRI sequence selected from SEQ ID NOs:158-170; (it) a light chain CDR2 sequence selected from SEQ ID N0s:171-179; (iii) a light chain CDR3 sequence selected from SEQ ID NOs: 180-194; and (b) a heavy chain variable domain comprising: (i) a heavy chain CDRI sequence selected from SEQ ID NOs:I21-13l; (ii) a heavy chain CDR2 sequence selected from SEQ ID NOs: 132-144; and (iii) a heavy chain CDR3 sequence selected from SEQ ID NOs: 145-157; or (c) the light chain variable domain of (a) and the heavy chain variable domain of (b), wherein the antigen binding protein specifically binds (i) β-Klotho; (ii) FGFRIc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRIc, FGFR2c, FGFR3c, and FGFR4.

In a further embodiment the provided antigen binding proteins comprise either: (a) a light chain variable domain sequence selected from the group consisting of: (i) amino acids having a sequence at least 80% identical to a light chain variable domain sequence selected from Vl, 1 -V[ 18, SEQ ID NOs:48-65; (ii) a sequence of amino acids encoded by a polynucleotide sequence that is at least 80% identical to a polynucleotide sequence encoding the light chain variable domain sequence of Vl.1-Vi.18, SEQ ID NOs:48-65; (b) a heavy chain variable domain sequence selected from the group consisting of: (i) a sequence of amino acids that is at least 80% identical to a heavy chain variable domain sequence of Vh1-Vh18 of SEQ ID NOs:66-84; (ii) a sequence of amino acids encoded by a polynucleotide sequence that is at least 80% identical to a polynucleotide sequence encoding the heavy chain variable domain sequence of Vnl-Vnl8, SEQ ID NOs:66-84; or (c) the light chain variable domain of (a) and the heavy chain variable domain of (b); wherein the antigen binding protein specifically binds (i) β-Klotho: (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-KIotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4. In particular embodiments the provided antigen binding proteins comprise cither: (a) a light chain variable domain sequence selected from the group consisting of: V1J-V1J8 of SEQ ID NOs:48-65; (b) a heavy chain variable domain sequence selected from the group consisting of: Vnl-Vn 18 of SEQ ID NOs:66-84; or (c) the light chain variable domain of (a) and the heavy chain variable domain of (b), wherein the antigen binding protein specifically binds (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4. In other particular embodiments, the provided antigen binding proteins the light chain variable domain and a heavy chain variable domain are selected from the group of combinations consisting of: Vs IV,,], V, 2Vn2, V,3Va3, VL3V„4, Vr4V„5, VL5V„6, VL6V„7, VL7V„8, V,.8V„8, V,9VH9, Vl9Vh10, VlIOVuU, V,2IV„11, VL12V„ 12T VL13V„13, V, I4V„14, V,.15V„15, V,J6V„16, Vr. 17Vn 17, and Vl18Vi,18, wherein the antigen binding protein specifically binds (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Kfotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4. In still further embodiments the provided antigen binding proteins further comprise: (a) the light chain constant sequence of SEQ ID NO: 10; (b) the light chain constant sequence of SEQ ID ΝΟ:11; (c) the heavy chain constant sequence of SEQ ID NO: 9; or (d) the light chain constant sequence of SEQ ID NO: 10 or SEQ ID NO:l 1 and the heavy chain constant sequence of SEQ ID NO: 9.

The provided antigen binding proteins can take many forms and can be, for example, a human antibody, a humanized antibody, chimeric antibody, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, an antigen-binding antibody fragment, a single chain antibody, a diabody, a triabody, a tetrabody, a Fab fragment, an Fffab’h fragment, a domain antibody, an IgD antibody, an IgE antibody, an IgM antibody, an lgGl antibody, an IgG2 antibody, an lgG3 antibody, an IgG4 antibody, or an IgG4 antibody having at least one mutation in the hinge region.

In another embodiment, the provided antigen binding proteins when bound to (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4: (a) bind to (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4, with substantially the same Kd as a reference antibody; (b) induce FGF21-likc signaling of 10 % or greater than the signaling induced by a wild-type FGF21 standard comprising the mature form of SEQ ID NO:2 as measured in an ELK-iucifcrase reporter assay; (c) exhibit an EC50 of lOnM or less of FGF21-likc signaling in an assay selected from the group consisting of: (i) a FGFRlc^-Klotho-mcdiatcd in vitro recombinant cell-based assay; and (ii) an in vitro human adipocyte functional assay; (d) exhibit an EC50 of less than 1 OnM of agonistic activity on FGFRlc in the presence of β-KIotho in an in vitro recombinant FGFRlc receptor mediated reporter assay: and (e) exhibit an EC'50 of greater than 1 μΜ of agonistic activity on FGFRlc in the absence of β-Klotho in an in vitro recombinant FGFRlc receptor mediated reporter assay; or (f) competes for binding with a reference antibody to (i) β-KIotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-KIotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4, wherein the reference antibody comprises a combination of light chain and heavy chain variable domain sequences selected from the group consisting of VL1V„1, VL2V„2, VL3Vn3, VL3Vn4, V,4Vn5, VL5Vu6, Vl/)Vh7, VL7Vn8, Vl8Vh8, Vi9Vh9, Vi.9VH10, ν, ΙΟν,,Π, Vr. 11VH11, VL12Vn12, Vr..l3VH13, Vl14Vh14, V[,15Vm]5, Vs I6Vh16, Vi„17Vnl7, and Vj 18Vnl8. In other embodiments the provided antigen binding proteins can when bound to (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4: (a) lower blood glucose in an animal model; (b) lower scrum lipid levels in an animal model; (c) lower insulin levels in an animal model; or (d) two or more of (a) and (b) and (c).

In specific embodiments the provided antigen binding proteins comprise: (a) a heavy chain comprising one of SEQ ID NOs:31, 32, 390-401,404-405; (b) a light chain comprising one of SEQ ID NO: 13, 14, 385-389, 402-403; or (c) a combination comprising a heavy chain of (a) and a light chain of (b).

Also provided are antigen binding proteins that are capable of binding wild type human β-KIotho (SEQ ID NO:7) but which doesn’t bind to a chimeric form of β-Klotho wherein the chimeric form of β-Klotho comprises a human β-Klotho framework wherein murine β-Klotho sequences replace the wild type human residues at at least one of (a) positions 1-80; (b) positions 303-522; (c) positions 852-1044; and (d) combinations thereof.

In another aspect, the present disclosure provides antigen binding proteins that are capable of binding wild type human β-Klotho (SEQ ID NO:7) at at least one of (a) positions I-80; (b) positions 303-522; (c) positions 852-1044; and (d) combinations thereof.

In still another aspect, the present disclosure provides antigen binding proteins that arc capable of competing with an antigen binding protein of claims 8 or 13 for binding to human wild type β-Klotho residues at at least one of (a) positions 1-80; (b) positions 303-522; (c) positions 852-1044; and (d) combinations thereof.

Also provided is a pharmaceutical composition comprising one or more antigen binding proteins provided herein, in admixture with a pharmaceutically acceptable carrier thereof.

In a further aspect, also provided arc isolated nucleic acid molecules that encode the antigen binding proteins disclosed herein. In some instances, the isolated nucleic acid molecules arc opcrably-linked to a control sequence. In embodiments, such nucleic acids comprise a polynucleotide sequence encoding the light chain variable domain, the heavy chain variable domain, or both, of an antigen binding protein provided herein. In particular embodiments the nucleic acids comprise (a) V,J-Vr 18 (SEQ ID NOs:48-65); (b) V„l-V„18 (SEQ ID NOs:66-84); or (c) one or more sequences of (a) and one or more sequences of (b).

In another aspect, also provided arc expression vectors and host cells transformed or transfected with the expression vectors that comprise the aforementioned isolated nucleic acid molecules that encode the antigen binding proteins disclosed herein.

In another aspect, also provided are methods of preparing antigen binding proteins that specifically or selectively bind (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4 and comprises the step of preparing the antigen binding protein from a host cel! that secretes the antigen binding protein.

Other embodiments provide a method of preventing or treating a condition in a subject in need of such treatment comprising administering a therapeutically effective amount of a pharmaceutical composition provided herein to a subject, wherein the condition is treatable by lowering blood glucose, insulin or serum lipid levels. In embodiments, the condition is type 2 diabetes, obesity, dyslipidemia, NASH, cardiovascular disease or metabolic syndrome.

These and other aspects are described in greater detail herein. Each of the aspects provided can encompass various embodiments provided herein. It is therefore anticipated that each of the embodiments involving one element or combinations of elements can be included in each aspect described, and all such combinations of the above aspects and embodiments are expressly considered. Other features and advantages of the disclosed antigen binding proteins and associated methods and compositions are apparent in the detailed description that follows.

Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A-1B is an alignment showing the sequence homology between human FGFRlc (GenBank Accession No PI 1362; SEQ ID NO: 356) and murine FGFRlc (GenBank Accession No NP_034336; SEQ ID NO: 357); various features are highlighted, including the signal peptide, transmembrane sequence, heparin binding region, and a consensus sequence (SEQ ID NO: 358) is provided.

Figure 2a-2c is an alignment showing the sequence homology between human β-Klotho (GenBank Accession No NP_783864; SEQ ID NO: 359) and murine β-Klotho (GenBank Accession No NP_112457; SEQ ID NO: 360); various features are highlighted, including the transmembrane sequence and two glycosyl hydrolase domains, and a consensus sequence (SEQ ID NO: 361) is provided.

Figure 3 is a flow cytometry profile of cells stained with FGF21-Alexa 647 that were used as an immunogen to generate antigen binding proteins; the figure shows the expression level of an FGF21R (a complex comprising FGFRlc and β-Klotho) and binding to FGF21.

Figure 4 is a sequence (SEQ ID NO: 362) showing an Fc fusion protein that was used as an immunogen to generate antigen binding proteins; the immunogen comprises the extracellular domain (ECD) of human FGFRlc fused to an IgGl Fc via a Glys linker (SEQ ID NO: 379); the FGFRlc component is in capitals, the linker is italic and underlined and the Fc is in lower case letters._

Figure 5 is a sequence (SEQ ID NO: 363) showing an Fc fusion protein that was used as an immunogen to generate antigen binding proteins: the immunogen comprises the extracellular domain (ECD) of human β-Klotho fused to an IgGl Fc via a Gly5 linker {SEQ ID NO: 379); the β-KIotho component is in capitals, the linker is italic and underlined and the Fc is in lower case letters.

Figure 6 is a SDS PAGE gel showing the level of purity achieved from preparations of a soluble FGF21 receptor complex comprising FGFRlc ECD-Fc and β-Klotho ECD-Fc, which was employed as an immunogen to generate antigen binding proteins.

Figure 7 is a scries of plots generated from an ELK-Iuciferasc reporter assay as described herein performed on recombinant CHO clone 2E10, demonstrating the ability of some of the antigen binding proteins to induce FGF21 -like signaling in recombinant CHO cells expressing a FGF21 receptor complex comprising FGFR lc and β-Klotho.

Figure 8 is a scries of plots generated from an ERK1/2 phosphorylation assay as described herein, demonstrating the ability of some of the antigen binding proteins to induce FGF21 -like signaling in rat L6 cells. The X-axis is the concentrations of the antigen binding proteins and the Y-axis is the percentage of phosphorylatcd ERK1/2 of total ERKi/2.

Figure 9 is a series of plots generated from an ERK.1/2 phosphorylation assay as described herein, demonstrating that antigen binding protein-mediated FGF2i-like signaling in L6 cells is FGFRlc.^-Klotho specific.

Figure 10 is a series of plots generated from an ERK phosphorylation assay as described herein, demonstrating that some antigen binding proteins are able to induce FGF21-likc signaling in human adipocyte cells.

Figure 1 la is a scries of binding sensorgrams (response units vs time) demonstrating that some of the antigen binding proteins that induce FGF21-mediated signaling bind to human β-Klotho at two different but partially overlapping binding sites represented by 24H11 (Group A) and 17D8 (Group B), while antigen binding proteins that do not induce FGF21-mediated signaling (2G10, 1A2) do not bind to these sites.

Figure 11b is a series of binding sensorgrams (response units vs time) demonstrating a third binding site on human β-Klotho that was identified for Group C antigen binding proteins represented by 39F7.

Figure 12 is a series of binding sensorgrams (response units vs time) demonstrating that some of the antigen binding proteins (12E4, 24H11, 17C3, 18BI1) that induce FGF21-mediated signaling interfere with β-Klotho binding to FGF21, while other antigen binding proteins (21H2, 17D8. 18G1) do not.

Figure 13 is an alignment of the variable regions of some of the antigen binding proteins that were generated; the framework and CDR regions are identified. Figure 13 discloses SEQ ID NOS: 364, 59, 365, 60, 366, 61, 367, 62, 368, 57, 369, 55, 51-52, 56, 56, 53-54, 63-65, 370, 58, 371, 50, 50, 49, 48, 372, 78, 373, 66-69, 79, 374, 76, 81, 375, 70, 73, 73, 71-72, 376, 83, 82, 84, 377, 80,378, 75 and 74, respectively, in order of appearance.

Figure 14 is a diagram graphically depicting the study design for a 68 days study performed in obese cynomolgus monkeys.

Figure 15 is a plot depicting the effects of vehicle and 16H7 on AM meal food intake of the obese cynomolgus monkeys studied.

Figure 16 is two plots depicting the effects of vehicle and 16H7 on fruit intake and PM food intake of the obese cynomolgus monkeys studied.

Figure 17 is a plot depicting the effects of vehicle and 16H7 on body weight of the obese cynomolgus monkeys studied.

Figure 18 is a plot showing the effects of vehicle and 16H7 on body mass index (BM1) of the obese cynomolgus monkeys studied.

Figure 19 is a plot showing the effects of vehicle on abdominal circumference (AC) of the obese cynomolgus monkeys studied.

Figure 20 is a plot showing the effects of vehicle and 16H7 on skin fold thickness (SFT) of the obese cynomolgus monkeys studied.

Figure 21 is a plot showing the effects of vehicle and 16H7 on glucose levels during glucose tolerance tests of the obese cynomolgus monkeys studied.

Figure 22 is a plot showing the effects of vehicle and 16H7 on plasma insulin levels during glucose tolerance tests of the obese cynomolgus monkeys studied.

Figure 23 is a plot showing the effects of vehicle and 16H7 on fasting plasma glucose levels of the obese cynomolgus monkeys studied.

Figure 24 is a plot showing the effects of vehicle and I6H7 on fasting plasma insulin levels of the obese cynomolgus monkeys studied.

Figure 25 is a plot showing the effects of vehicle and 16H7 on fed plasma glucose levels of the obese cynomolgus monkeys studied.

Figure 26 is a plot showing the effects of vehicle and 16H7 on fed plasma insulin levels of the obese cynomolgus monkeys studied.

Figure 27 is a plot showing the effects of vehicle and 16H7 on fasting plasma triglyceride levels of the obese cynomolgus monkeys studied.

Figure 28 is a plot showing the effects of vehicle and 16H7 on fed plasma triglyceride levels of the obese cynomolgus monkeys studied.

Figure 29 is a schematic depicting human-mouse β-Klotho chimeras that were constructed and used to studying the binding of antigen binding proteins.

Figure 30 is a schematic depicting the human-mouse β-Klotho chimeras that were constructed and also includes qualitative binding data for FGF21, 16H7, 37D3 and 39F7.

Figure 31A-C is a series of plots depicting binding data for eight of the 16H7 and 22H5 variants that were constructed, as well as for 22H5 and I6H7.

Figures 32A-C is a scries of plots depicting the results of ELISA assays that were used to demonstrate that several of the 22H5 and 16H7 variants have binding ability.

Figure 33 is a bar graph comparing off-rates for several 22H5 and I7H7 variants that were generated.

Figure 34 is two plots that depict binding curves for 39F11 when titrated with FGF21 and for FGF21 when titrated with 39FI1; the plots demonstrate an additive effect.

Figure 35 is two plots that depict binding curves for 16H7 when titrated with 39F11 and 39FI1 when it is titrated with 16H7; the plots demonstrate an additive effect.

DETAILED DESCRIPTION

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that arc commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein arc those well known and commonly used in the art. The methods and techniques of the present application arc generally performed according to conventional methods well known in the art and as described in various general and more specific references that arc cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Sambrook et a/,, Molecular Cloning: A Laboratory Manual, 3rd ed,, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001), Ausubel et a!., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), and Harlow and Lane Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990), which are incosporated herein by reference. Enzymatic reactions and purification techniques arc performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The terminology used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques can be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

It should be understood that the instant disclosure is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure.

Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term ’‘about.” The term “about” when used in connection with percentages can mean ±5%, e.g., 1%, 2%, 3%, or 4%.

I. DEFINITIONS

As used herein, the terms “a” and “an” mean “one or more” unless specifically stated otherwise.

An “antigen binding protein” is a protein comprising a portion that binds to an antigen or target and, optionally, a scaffold or framework portion that allows the antigen binding portion to adopt a conformation that promotes binding of the antigen binding protein to the antigen. Examples of antigen binding proteins include a human antibody, a humanized antibody; a chimeric antibody; a recombinant antibody; a single chain antibody; a diabody; a triabody; a tetrabody; a Fab fragment; a Ffab'b fragment; an IgD antibody; an IgE antibody; an IgM antibody; an IgGl antibody; an igG2 antibody; an lgG3 antibody; or an IgG4 antibody, and fragments thereof. The antigen binding protein can comprise, for example, an alternative protein scaffold or artificial scaffold with grafted CDRs or CDR derivatives. Such scaffolds include, but are not limited to, antibody-derived scaffolds comprising mutations introduced to, for example, stabilize the three-dimensional structure of the antigen binding protein as well as wholly synthetic scaffolds comprising, for example, a biocompatiblc polymer. See, e.g., Komdorfer et al„ 2003, Proteins: Structure, Function, and Bioinformatics, 53(1):121-129 (2003); Roque ct al„ Biotechnoi. Prog. 20:639-654 (2004). In addition, peptide antibody mimctics ("PAiVls”) can be used, as well as scaffolds based on antibody mimctics utilizing fibroncctin components as a scaffold.

An antigen binding protein can have, for example, the structure of a naturally occurring immunoglobulin. An “immunoglobulin” is a tctrameric molecule. In a naturally occurring immunoglobulin, each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as kappa and lambda light chains. Heavy chains arc classified as mu, delta, gamma, alpha, or epsilon, and define the antibody’s isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions arc joined by a “J” region of about 12 or more amino acids, with the heavy chain also including a “D” region of about 10 more amino acids. See generally, Fundamental Immunology C'h. 7 (Paul, W„ ed., 2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference in its entirety for all purposes). The variable regions of each light/hcavy chain pair form the antibody binding site such that an intact immunoglobulin has two binding sites.

Naturally occurring immunoglobulin chains exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs, From N-terminus to C-tcmiinus, both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain can be done in accordance with the definitions of Rabat et al. in Sequences of Proteins of Immunological Interest, 5Ul Ed., US Dept, of Health and Human Services, PHS, ΜΗ, NIH Publication no. 91-3242, 1991. As desired, the CDRs can also be redefined according an alternative nomenclature scheme, such as that of Chothia (sec Chothia &amp; Lesk, 1987,./. Mol. Biol. I%:901-917; Chothia et a!.. 1989, Nature 342:878-883 or Honegger &amp; Pluckthun, 2001,,/. Mol. Biol. 309:657-670).

In the context of the instant disclosure an antigen binding protein is said to ‘‘specifically bind” or “selectively bind” its target antigen when the dissociation constant (Kd) is <10's M. The antibody specifically binds antigen with “high affinity” when the Kn is <5x 10’9 M, and with “very high affinity” when the Kd is <5x 10'IW M. In one embodiment, the antibodies will bind to FGFRlc, β-KIotho, both FGFRlc and β-Klotho ora complex comprising FGFRIc and β-KIotho, including human FGFRlc, human β-Klotho or both human FGFRlc and human β-Klotho, with a Kd of between about 10" M and 10‘12 M, and in yet another embodiment the antibodies will bind with a Kd <5,k 10'9.

An “antibody” refers to an intact immunoglobulin or to an antigen binding portion thereof that competes with the intact antibody for specific binding, unless otherwise specified. Antigen binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Antigen binding portions include, inter alia, Fab, Fab’, F(ab’)2, Fv, domain antibodies (dAbs), fragments including complementarity determining regions (CDRs), single-chain antibodies (scFv), chimeric antibodies, diabodies, triabodies, tetrabodies, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide. A Fab fragment is a monovalent fragment having the VL, Vn, Cl and Cnl domains; a F(ab’)2 fragment is a bivalent fragment having two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment has the VH and Cnl domains; an Fv fragment has the Vj, and Vn domains of a single arm of an antibody; and a dAb fragment has a VH domain, a V), domain, or an antigen-binding fragment of a Vn or Vi, domain (US Pat. Nos. 6,846,634, 6,696,245, US App. Pub. Nos. 05/0202512, 04/0202995, 04/0038291, 04/0009507, 03/0039958, Ward ct al., Nature 341:544-546(1989)). A single-chain antibody (scFv) is an antibody in which a Vr„ and a Vh region are joined via a linker (e.g., a synthetic sequence of amino acid residues) to form a continuous protein chain wherein the linker is long enough to allow the protein chain to fold back on itself and form a monovalent antigen binding site (see, e.g., Bird ct al.. Science 242:423-26 ¢1988) and Huston et al., 1988, Proc. Natl, Acad. Sci. USA 85:5879-83 (1988)). Diabodies are bivalent antibodies comprising two polypeptide chains, wherein each polypeptide chain comprises Vh and VL domains joined by a linker that is too short to allow for pairing between two domains on the same chain, thus allowing each domain to pair with a complementary domain on another polypeptide chain (see, e.g., Holligeret ai„ 1993, Proc. Natl. Acad. Sci. USA 90:6444-48 (1993), and Poljak et al., Structure 2:1121-23 (1994)). If the two polypeptide chains of a diabody are identical, then a diabody resulting from their pairing will have two identical antigen binding sites. Polypeptide chains having different sequences can be used to make a diabody with two different antigen binding sites. Similarly, tribodics and tctrabodics are antibodies comprising three and four polypeptide chains, respectively, and forming three and four antigen binding sites, respectively, which can be the same or different.

Complementarity determining regions (CDRs) and framework regions (FR) of a given antibody can be identified using the system described by Rabat et al. in Sequences of Proteins of Immunological Interest, 5th Ed., US Dept, of Health and Human Services, PHS, NJH, NIH Publication no. 91-3242, 1991 As desired, the CDRs can also be redefined according an alternative nomenclature scheme, such as that of Chothia (see Cholhia &amp; Lesk, 1987,,/. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342:878-883 or Honegger &amp; Pluckthun, 2001,,/. Mol. Biol. 309:657-670. One or more CDRs can be incorporated into a molecule either covalently or noncovalently to make it an antigen binding protein. An antigen binding protein can incorporate the CDR(s) as part of a larger polypeptide chain, can covalently link the CDR(s) to another polypeptide chain, or can incorporate the CDR(s) noncovalently. The CDRs permit the antigen binding protein to specifically bind to a particular antigen of interest.

An antigen binding protein can have one or more binding sites, if there is more than one binding site, the binding sites can be identical to one another or can be different. For example, a naturally occurring human immunoglobulin typically has two identical binding sites, while a “bispecific”or “bifunctional” antibody has two different binding sites.

The term “human antibody” includes all antibodies that have one or more variable and constant regions derived from human immunoglobulin sequences. In one embodiment, all of the variable and constant domains are derived from human immunoglobulin sequences (a fully human antibody). These antibodies can be prepared in a variety of ways, examples of which are described below, including through the immunization with an antigen of interest of a mouse that is genetically modified to express antibodies derived from human heavy and/or fight chainencoding genes, such as a mouse derived from a XcnomouseflP, UltiMab™, or Velocimmunc(R> system. Phage-based approaches can also be employed. A humanized antibody has a sequence that differs from the sequence of an antibody derived from a non-human species by one or more amino acid substitutions, deletions, and/or additions, such that the humanized antibody is less likely to induce an immune response, and/or induces a less severe immune response, as compared to the non-human species antibody, when it is administered to a human subject. In one embodiment, certain amino acids in the framework and constant domains of the heavy and/or light chains of the non-human species antibody arc mutated to produce the humanized antibody. In another embodiment, the constant domain(s) from a human antibody arc fused to the variable domain(s) of a non-human species. In another embodiment, one or more amino acid residues in one or more CDR sequences of a non-human antibody arc changed to reduce the likely immunogenicity of the non-human antibody when it is administered to a human subject, wherein the changed amino acid residues cither arc not critical for immunospecific binding of the antibody to its antigen, or the changes to the amino acid sequence that are made are conservative changes, such that the binding of the humanized antibody to the antigen is not significantly worse than the binding of the non-human antibody to the antigen. Examples of how to make humanized antibodies can be found in U.S. Pat. Nos. 6,054,297, 5,886,152 and 5,877,293.

The term “chimeric antibody” refers to an antibody that contains one or more regions from one antibody and one or more regions from one or more other antibodies. In one embodiment, one or more of the CDRs are derived from a human antibody that binds (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (Hi) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4. In another embodiment, all of the CDRs arc derived from a human antibody that binds (i) β-Klotho; (ii) FGFRlc. FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFRSc, and FGFR4. In another embodiment, the CDRs from mom than one human antibody that binds (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4 are mixed and matched in a chimeric antibody. For instance, a chimeric antibody can comprise a CDR1 from the light chain of a first human antibody that binds (i) β-ICiotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4, a CDR2 and a CDR3 from the light chain of a second human antibody that binds (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4, and the CDRs from the heavy chain from a third antibody that binds (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4. Further, the framework regions can be derived from one of the same antibodies that bind (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4, from one or more different antibodies, such as a human antibody, or from a humanized antibody. In one example of a chimeric antibody, a portion of the heavy and/or light chain is identical with, homologous to, or derived from an antibody from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is/are identical with, homologous to, or derived from an antibody or antibodies from another species or belonging to another antibody class or subclass. Also included are fragments of such antibodies that exhibit the desired biological activity (e.g., the ability to specifically bind (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4).

The term “light chain” includes a full-length light chain and fragments thereof having sufficient variable region sequence to confer binding specificity. A full-length light chain includes a variable region domain, V|„, and a constant region domain, Ci„. The variable region domain of the light chain is at the amino-terminus of the polypeptide, Light chains include kappa (“κ”) chains and lambda (“λ”) chains.

The term “heavy chain” includes a full-length heavy chain and fragments thereof having sufficient variable region sequence to confer binding specificity. A full-length heavy chain includes a variable region domain, Vn, and three constant region domains, CuL Cu2, and Cn3,

The Vj] domain is at the amino-terminus of the polypeptide, and the Cu domains arc at the carboxyl-terminus, with the Ch3 being closest to the carboxy-terminus of the polypeptide. Heavy chains can be of any isotype, including IgG (including IgGl, 3gG2, !gG3 and IgG4 subtypes), IgA (including IgA I and IgA2 subtypes), IgM and lgE.

The term “immunologically functional fragment” (or simply “fragment”) of an antigen binding protein, e.g., an antibody or immunoglobulin chain (heavy or light chain), as used herein, is an antigen binding protein comprising a portion (regardless of how that portion is obtained or synthesized) of an antibody that lacks at least some of the amino acids present in a full-length chain but which is capable of specifically binding to an antigen. Such fragments are biologically active in that they bind specifically to the target antigen and can compete with other antigen binding proteins, including intact antibodies, for specific binding to a given epitope. In one aspect, such a fragment will retain at least one CDR present in the full-length light or heavy chain, and in some embodiments will comprise a single heavy chain and/or light chain or portion thereof. These biologically active fragments can be produced by recombinant DNA techniques, or can be produced by enzymatic or chemical cleavage of antigen binding proteins, including intact antibodies. Immunologically functional immunoglobulin fragments include, but are not limited to, Fab, Fab’, F(ab’)2, Fv, domain antibodies and single-chain antibodies, and can be derived from any mammalian source, including but not limited to human, mouse, rat, camelid or rabbit. It is contemplated further that a functional portion of the antigen binding proteins disclosed herein, for example, one or more CDRs, could be covalently bound to a second protein or to a small molecule to create a therapeutic agent directed to a particular target in the body, possessing bifunctional therapeutic properties, or having a prolonged scrum half-life.

An “Fc” region contains two heavy chain fragments comprising the Cn2 and Ch3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the Ch3 domains.

An “Fab’ fragment” contains one light chain and a portion of one heavy chain that contains the V» domain and the Cul domain and also the region between the C»! and Cn2 domains, such that an interchain disulfide bond can be formed between the two heavy chains of two Fab’ fragments to form an F(ab')2 molecule.

An “F(ab’)2 fragment” contains two light chains and two heavy chains containing a portion of the constant region between the C»! and Cn2 domains, such that an interchain disulfide bond is formed between the two heavy chains. A Ffab’b fragment thus is composed of two Fab’ fragments that are held together by a disulfide bond between the two heavy chains.

The “Fv region” comprises the variable regions from both the heavy and light chains, but lacks the constant regions. A “domain antibody” is an immunologically functional immunoglobulin fragment containing only the variable region of a heavy chain or the variable region of a light chain. In some instances, two or more Vu regions are covalently joined with a peptide linker to create a bivalent domain antibody. The two Vu regions of a bivalent domain antibody can target the same or different antigens. A “hemibody” is an immunologically functional immunoglobulin construct comprising a complete heavy chain, a complete light chain and a second heavy chain Fc region paired with the Fc region of the complete heavy chain. A linker can, but need not, be employed to join the heavy chain Fc region and the second heavy chain Fc region. In particular embodiments a hemibody is a monovalent form of an antigen binding protein disclosed herein. In other embodiments, pairs of charged residues can be employed to associate one Fc region with the second Fc region. The second heavy chain Fc region can comprise, for example, SEQ ID NO:44l and can be joined to the light chain via a linker fe.g., SEQ ID NO:440) An exemplary hemibody heavy chain comprises the sequence SEQ ID NO:453. A “bivalent antigen binding protein” or “bivalent antibody” comprises two antigen binding sites. In some instances, the two binding sites have the same antigen specificities. Bivalent antigen binding proteins and bivalent antibodies can be bispecific, as described herein. A multispccific antigen binding protein” or “multispccific antibody” is one that targets more than one antigen or epitope. A “bispccific,” “dual-specific” or “bifunctional” antigen binding protein or antibody is a hybrid antigen binding protein or antibody, respectively, having two different antigen binding sites. Bispccific antigen binding proteins and antibodies arc a species of multispccific antigen binding protein or multispccific antibody and can be produced by a variety of methods including, but not limited to, fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai and Lachmann, 1990, Clin. Exp. Immunol. 79:315-321; Kostclny eta!., 1992,,/. Immunol. 148:1547-1553. The two binding sites of a bispecific antigen binding protein or antibody will bind to two different epitopes, which can reside on the same or different protein targets.

The terms “FGF2i-like signaling” and “induces FGF21-like signaling,” when applied to an antigen binding protein of the present disclosure, means that the antigen binding protein mimics, or modulates, an in vivo biological effect induced by the binding of (i) β-Klotho: (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4 and induces a biological response that otherwise would result from FGF21 binding to (i) β-Klotho; {ii) FGFRlc, FGFR2c. FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFR1 c, FGFR2c, FGFR3c, and FGFR4 in vivo. In assessing the binding and specificity of an antigen binding protein, e.g., an antibody or immunologically functional fragment thereof, an antibody or fragment is deemed to induce a biological response when the response is equal to or greater than 5%, and preferably equal to or greater than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%, of the activity of a wild type FGF21 standard comprising the mature form of SEQ ID NO:2 {/.<?., the mature form of the human FGF21 sequence) and has the following properties: exhibiting an efficacy level of equal to or more than 5% of an FGF21 standard, with an EC50 of equal to or less than lOOnM, e.g., 90 nM, 80 nM, 70nM, 60nM, 50nM, 40nM, 30nM, 20nM or 10 nM in (1) the recombinant FGF21 receptor mediated luciferase-reporter cell assay of Example 5; (2) ERK-phosphorylation in the recombinant FGF21 receptor mediated cell assay of Example 5; and (3) ERK-phosphorylation in human adipocytes as described in Example 7. The “potency” of an antigen binding protein is defined as exhibiting an EC50 of equal to or less than lOOnM, e.g,, 90nM, 80nM, 70nM, 60nM, 50nM, 40nM, 30nM, 20nM, 10 nM and preferably less than lOnM of the antigen binding protein in the following assays: (1) the recombinant FGF2I receptor mediated iuciferasc-reportcr cell assay of Example 5; (2) the ERK-phosphorylation in. the recombinant FGF21 receptor mediated cell assay of Example 5; and (3) ERK-phosphorylation in human adipocytes as described in Example 7.

It is noted that not all of the antigen binding proteins of the present disclosure induce FGF21-mediated signaling, nor is this property desirable in all circumstances. Nevertheless, antigen binding proteins that do not induce FGF21-mediated signaling form aspects of the present disclosure and may be useful as diagnostic reagents or other applications.

As used herein, the term “FGF21R” means a multimeric receptor complex that FGF2I is known or suspected to form in vivo. In various embodiments, FGF21R comprises (i) an FGFR, e.g., FGFRlc, FGFR2c, FGFR3c or FGFR4, and (ii) β-Klotho.

The term “polynucleotide” or “nucleic acid” includes both single-stranded and double-stranded nucleotide polymers. The nucleotides comprising the polynucleotide can be ribonucleotides or dcoxyribonucleotidcs or a modified form of cither type of nucleotide. Said modifications include base modifications such as bromouridine and inosine derivatives, ribosc modifications such as 2', 3’-didcoxyribosc, and intcrnuclcotidc linkage modifications such as phosphorothioatc, phosphorodithioate, phosphorosclenoate, phosphorodisclcnoatc, phosphoroanilothioate, phoshoraniladate and phosphoroamidatc.

The term “oligonucleotide” means a polynucleotide comprising 200 or fewer nucleotides. In some embodiments, oligonucleotides are 10 to 60 bases in length. In other embodiments, oligonucleotides are 12, 13, 14, 15, 16, 17, 18, 39, or 20 to 40 nucleotides in length. Oligonucleotides can be single stranded or double stranded, e.g., for use in the construction of a mutant gene. Oligonucleotides can be sense or antisense oligonucleotides. An oligonucleotide can include a label, including a radio label, a fluorescent label, a hapten or an antigenic label, for detection assays. Oligonucleotides can be used, for example, as PCR primers, cloning primers or hybridization probes.

An “isolated nucleic acid molecule” means a DNA or RNA of genomic, mRNA, cDNA, or synthetic origin or some combination thereof which is not associated with all or a portion of a polynucleotide in which the isolated polynucleotide is found in nature, or is linked to a polynucleotide to which it is not linked in nature. For purposes of this disclosure, it is understood that “a nucleic acid molecule comprising” a particular nucleotide sequence does not encompass intact chromosomes. Isolated nucleic acid molecules “comprising” specified nucleic acid sequences can include, in addition to the specified sequences, coding sequences for up to ten or even up to twenty other proteins or portions thereof, or can include operably linked regulatory sequences that control expression of the coding region of the recited nucleic acid sequences, and/or can include vector sequences.

Unless specified otherwise, the left-hand end of any single-stranded polynucleotide sequence discussed herein is the 5’ end; tlic left-hand direction of double-stranded polynucleotide sequences is referred to as the 5’ direction. The direction of 5’ to 3' addition of nascent RNA transcripts is referred to as the transcription direction; sequence regions on the DNA strand having the same sequence as the RNA transcript that are 5’ to the 5’ end of the RNA transcript are referred to as “upstream sequences;” sequence regions on the DNA strand having the same sequence as the RNA transcript that are 3' to the 3’ end of the RNA transcript are referred to as “downstream sequences.”

The term “control sequence” refers to a polynucleotide sequence that can affect the expression and processing of coding sequences to which it is ligated. The nature of such control sequences can depend upon the host organism. In particular embodiments, control sequences for prokaryotes can include a promoter, a ribosomal binding site, and a transcription termination sequence. For example, control sequences for eukaryotes can include promoters comprising one or a plurality of recognition sites for transcription factors, transcription enhancer sequences, and transcription termination sequence. “Control sequences” can include leader sequences and/or fusion partner sequences.

The term “vector” means any molecule or entity fe.g., nucleic acid, plasmid, bacteriophage or virus) used to transfer protein coding information into a host cell.

The term “expression vector” or “expression construct” refers to a vector that is suitable for transformation of a host cell and contains nucleic acid sequences that direct and/or control (in conjunction with the host ceil) expression of one or more heterologous coding regions operatively linked thereto. An expression construct can include, but is not limited to, sequences that affect or control transcription, translation, and, if nitrons are present, affect RNA splicing of a coding region operably linked thereto.

As used herein, “operably linked” means that the components to which the term is applied are in a relationship that allows them to cany out their inherent functions under suitable conditions. For example, a control sequence in a vector that is "operably linked" to a protein coding sequence is ligated thereto so that expression of the protein coding sequence is achieved under conditions compatible with the transcriptional activity of the control sequences.

The term “host cell” means a cell that has been transformed, or is capable of being transformed, with a nucleic acid sequence and thereby expresses a gene of interest. The term includes the progeny of the parent cell, whether or not the progeny is identical in morphology or in genetic make-up to the original parent cell, so long as the gene of interest is present.

The term “transduction” means the transfer of genes from one bacterium to another, usually by bacteriophage. “Transduction” also refers to the acquisition and transfer of eukaryotic cellular sequences by replication-defective retroviruses.

The term “transfection” means the uptake of foreign or exogenous DNA by a ceil, and a ceil has been “transfected” when the exogenous DNA has been introduced inside the cell membrane. A number of transfection techniques are well known in the art and arc disclosed herein. See, e.g., Graham el al., (1973) Virology 52:456: Sambrook et a/., (2001) Molecular Cloning: A Laboratory Manual, supra; Davis el al., (1986) Basic Methods in Molecular Biology, Elsevier; Chu et al., (1981) Gene J_3:197. Such techniques can be used to introduce one or more exogenous DNA moieties into suitable host cells.

The term “transformation” refers to a change in a cell's genetic characteristics, and a cell has been transformed when it has been modified to contain new DNA or RNA. For example, a cell is transformed where it is genetically modified from its native state by introducing new genetic material via transfection, transduction, or other techniques. Following transfection or transduction, the transforming DNA can recombine with that of the cell by physically integrating into a chromosome of the cell, or can be maintained transiently as an episomal element without being replicated, or can replicate independently as a plasmid. A cell is considered to have been “stably transformed” when the transforming DNA is replicated with the division of the cell.

The terms “polypeptide” or “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms also apply to amino acid polymers in which one or more amino acid residues is an analog or mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The terms can also encompass amino acid polymers that have been modified, e.g., by the addition of carbohydrate residues to form glycoproteins, or phosphorylated. Polypeptides and proteins can be produced by a naturally-occurring and non-recombinant cell, or polypeptides and proteins can be produced by a genetically-engineered or recombinant cell. Polypeptides and proteins can comprise molecules having the amino acid sequence of a native protein, or molecules having deletions from, additions to, and/or substitutions of one or more amino acids of the native sequence. The terms “polypeptide” and “protein” encompass antigen binding proteins that specifically or selectively bind (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4, or sequences that have deletions from, additions to, and/or substitutions of one or more amino acids of an antigen binding protein that specifically or selectively binds (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4. The term “polypeptide fragment” refers to a polypeptide that has an amino-terminal deletion, a carboxyl-terminal deletion, and/or an internal deletion as compared with the full-length protein. Such fragments can also contain modified amino acids as compared with the full-length protein. In certain embodiments, fragments arc about five to 500 amino acids long. For example, fragments can be at least 5, 6, 8, 10, 14. 20, 50, 70, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids long. Useful polypeptide fragments include immunologically functional fragments of antibodies, including binding domains. In the case of an antigen binding protein that binds to (i) β-Kiolho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4, useful fragments include but are not limited to a CDR region, a variable domain of a heavy or light chain, a portion of an antibody chain or just its variable region including two CDRs, and the like.

The term “isolated protein” referred means that a subject protein (1) is free of at least some other proteins with which it would normally be found, (2) is essentially free of other proteins from the same source, e.g., from the same species, (3) is expressed by a cell from a different species, (4) has been separated from at least about 50 percent of polynucleotides, lipids, carbohydrates, or other materials with which it is associated in nature, (5) is opcrably associated (by covalent or noncovalcnt interaction) with a polypeptide with which it is not associated in nature, or (6) does not occur in nature. Typically, an “isolated protein” constitutes at least about 5%, at least about 10%, at least about 25%, or at least about 50% of a given sample. Genomic DNA, cDNA, mRNA or other RNA, of synthetic origin, or any combination thereof can encode such an isolated protein. Preferably, the isolated protein is substantially free from proteins or polypeptides or other contaminants that are found in its natural environment that would interfere with its therapeutic, diagnostic, prophylactic, research or other use. A “variant” of a polypeptide (e.g., an antigen binding protein, or an antibody) comprises an amino acid sequence wherein one or more amino acid residues are inserted into, deleted from and/or substituted into the amino acid sequence relative to another polypeptide sequence. Variants include fusion proteins. A “derivative” of a polypeptide is a polypeptide (e.g., an antigen binding protein, or an antibody) that has been chemically modified in some manner distinct from insertion, deletion, or substitution variants, e.g., by conjugation to another chemical moiety.

The term “naturally occurring” as used throughout the specification in connection with biological materials such as polypeptides, nucleic acids, host cells, and the like, refers to materials which arc found in nature. “Antigen binding region” means a protein, or a portion of a protein, that specifically binds a specified antigen, e.g., FGFRlc, β-Klotho or both FGFRlc and β-Klotho. For example, that portion of an antigen binding protein that contains the amino acid residues that interact with an antigen and confer on the antigen binding protein its specificity and affinity for the antigen is referred to as “antigen binding region.” An antigen binding region typically includes one or more “complementary binding regions” (“CDRs”). Certain antigen binding regions also include one or more “framework” regions. A “CDR” is an amino acid sequence that contributes to antigen binding specificity and affinity. “Framework” regions can aid in maintaining the proper conformation of the CDRs to promote binding between the antigen binding region and an antigen.

In certain aspects, recombinant antigen binding proteins that bind (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4, arc provided. In this context, a “recombinant protein” is a protein made using recombinant techniques, i.e., through the expression of a recombinant nucleic acid as described herein. Methods and techniques for the production of recombinant proteins arc well known in the art.

The term “compete” when used in the context of antigen binding proteins (e.g., neutralizing antigen binding proteins, neutralizing antibodies, agonistic antigen binding proteins, agonistic antibodies and binding proteins that bind to (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4) that compete for the same epitope or binding site on a target means competition between antigen binding proteins as determined by an assay in which the antigen binding protein (e.g., antibody or immunologically functional fragment thereof) under study prevents or inhibits the specific binding of a reference molecule (e.g., a reference ligand, or reference antigen binding protein, such as a reference antibody) to a common antigen (e.g., FGFRlc, FGFR2c, FGFR3c, FGFR4, β-Klotho or a fragment thereof)- Numerous types of competitive binding assays can be used to determine if a test molecule competes with a reference molecule for binding. Examples of assays that can be employed include solid phase direct or indirect radioimmunoassay (R1A), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see, e.g., Stahli el al., (1983) Methods in Enzymohgy 2:242-253); solid phase direct biotin-avidin EIA (see, e.g., Kirkland et ah, (1986),/. Immunol. 137:3614-3619) solid phase direct labeled assay, solid phase direct labeled sandwich assay (see, e.g., Harlow and Lane, (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid phase direct label RIA using 1-125 label (see, e.g., Morel et a/., (1988) Moke. Immunol. 25:7-15); solid phase direct biotin-avidin EIA (see, e.g., Cheung, et al., (1990) Virology 176:546-552); and direct labeled RIA (Moldcnhauer et al., (1990) Scand. J. Immunol. 32:77-82). Typically, such an assay involves the use of a purified antigen bound to a solid surface or cells bearing either of an unlabciled test antigen binding protein or a labeled reference antigen binding protein. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test antigen binding protein, Usually the test antigen binding protein is present in excess. Antigen binding proteins identified by competition assay (competing antigen binding proteins) include antigen binding proteins binding to the same epitope as the reference antigen binding proteins and antigen binding proteins binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference antigen binding protein for steric hindrance to occur. Additional details regarding methods for determining competitive binding are provided in the examples herein. Usually, when a competing antigen binding protein is present in excess, it will inhibit specific binding of a reference antigen binding protein to a common antigen by at least 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75%. In some instance, binding is inhibited by at least 80%, 85%, 90%, 95%, or 97% or more.

The term “antigen” refers to a molecule or a portion of a molecule capable of being bound by a selective binding agent, such as an antigen binding protein (including, e.g., an antibody or immunological functional fragment thereof), and may also be capable of being used in an animal to produce antibodies capable of binding to that antigen. An antigen can possess one or more epitopes that are capable of interacting with different antigen binding proteins, e.g., antibodies.

The term “epitope” means the amino acids of a target molecule that are contacted by an antigen binding protein (for example, an antibody) when the antigen binding protein is bound to the target molecule. The term includes any subset of the complete list of amino acids of the target molecule that are contacted when an antigen binding protein, such as an antibody, is bound to the target molecule. An epitope can be contiguous or non-contiguous (e.g., (i) in a single-chain polypeptide, amino acid residues that are not contiguous to one another in the polypeptide sequence but that within in context of the target molecule are bound by the antigen binding protein, or (ii) in a multimcric receptor comprising two or more individual components, e.g., (i) FGFRlc, FGFR2c, FGFR3c or FGFR4, and (ii) β-Klotho, amino acid residues that are present on one or more of the individual components, but which arc still bound by the antigen binding protein). In certain embodiments, epitopes can be mimetic in that they comprise a three dimensional structure that is similar to an antigenic epitope used to generate the antigen binding protein, yet comprise none or only some of the amino acid residues found in that epitope used to generate the antigen binding protein. Most often, epitopes reside on proteins, but in some instances can reside on other kinds of molecules, such as nucleic acids. Epitope determinants can include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and can have specific three dimensional structural characteristics, and/or specific charge characteristics. Generally, antigen binding proteins specific for a particular target molecule will preferentially recognize an epitope on the target molecule in a complex mixture of proteins and/or maciOmolccuics.

The term “identity” refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. “Percent identity” means the percent of identical residues between the amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest of the molecules being compared. For these calculations, gaps in alignments (if any) must be addressed by a particular mathematical model or computer program (i.e., an “algorithm”). Methods that can be used to calculate the identity of the aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, A. M., ed.), (1988) New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, D. W., ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, A. M., and Griffin, H. G„ eds.), 1994, New Jersey: Humana Press; von Heinjc, G., (1987) Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et a!., (1988) SIAM.]. Applied Math. 48:1073.

In calculating percent identity, the sequences being compared are aligned in a way that gives the largest match between the sequences. The computer program used to determine percent identity is the GCG program package, which includes GAP (Dcvereux et a 1., (1984) Nucl. Acid Res. 12:387; Genetics Computer Group, University of Wisconsin, Madison, WI). The computer algorithm GAP is used to align the two polypeptides or polynucleotides for which the percent sequence identity is to be determined. The sequences arc aligned for optimal matching of their respective amino acid or nucleotide (the “matched span”, as determined by the algorithm). A gap opening penalty (which is calculated as 3x the average diagonal, wherein the “average diagonal” is the average of the diagonal of the comparison matrix being used; the “diagonal” is the score or number assigned to each perfect amino acid match by the particular comparison matrix) and a gap extension penalty (which is usually 1/10 times the gap opening penalty), as well as a comparison matrix such as PAM 250 or BLOSUM 62 are used in conjunction with the algorithm. In certain embodiments, a standard comparison matrix (see, Dayhoff et a/., (1978) Atlas of Protein Sequence and Structure 5:345-352 for the PAM 250 comparison matrix; Henikoff et ai., (1992) Proc. Natl. Acad. Sci. U.S.A. 89:10915-10919 for the BLOSUM 62 comparison matrix) is also used by the algorithm.

Recommended parameters for determining percent identity for polypeptides or nucleotide sequences using the GAP program are the following:

Algorithm: Nccdleman etal., 1970,./. Mol. Biol, 48:443-453:

Comparison matrix: BLOSUM 62 from Henikoffe/·#/., 1992, supra:

Gap Penalty: 12 (but with no penalty for end gaps)

Gap Length Penalty: 4 Threshold of Similarity: 0

Certain alignment schemes for aligning two amino acid sequences can result in matching of only a short region of the two sequences, and this small aligned region can have very high sequence identity even though there is no significant relationship between the two full-length sequences. Accordingly, the selected alignment method (e.g., the GAP program) can be adjusted if so desired to result in an alignment that spans at least 50 contiguous amino acids of the target polypeptide.

As used herein, “substantially pure” means that the described species of molecule is the predominant species present, that is, on a molar basis it is more abundant than any other individual species in the same mixture. In certain embodiments, a substantially pure molecule is a composition wherein the object species comprises at least 50% (on a molar basis) of ail macromolecular species present. In other embodiments, a substantially pure composition will comprise at least 80%, 85%, 90%, 95%, or 99% of all macromolecular species present in the composition, in other embodiments, the object species is purified to essential homogeneity wherein contaminating species cannot be detected in the composition by conventional detection methods and thus the composition consists of a single detectable macromolecular species.

The terms “treat” and “treating” refer to any indicia of success in the treatment or amelioration of an injury, pathology or condition, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the injury, pathology or condition more tolerable to the patient; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating; improving a patient's physical or mental well-being. The treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of a physical examination, ncuropsychiatric exams, and/or a psychiatric evaluation. For example, certain methods presented herein can be employed to treat Type 2 diabetes, obesity and/or dysiipidemia, cither prophylactically or as an acute treatment, to decrease plasma glucose levels, to decrease circulating triglyceride levels, to decrease circulating cholesterol levels and/or ameliorate a symptom associated with type 2 diabetes, obesity and dysiipidemia.

An “effective amount” is generally an amount sufficient to reduce the severity and/or frequency of symptoms, eliminate the symptoms and/or underlying cause, prevent the occurrence of symptoms and/or their underlying cause, and/or improve or remediate the damage that results from or is associated with diabetes, obesity and dysiipidemia. In some embodiments, the effective amount is a therapeutically effective amount or a prophylactically effective amount. A “therapeutically effective amount” is an amount sufficient to remedy a disease state (e.g., diabetes, obesity or dysiipidemia) or symptoms, particularly a state or symptoms associated with the disease state, or otherwise prevent, hinder, retard or reverse the progression of the disease state or any other undesirable symptom associated with the disease in any way whatsoever. A "prophylactically effective amount” is an amount of a pharmaceutical composition that, when administered to a subject, will have the intended prophylactic effect, e.g., preventing or delaying the onset (or reoccurrence) of diabetes, obesity or dysiipidemia, or reducing the likelihood of the onset (or reoccurrence) of diabetes, obesity or dyslipidemia or associated symptoms. The full therapeutic or prophylactic effect does not necessarily occur by administration of one dose, and can occur only after administration of a scries of doses. Thus, a therapeutically or prophylactically effective amount can be administered in one or more administrations. “Amino acid” takes its normal meaning in the art. The twenty naturally-occurring amino acids and their abbreviations follow conventional usage. See, Immunology-A Synthesis, 2nd Edition, (E. S. Golub and D. R. Green, eds.), Sinauer Associates: Sunderland, Mass. (1991), incorporated herein by reference for any purpose. Stereoisomers (e.g., D-amino acids) of the twenty conventional amino acids, unnatural or non-naturally occurring amino acids such as α-,α-disubstituted amino acids, N-atkyl amino acids, and other unconventional amino acids can also be suitable components for polypeptides and arc included in the phrase “amino acid.” Examples of non-naturalfy amino acids (which can be substituted for any naturally-occurring amino acid found in any sequence disclosed herein, as desired) include; 4-hydroxyproline, γ-carboxyglutamatc, i>N,N,N-trimcthyllysine, e-N-acetyl lysine, O-phosphoscrinc, N-acctylscrinc, N-formylmethionine, 3-mcthylhistidine, 5-hydroxylysine, σ-Ν-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). in the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxyl-terminal direction, in accordance with standard usage and convention. A non-limiting lists of examples of non-naturally occurring amino acids that can be inserted into an antigen binding protein sequence or substituted for a wild-type residue in an antigen binding sequence include β-amino acids, homoamino acids, cyclic amino acids and amino acids with derivatized side chains. Examples include (in the L-form or D-form; abbreviated as in parentheses): citruliine (Cit), homocitrulline (hCit), Na-methylcitrulline (NMeCit), Ncx-methylhomocitrulline (Na-MeHoCit), ornithine (Om), Na-Mcthylornithine (Na-McOrn or NMcOrn), sarcosine (Sar), homolysinc (hLys or hit), homoargininc (hArg or hR), homoglutamine (hQ), Na-mcthylargininc (NMeR), Να-methyllcucine (Na-McL or NMeL), N-methylhomoiysinc (NMeHoK), Na-mcthylglutaminc (NMcQ), norleucine (NIc), norvaline (Nva), 1,2,3,4-tctrahydroisoquinolinc (Tic), Octahydroindole-2-carboxylic acid (Oic), 3-(1-naphthyl )alanine (1-Nai), 3-(2- naphthyl)alanine (2-Nal), 1,2,3,4-tetrahydroisoquinoline (Tic), 2-indanylglycine (Igl), para-iodophenylalanine (pl-Phe), para-aminophenylaianine (4AmP or 4-A.mino-Phe), 4-guanidino phenylalanine (Guf), glycyllysine (abbreviated “KL(Nc-glycyf)” or “K(glycyl)” or “K(gly)”), nitrophcnylalanine (nitrophe), aminophenylalanine (aminophe or Amino-Phe), benzylphenylalanine (bcnzylphe), γ-carboxyglutamic acid (γ-carboxygiu), hydroxyproiine (hydroxypro), p-carboxyl-phenylalaninc (Cpa), α-aminoadipic acid (Aad), Να-mcthyl valine (NMcVal), Ν-α-mcthyl tcucinc (NMeLcu), Na-methylnorlcucine (NMcNle), cyclopentylglycine (Cpg), cyclohcxylglycinc (Chg), acetyl arginine (acctylarg), a, β-diaminopropionoic acid (Dpr), a, γ-diaminobutyric acid (Dab), diaminopropionic acid (Dap), cyclohcxylalaninc (Cha), 4-methyl-phenylalaninc (McPhc), β, β-diphcnyl-alanine (BiPhA), aminobutyric acid (Abu), 4-phcnyl-phcnylalanine (or biphenylalanine; 4Bip), «-amino-isobutyric acid (Aib), beta-alanine, beta-aminopropionic acid, pipcridinic acid, aminocaprioic acid, aminohcptanoic acid, aminopimclic acid, desmosine, diaminopimelic acid. N-ethylglycine, N-cthylaspargine, hydroxylysinc, alio-hydroxylysine, isodcsmosinc, allo-isolcucine, N-mcthylglycinc, N-mcthylisolcucinc, N-mcthylvalinc, 4-hydroxyprolinc (Hyp), γ-carboxyglutamatc, ε-Ν,Ν,Ν-trimethyllysinc, ε-Ν-acctyl lysine, O-phosphoserine, N-acetylscrine, N-formy I methionine, 3-mcihyIhistidinc, 5-hydroxyiysinc, ω-rncthylargininc, 4-Amino-O-Phthaiic Acid (4APA), and other similar amino acids, and derivatized forms of any of those specifically listed.

H. GENERAL OVERVIEW

Antigen-binding proteins that bind (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (Hi) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4, are provided herein. A unique property of the antigen binding proteins disclosed herein is the agonistic nature of these proteins, specifically the ability to mimic the in vivo effect of FGF2I and to induce FGF21-like signaling. More remarkably and specifically, some of the antigen binding proteins disclosed herein induce FGF21-likc signaling in several in vitro cell-based assay, including the ELK-luciferase reporter assay of Example 5 under the following conditions: (1) the binding to and activity of the FGF21 receptor is β-Klotho dependent; (2) the activity is selective to FGFRIc^Klotho complex; (3) the binding to the FGFR Ιο/βΚΙοίΙιο triggers FGF21-like signaling pathways; and (4) the potency (EC50) is comparable to a wild-type FGF21 standard comprising the mature form of SEQ ID NO:2, as measured in the following cell-based assays: (1) the recombinant FGF21 receptor mediated luciferasc-reporter cell assay of Example 5; (2) the ERK-phosphorylation in the recombinant FGF21 receptor mediated cell assay of Example 5; and (3) ERK-phosphorylation in human adipocytes as described in more details in

Example 7. The disclosed antigen binding proteins, therefore, are expected to exhibit activities in vivo that are consistent with the natural biological function of FGF21. This property makes the disclosed antigen binding proteins viable therapeutics for the treatment of metabolic diseases such as type 2 diabetes, obesity, dyslipidcmia, NASH, cardiovascular disease, metabolic syndrome and broadly any disease or condition in which it is desirable to mimic or augment the in vivo effects of FGF2].

In some embodiments of the present disclosure the antigen binding proteins provided can comprise polypeptides into which one or more complementary determining regions (CDRs) can be embedded and/or joined. In such antigen binding proteins, the CDRs can be embedded into a “framework” region, which orients the CDR(s) such that the proper antigen binding properties of the CDR(s) is achieved. In general, such antigen binding proteins that are provided can facilitate or enhance the interaction between FGFRIc and β-Klotho, and can substantially induce FGF21-likc signaling.

Certain antigen binding proteins described herein arc antibodies or are derived from antibodies. In certain embodiments, the polypeptide structure of the antigen binding proteins is based on antibodies, including, but not limited to, monoclonal antibodies, bispccific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as “antibody mimetics”), chimeric antibodies, humanized antibodies, human antibodies, antibody fusions (sometimes referred to herein as “antibody conjugates”), hemibodies and fragments thereof. The various structures are further described herein below.

The antigen binding proteins provided herein have been demonstrated to bind to (i) β-Klolho; (ii) FGFRIc, FGFR2c, FGFR3c or FGFR4; or (in) a complex comprising β-Klotho and one of FGFRIc, FGFR2c, FGFR3c, and FGFR4, and particularly to (i) human β-Klotho; (ii) human FGFRIc, human FGFR2c, human FGFR3c or human FGFR4; or (iii) a complex comprising human β-Klotho and one of human FGFRIc, human FGFR2c, human FGFR3c, and human FGFR4. As described and shown in the Examples presented herein, based the Western blot results, commcrciaiiy-availablc πηίι-β-ΚΙοΐΙιο or anti-FGFRIc antibodies bind to denatured β-Klotho or FGFRIc whereas the antigen binding protein (agonistic antibodies) do not. Conversely, the provided antigen binding proteins recognize the native structure of the FGFRIc and β-Klotho on the celt surface whereas the commercial antibodies do not, based on the FACS results provided. See Example 9. The antigen binding proteins that are provided therefore mimic the natural in vivo biological activity of FGF21. As a consequence, the antigen binding proteins provided herein are capable of activating FGF21-like signaling activity. In particular, the disclosed antigen binding proteins can have one or more of the following activities in vivo: induction of FGF21-like signal transduction pathways, lowering blood glucose levels, lowering circulating lipid levels, improving metabolic parameters and other physiological effects induced in vivo by the formation of the ternary complex of FGFRlc, β-Klotho and FGF21, for example in conditions such as type 2 diabetes, obesity, dyslipidemia, NASH, cardiovascular disease, and metabolic syndrome.

The antigen binding proteins that specifically bind to (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4: or (iii) a complex comprising β-KLlotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4 that are disclosed herein have a variety of utilities. Some of the antigen binding proteins, for instance, are useful in specific binding assays, in the affinity purification of (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4, including the human forms of these disclosed proteins, and in screening assays to identify other agonists of FGF21 -like signaling activity.

The antigen binding proteins that specifically bind (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4 that arc disclosed herein can be used in a variety of treatment applications, as explained herein. For example, certain antigen binding proteins are useful for treating conditions associated with FGF21-!i.ke signaling processes in a patient, such as reducing, alleviating, or treating type 2 diabetes, obesity, dyslipidemia, NASH, cardiovascular disease, and metabolic syndrome. Other uses for the antigen binding proteins include, for example, diagnosis of diseases or conditions associated with β-Klotho, FGFRlc, FGFR2c, FGFR3c, FGFR4 or FGF21, and screening assays to determine the presence or absence of these molecules. Some of the antigen binding proteins described herein can be useful in treating conditions, symptoms and/or the pathology associated with decreased FGF21-likc signaling activity. Exemplary conditions include, but arc not limited to, diabetes, obesity, NASH and dyslipidemia. FGF21

The antigen binding proteins disclosed herein induce FGF21-mediated signaling, as defined herein. In vivo, the mature form of FGF21 is the active form of the molecule. The nucleotide sequence encoding full length FGF21 is provided; the nucleotides encoding the signal sequence are underlined. ATG GAC TCG GAC GAG ACC GGG TTC GAG CAC TC'A GGA CTG TGG GTT TCT GTG CTG GCT GGT CTT CTG CTG GGA GCC TGC GAG GCA CAC CCC ATC CCT GAC TCC AGT CCT CTC CTG CAA TTC GGG GGC CAA GTC CGG CAG CGG TAC CTC TAC ACA GAT GAT GCC CAG CAG ACA GAA GCC CAC CTG GAG ATC AGG GAG GAT GGG AC'G GTG GGG GGC GCT GCT GAC CAG AGC CCC GAA AGT CTC CTG CAG CTG AAA GCC TTG AAG CCG GGA GTT ATT CAA ATC TTG GGA GTC AAG ACA TCC AGG TTC CTG TGC CAG CGG CCA GAT GGG GCC CTG TAT GGA TCG CTC CAC TTT GAC CCT GAG GCC TGC AGC TTC CGG GAG CTG CTT CTT GAG GAC GGA TAC AAT GTT TAC CAG TCC GAA GCC CAC GGC CTC CCG CTG CAC CTG CCA GGG AAC AAG TCC CCA CAC CGG GAC CCT GCA CCC CGA GGA CCA GCT CGC TTC CTG CCA CTA CCA GGC CTG CCC CCC GCA CCC CCG GAG CCA CCC GGA ATC CTG GCC CCC CAG CCC CCC GAT GTG GGC TCC TCG GAC CCT CTG AGC ATG GTG GGA CCT TCC CAG GGC CGA AGC CCC AGC TAC GCT TCC TGA (SEQ ID NO: 1)

The amino acid sequence of full length FGF21 is provided; the amino acids that make up the signal sequence arc underlined: MDSDETGFEHSGLWVSVLAGLLLGACOAHPIPDSSPLLOFGCO VRQRYLYTDDAQQTEAHLE1REDGTVGGAADQSPESLLQLKA LKPGVJQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLE DGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPP APPEPPG1LAPQPPDVGSSDPLSMVGPSQGRSPSYAS (SEQ ID NO: 2) FGFRlc

The antigen binding proteins disclosed herein bind to FGFRlc, in particular human FGFRlc, when associated with β-Klotho. The nucleotide sequence encoding human FGFRlc (GenBunk Accession Number NM_023110) is provided:

ATGTGGAGCTGGAAGTGCCTCCTCTTCTGGGCTGTGCTGGTCACAGCC

ACACTCTGCACCGCTAGGCCGTCCCCGACCTTGCCTGAACAAGCCCAG

CCCTGGGGAGCCCCTGTGGAAGTGGAGTCCTTCCTGGTCC'ACCCCGGT

GACCTGCTGCAGCTTCGC'TGTCGGCTGCGGGACGATGTGCAGAGCATC

AACTGGCTGC'GGGACGGGGTGCAGCTGGCGGAAAGCAACCGCACCCG

CATCACAGGGGAGGAGGTGGAGGTGCAGGACTCCGTGCCCGCAGACT

CCGGCCTCTATGCTTGCGTAACCAGCAGCCCCTCGGGCAGTGACACCA

CCTACTTCTCCGTCAATGTTTCAGATGCTCTCCCCTCCTCGGAGGATGA

TGATGATGATGATGACTCCTCTTCAGAGGAGAAAGAAACAGATAACA

CCAAACCAAACCGTATGCCCGTAGCTCCATATTGGACATCACCAGAAA

AGATGGAAAAGAAATTGCATGCAGTGCCGGCTGCCAAGACAGTGAAG

TTCAAATGCCCTTCCAGTGGGACACCAAACCCAACACTGCGCTGGTTG

AAAAATGGCAAAGAATTCAAACCTGACCACAGAATTGGAGGCTACAA

GGTCCGTTATGCCACCTGGAGCATCATAATGGACTCTGTGGTGCCCTC

TGACAAGGGCAACTACACCTGCATTGTGGAGAATGAGTACGGCAGCA

TCAACCACACATACCAGCTGGATGTCGTGGAGCGGTCCCCTCACCGGC

CCATCCTGCAAGCAGGGTTGCCCGCCAACAAAACAGTGGCCCTGGGT

AGCAACGTGGAGTTCATGTGTAAGGTGTACAGTGACCCGCAGCCGCAC

ATCCAGTGGCTAAAGCACATCGAGGTGAATGGGAGCAAGATTGGCCC

AGACAACCTGCCTTATGTCCAGATCTTGAAGACTGCTGGAGTTAATAC

CACCGACAAAGAGATGGAGGTGCTTCACTTAAGAAATGTCTCCTTTGA

GGACGCAGGGGAGTATACGTGCTTGGCGGGTAACTCTATCGGACTCTC

CCATCACTCTGCATGGTTGACCGTTCTGGAAGCCCTGGAAGAGAGGCC

GGCAGTGATGACCTCGCCCCTGTACCTGGAGATCATCATCTATTGCAC

AGGGGCCTTCCTCATCTCCTGCATGGTGGGGTCGGTCATCGTCTACAA

GATGAAGAGTGGTACCAAGAAGAGTGACTTCCACAGCCAGATGGCTG

TGCACAAGCTGGCCAAGAGCATCCCTCTGCGCAGACAGGTAACAGTG

TCTGCTGACTCCAGTGCATCCATGAACTCTGGGGTTCTTCTGGTTCGGC

CATCACGG CTCTCCTCCAGTG GG A CTCCC ATGCTAG C AG GGGTCTCTG

AGTATGAGCTTCCCGAAGACCCTCGCTGGGAGCTGCCTCGGGACAGAC

TGGTCTTAGGCAAACCCCTGGGAGAGGGCTGCTTTGGGCAGGTGGTGT

TGGCAGAGGCTATCGGGCTGGACAAGGACAAACCCAACCGTGTGACC

AAAGTGGCTGTGAAGATGTTGAAGTCGGACGCAACAGAGAAAGACTT

GTCAGACCTGATCTCAGAAATGGAGATGATGAAGATGATCGGGAAGC

ATAAGAATATCATCAACCTGCTGGGGGCCTGCACGCAGGATGGTCCCT

TGTATGTCATCGTGGAGTATGCCTCCAAGGGCAACCTGCGGGAGTACC

TGCAGGCCCGGAGGCCCCCAGGGCTGGAATACTGCTACAACCCCAGC

CACAACCCAGAGGAGCAGCTCTCCTCCAAGGACCTGGTGTCCTGCGCC

TACCAGGTGGCCCGAGGCATGGAGTATCTGGCCTCCAAGAAGTGCATA

CACCGAGACCTGGCAGCCAGGAATGTCCTGGTGACAGAGGACAATGT

GATGAAGATAGCAGACTTTGGCCTCGCACGGGACATTCACCACATCGA

CTACTATAAAAAGACAACCAACGGCCGACTGCCTGTGAAGTGGATGG

CACCCGAGGCATTATTTGACCGGATCTACACCCACCAGAGTGATGTGT

GGTCTTTCGGGGTGCTCCTGTGGGAGATCTTCACTCTGGGCGGCTCCCC

ATACCCCGGTGTGCCTGTGGAGGAACTTTTCAAGCTGCTGAAGGAGGG

TCACCGCATGGACAAGCCCAGTAACTGCACCAACGAGCTGTACATGAT

GATGCGGGACTGCTGGCATGCAGTGCCCTCACAGAGACCCACCTTCAA

GCAGCTGGTGGAAGACCTGGACCGCATCGTGGCCTTGACCTCCAACCA

GGAGTACCTGGACCTGTCCATGCCCCTGGACCAGTACTCCCCCAGCTT

TCCCGACACCCGGAGCTCTACGTGCTCCTCAGGGGAGGATTCCGTCTT

CTCTCATGAGCCGCTGCCCGAGGAGCCCTGCCTGCCCCGACACCCAGC CCAGCTTGCCAATGGCGGACTCAAACGCCGCTGA (SEQ ID NOG).

The amino acid sequence of human FGFRlc (GcnBank Accession Number NP_075598) is provided:

MWSWKCLLFWAVLVTATLCTARPSPTLPEQAQPWGAPVEVESFLVHPG

DLLQLRCRLRDDVQSINWLRDGVQLAESNRTR1TGEEVEVQDSVPADSGL

YACVTSSPSGSDTTYFSVNVSDALPSSEDDDDDDDSSSEEKETDNTKPNR

MPVAPYWTSPEKMEKKLHAVPAAKTVKFKCPSSGTPNPTLRWLKNGKEF

KPDHRIGGYKVRYATWSI1MDSVVPSDKGNYTC1VENEYGSINHTYQLDV

VERSPHRPILQAGLPANKTVALGSNVEFMCKVYSDPQPHIQWLKHIEVNG

SK1GPDNLPYVQILKTAGVNTTDK.EMEVLHLRNVSFEDAGEYTCLAGNSI

GLSHHSAWLTVLEALEERPAVMTSPLYLEII1YCTGAFLISCMVGSVIVYK

MKSGTKKSDFHSQMAVHK.LAKSIPLRRQVTVSADSSASMNSGVLLVRPS

RLSSSGTPMLAGVSEYELPEDPRWELPRDRLVLGKPLGEGCFGQVVLAEA

IGLDKDKPNRVTKVAVKMLKSDATEKDLSDLISEMEMMKM1GKHKN1IN

LLGACTQDGPLYVIVEYASKGNLREYLQARRPPGLEYCYNPSHNPEEQLS SKDLVSCAYQVARGMEYLASKKCIHRDLAARNVLVTEDNVMK1ADFGL ARDIHHJDYYKKTTNGRLPVKWMAPEALFDRIYTHQSDVWSFGVLLWEI FTLGGSPYPGVPVEELFKLLKEGHRMDKPSNCTNELYMMMRDCWHAVP SQRPTFKQLVEDLDR1VALTSNQEYLDLSMPLDQYSPSFPDTRSSTCSSGE DSVFSHEPLPEEPCLPRHPAQLANGGLKRR (SEQ ID NO: 4).

The antigen binding proteins described herein bind the extracellular portion of FGFRI c. An example of an extracellular region of FGFRlc is:

MWSWKCLLFWAVLVTATLCTARPSPTLPEQAQPWGAPVEVESFLVHPGDLLQL

RCRLRDDVQSINWLRDGVQLAESNRTRITGEEVEVQDSVPADSGLYACVTSSPS

GSDTTYFSVNVSDALPSSEDDDDDDDSSSEEKETDNTKPNRMPVAPYWTSPEKM ekklhavpaaktvkfkcpssgtpnptlrwlkngkefkpdhriggykvryatwsi 1MDSVVPSDKGNYTCIVENEYGS1NHTYQLDVVERSPHRPILQAGLPANKTVALG SNVEFMCKVYSDPQPHIQWLKH1EVNGSK1GPDNLPYVQILKTAGVNTTDKEME VLHLRNVSFEDAGEYTCLAGNSIGLSHHSAWLTVLEALEERPAVMTSPLY (SEQ ID NO: 5).

As described herein, FGFRlc proteins can also include fragments. As used herein, the terms arc used interchangeably to mean a receptor, in particular and unless otherwise specified, a human receptor, that upon association with β-Klotho and FGF21 induces FGF21 -like signaling activity.

The term FGFRlc also includes post-translational modifications of the FGFRlc amino acid sequence, for example, possible N-linked glycosylation sites. Thus, the antigen binding proteins can bind to or be generated from proteins glycosylated at one or more of the positions. β-Klotho

The antigen binding proteins disclosed herein bind to β-Klotho, in particular human β-Klotho. The nucleotide sequence encoding human β-Klotho (GenBank Accession Number NNM75737) is provided:

ATGAAGCCAGGCTGTGCGGCAGGATCTCCAGGGAATGAATGGATTTTC

TTCAGCACTGATGAAATAACCACACGCTATAGGAATACAATGTCCAAC

GGGGGATTGCAAAGATCTGTCATCCTGTCAGCACTTATTCTGCTACGA

GCTGTTACTGGATTCTCTGGAGATGGAAGAGCTATATGGTCTAAAAAT

CCTAATTTTACTCCGGTAAATGAAAGTCAGCTGTTTCTCTATGACACTT

TCCCTAAAAACTTTTTCTGGGGTATTGGGACTGGAGCATTGCAAGTGG

AAGGGAGTTGGAAGAAGGATGGAAAAGGACCTTCTATATGGGATCAT

TTCATCCACACACACCTTAAAAATGTCAGCAGCACGAATGGTTCCAGT

GACAGTTATATTTTTCTGGAAAAAGACTTATCAGCCCTGGATTTTATAG

GAGTTTCTTTTTATCAATTTTCAATTTCCTGGCCAAGGCTTTTCCCCGAT

GGAATAGTAACAGTTGCCAACGCAAAAGGTCTGCAGTACTACAGTACT

CTTCTGGACGCTCTAGTGCTTAGAAACATTGAACCTATAGTTACTTTAT

ACCACTGGGATTTGCCTTTGGCACTACAAGAAAAATATGGGGGGTGGA

AAAATGATACCATAATAGATATCTTCAATGACTATGCCACATACTGTT

TCCAGATGTTTGGGGACCGTGTCAAATATTGGATTACAATTCACAACC

CATATCTAGTGGCTTGGCATGGGTATGGGACAGGTATGCATGCCCCTG

GAGAGAAGGGAAATTTAGCAGCTGTCTACACTGTGGGACACAACTTG

ATCAAGGCTCACTCGAAAGTTTGGCATAACTACAACACACATTTCCGC

CCACATCAGAAGGGTTGGTTATCGATCACGTTGGGATCTCATTGGATC

GAGCCAAACCGGTCGGAAAACACGATGGATATATTCAAATGTCAACA

ATCCATGGTTTCTGTGCTTGGATGGTTTGCCAACCCTATCCATGGGGAT

GGCGACTATCCAGAGGGGATGAGAAAGAAGTTGTTCTCCGTTCTACCC

ATTTTCTCTGAAGCAGAGAAGCATGAGATGAGAGGCACAGCTGATTTC

TTTGCCTTTTCTTTTGGACCCAACAACTTCAAGCCCCTAAACACCATGG

CT AAA AT G GG AC A A A ATG TTT CACTTAATTT A AG AG A AGCG CT G A A CT

GGATTAAACTGGAATACAACAACCCTCGAATCTTGATTGCTGAGAATG

GCTGGTTCACAGACAGTCGTGTGAAAACAGAAGACACCACGGCCATC

TACATGATGAAGAATTTCCTCAGCCAGGTGCTTCAAGCAATAAGGTTA

GATGAAATACGAGTGTTTGGTTATACTGCCTGGTCTCTCCTGGATGGCT

TTGAATGGCAGGATGCTTACACCATCCGCCGAGGATTATTTTATGTGG

ATTTTAACAGTAAACAGAAAGAGCGGAAACCTAAGTCTTCAGCACACT

ACT ACA AA C AG ATC AT ACG AG AAAATG GTTTTTCTTTAAAAG AGTCCA

CGCCAGATGTGCAGGGCCAGTTTCCCTGTGACTTCTCCTGGGGTGTCA

CTGAATCTGTTCTTAAGCCCGAGTCTGTGGCTTCGTCCCCACAGTTCAG

CGATCCTCATCTGTACGTGTGGAACGCCACTGGCAACAGACTGTTGCA

CCGAGTGGAAGGGGTGAGGCTGAAAACACGACCCGCTCAATGCACAG

ATTTTGTAAACATCAAAAAACAACITGAGATGrrGGCAAGAA'rGAAA

GTCACCCACTACCGGTTTGCTCTGGATTGGGCCTCGGTCCTTCCCACTG

GCAACCTGTCCGCGGTGAACCGACAGGCCCTGAGGTACTACAGGTGC

GTGGTCAGTGAGGGGCTGAAGCTTGGCATCTCCGCGATGGTCACCCTG

TATTATCCGACCCACGCCCACCTAGGCCTCCCCGAGCCTCTGTTGCAT

GCCGACGGGTGGCTGAACCCATCGACGGCCGAGGCCTTCCAGGCCTA

CGCTGGGCTGTGCTTCCAGGAGCTGGGGGACCTGGTGAAGCTCTGGAT

CACCATCAACGAGCCTAACCGGCTAAGTGACATCTACAACCGCTCTGG

CAACGACACCTACGGGGCGGCGCACAACCTGCTGGTGGCCCACGCCC

TGGCCTGGCGCCTCTACGACCGGCAGTTCAGGCCCTCACAGCGCGGGG

CCGTGTCGCTGTCGCTGCACGCGGACTGGGCGGAACCCGCCAACCCCT

ATGCTGACTCGCACTGGAGGGCGGCCGAGCGCTTCCTGCAGTTCGAGA

TCGCCTGGTTCGCCGAGCCGCTCTTCAAGACCGGGGACTACCCCGCGG

CCATGAGGGAATACATTGCCTCCAAGCACCGACGGGGGCTTTCCAGCT

CGGCCCTGCCGCGCCTCACCGAGGCCGAAAGGAGGCTGCTCAAGGGC

ACGGTCGACTTCTGCGCGCTCAACCACTTCACCACTAGGTTCGTGATG

CACGAGCAGCTGGCCGGCAGCCGCTACGACTCGGACAGGGACATCCA

GTTtCTGCAgGACATCACCCGCCTGAgCTCCCCcACGCgCCTGGCTGT

GATTCCCTGGGGGGTGCGCAAGCTGCTGCGGTGGGTCCGGAGGAACT

ACGGCGACATGGACATTTACATCACCGCCAGTGGCATCGACGACCAG

GCTCTGGAGGATGACCGGCTCCGGAAGTACTACCTAGGGAAGTACCTT

C AGG AGGTGCTG A A AGC ATACCTG ATTG ATA A A GTC A G A ATC A A AG G

CTATTATGCATTCAAACTGGCTGAAGAGAAATCTAAACCCAGATTTGG

ATTCTTCACATCTGATTTTAAAGCTAAATCCTCAATACAATTTTACAAC

AAAGTGATCAGCAGCAGGGGCTTCCCTTTTGAGAACAGTAGTTCTAGA

TGCAGTCAGACCCAAGAAAATACAGAGTGCACTGTCTGCTTATTCCTT

GTGCAGAAGAAACCACTGATATTCCTGGGTTGTTGCTTCTTCTCCACCC

TGGTT CT ACT CTT AT C A ATTGCCATTTTT C AAAGGCAG A AGAG AAG AA agtttt g g a aag c a a aa aacttacaacacataccatt a aag a aag gc AAGAGAGTTGTTAGCTAA (SEQ ID NO: 6).

The amino acid sequence of full length human β-Klotho (GenBank Accession Number NP_783864) is provided:

MKPGCAAGSPGNEWIFFSTDE1TTRYRNTMSNGGLQRSV1LSALILLRAVT

GFSGDGRASWSKNPNFTPVNESQLFLYDTFPKNFFWGIGTGALQVEGSWK

KDGKGPSIWDHF1HTHLKNVSSTNGSSDSYIFLEKDLSALDFIGVSFYQFSI

SWPRLFPDG1VTVANAKGLQYYSTLLDALVLRN1EP1VTLYHWDLPLALQ

EKYGGWKNDTIID1FNDYATYCFQMFGDRVKYWIT1HNPYLVAWHGYGT

GMHAPGEKGNLAAVYTVGHNLIKAHSKVWHNYNTHFRPHQKGWLS1TL

GSHWJEPNRSENTMDIFKCQQSMVSVLGWFANPIHGDGDYPEGMRKKLF

SVLP1FSEAEKHEMRGTADFFAFSFGPNNFKPLNTMAKMGQNVSLNLREA

LNWIKLEYNNPRILIAENGWFTDSRVKTEDTTAIYMMKNFLSQVLQA1RL

DEIRVFGYTAWSLLDGFEWQDAYTIRRGLFYVDFNSKQKERKPKSSAHY

YKQ1IRENGFSLKESTPDVQGQFPCDFSWGVTESVLKPESVASSPQFSDPH

LYVWNATGNRLLHRVEGVRLKTRPAQCTDFVNIKKQLEMLARMICVTHY

RFALDWASVLPTGNLSAVNRQALRYYRCVVSEGLKLGISAMVTLYYPTH

ABLGLPEPLLHADGWLNPSTAEAFQAYAGLCFQELGDLVKLW1T1NEPNR

ESDI YNRSGNDTYGAAHNLLVAH ALA WRLYDRQFRPSQRGAVSLSLHAD

WAEPANPYADSHWRAAERFLQFE1AWFAEPLFKTGDYPAAMREYIASKH

RRGLSSSALPRLTEAERRLLKGTVDFCALNHFTTRFVMHEQLAGSRYDSD

RDIQFLQDITRLSSPTRLAVIPWGVRKLLRWVRRNYGDMDIYITASG1DDQ

ALEDDRLRKYYLGKYLQEVLKAYLJDKVRIKGYYAFKLAEEKSKPRFGFF

TSDFKAKSSIQFYNKVISSRGFPFENSSSRCSQTQENTECTVCLFLVQKKPL

IFLGCCFFSTLVLLLSIAIFQRQKRRKFWKAKNLQHIPLKKGKRVVS (SEQ ID NO: 7).

The antigen binding proteins described herein bind the extracellular portion of β-Klotho. An example of an extracellular region of β-Klotho is:

MKPGCAAGSPGNEWIFFSTDE1TTRYRNTMSNGGLQRSV1LSALILLRAVTGFSG DGRA1WSKNPNFTPVNESQLFLYDTFPKNFFWGIGTGALQVEGSWKKDGKGPS1

WDHFIHTHLKNVSSTNGSSDSYIFLEKDLSALDFIGVSFYQFS1SWPRLFPDGIVTV

ANAKGLQYYSTLLDALVLRNIEPIVTLYHWDLPLALQEKYGGWKNDT11DIFNDY

ATYCFQMFGDRVKYWITIHNPYLVAWHGYGTGMHAPGEKGNLAAVYTVGHNL

1KAHSKVWHNYNTHFRPHQKGWLS1TLGSHWIEPNRSENTIV1D1FKCQQSMVSVL

GWFANPIHGDGDYPEGMRKKLFSVLPIFSEAEKHEMRGTADFFAFSFGPNNFKPL

NTMAKMGQNVSLNLREALNWIKLEYNNPRILIAENGWFTDSRVKTEDTTAIYM

MKNFLSQVLQA1RLDEIRVFGYTAWSLLDGFEWQDAYTIRRGLFYVDFNSKQKE

RKPKSSAHYYKQIIRENGFSLRESTPDVQGQFPCDFSWGVTESVLKPESVASSPQF

SDPHLYVWNATGNRLLHRVEGVRLKTRPAQCTDFVNIKKQLEMLARMKVTHY

RFALDWASVLPTGNLSAVNRQALRYYRCVVSEGLKLG1SAMVTLYYPTHAHLG

LPEPLLHADGWLNPSTAEAFQAYAGLCFQELGDLVKLWITINEPNRLSD1YNRSG

NDTYGAAHNLLVAHALAWRLYDRQFRPSQRGAVSLSLHADWAEPANPYADSH

WRAAERFLQFEIAWFAEPLFKTGDYPAAMREYIASKHRRGLSSSALPRLTEAERR

LLKGTVDFCALNHFTTRFVMHEQLAGSRYDSDRDIQFLQDITRLSSPTRLAVIPW GVRKLLRWVRRNYGDiVlDIYITASGlDDQALEDDRLRKYYLGKYLQEVLKAYLl

DK.VR1KGYYAFKLAEEKSKPRFGFFTSDFKAKSSIQFYNKVISSRGFPFENSSSRCS

QTQENTECTVCLFLVQKKP (SEQ ID NO: 8.)

The murine form of β-Klotho, and fragments and subsequences thereof, can be of use in studying and/or constructing the molecules provided herein. The nucleotide sequence encoding murine β-Klotho (GcnBank Accession Number NM_031180) is provided:

ATGAAGACAGGCTGTGCAGCAGGGTCTCCGGGGAATGAATGGATTTTCTTCA

GCTCTGATGAAAGAAACACACGCTCTAGGAAAACAATGTCCAACAGGGCACT

GCAAAGATCTGCCGTGCTGTCTGCGTTTGTTCTGCTGCGAGCTGTTACCGGCT

TCTCCGGAGACGGGAAAGCAATATGGGATAAAAAACAGTACGTGAGTCCGG

TAAACCCAAGTCAGCTGTTCCTCTATGACACTTTCCCTAAAAACTTTTCCTGG

GGCGTTGGGACCGGAGCATTTCAAGTGGAAGGGAGTTGGAAGACAGATGGA

AGAGGACCCTCGATCTGGGATCGGTACGTCTACTCACACCTGAGAGGTGTCA

ACGGCACAGACAGATCCACTGACAGTTACATCTTTCTGGAAAAAGACTTGTT

GGCTCTGGATTTTTTAGG AGTTT CTTTTf ATCAGTT CT CA AT CT CCTGGCCACG

GTTGTTTCCCAATGGAACAGTAGCAGCAGTOAATGCGCAAGGTCTCCGGTAC

TACCGTGCACTTCTGGACTCGCTGGTACTTAGGAATATCGAGCCCATTGTTAC

CTTGTACCATTGGGATTTGC'CTCTGACGCTCCAGGAAGAATATGGGGGCTGG

AAAAATGCAACTATGATAGATCTCTTCAACGACTATGCCACATACTGCTTCCA

GACCTTTGGAGACCGTGTCAAATATTGGATTACAATTCACAACCCTTACCTTG

TTGCTTGGCATGGGTTTGGCACAGGTATGCATGCACCAGGAGAGAAGGGAAA

TTTAACAGCTGTCTACACTGTGGGACACAACCTGATCAAGGCACATTCGAAA gtgtggcataactacgacaaaaacttccgccctcatcagaagggttggctct

CCATCACCTTGGGGTCCCATTGGATAGAGCCAAACAGAACAGACAACATGGA

GGACGTGATCAACTGCCAGCACTCCATGTCCTCTGTGCTTGGATGGTTCGCCA

ACCCCATCCACGGGGACGGCGACTACCCTGAGTTCATGAAGACGGGCGCCAT

GATCCCCGAGTTCTCTGAGGCAGAGAAGGAGGAGGTGAGGGGCACGGCTGA

TTTCTTTGCCTTTTCCTTCGGGCCCAACAACTTCAGGCCCTCAAACACCGTGG

TGAAAATGGGACAAAATGTATCACTCAACTTAAGGCAGGTGCTGAACTGGAT

TAAACTGGAATACGATGACCCTCAAATCTTGATTTCGGAGAACGGCTGGTTC

ACAGATAGCTATATAAAGACAGAGGACACCACGGCCATCTACATGATGAAG

AATTTCCTAAACCAGGTTCTTCAAGCAATAAAATTTGATGAAATCCGCGTGTT

TGGTTATACGGCCTGGACTCTCCTGGATGGCTTTGAGTGGCAGGATGCCTATA

CGACCCGACGAGGGCTGTTTTATGTGGACTTTAACAGTGAGCAGAAAGAGAG

GAAACCCAAGTCCTCGGCTCATTACTACAAGCAGATCATACAAGACAACGGC

TTCCCTTTGAAAGAGTCCACGCCAGACATGAAGGGTCGGTTCCCCTGTGATTT

CTCTTGGGGAGTC’ACTGAGTCTGTTCTTAAGCCCGAGTTTACGGTCTCCTCCC

CGCAGTTTACCGATCCTCACCTGTATGTGTGGAATGTCACTGGCAACAGATTG

CTCTACCGAGTGGAAGGGGTAAGGCTGAAAACAAGACCATCCCAGTGCACA

GATTAT GTG A G CAT C A AA A A ACG A GTT G A AATGTT G GC A A AAAT G AAAGT C A

CCCACTACCAGTTTGCTCTGGACTGGACCTCTATCCTTCCCACTGGCAATCTG

TCCAAAGTTAACAGACAAGTGTTAAGGTACTATAGGTGTGTGGTGAGCGAAG

GACTGAAGCTGGGCGTCTTCCCCATGGTGACGTTGTACCACCCAACCCACTCC

CATCTCGGCCTCCCCCTGCCACTTCTGAGCAGTGGGGGGTGGCTAAACATGA

ACACAGCCAAGGCCTTCCAGGACTACGCTGAGCTGTGCTTCCGGGAGTTGGG

GGACTTGGTGAAGCTCTGGATCACCATCAATGAGCCTAACAGGCTGAGTGAC

ATGTACAACCGCACGAGTAATGACACCTACCGTGCAGCCCACAACCTGATGA

TCGCCCATGCCCAGGTCTGGCACCTCTATGATAGGCAGTATAGGCCGGTCCA

GCATGGGGCTGTGTCGCTGTCCTTACATTGCGACTGGGCAGAACCTGCCAAC

CCCTTTGTGGATTCACACTGGAAGGCAGCCGAGCGCTTCCTCCAGTTTGAGAT

CGCCTGGTTTGCAGATCCGCTCTTCAAGACTGGCGACTATCCATCGGTTATGA

AGGAATACATCGCCTCCAAGAACCAGCGAGGGCTGTCTAGCTCAGTCCTGCC

GCGCTTCACCGCGAAGGAGAGCAGGCTGGTGAAGGGTACCGTCGACTTCTAC

GCACTGAACCACTTCACTACGAGGTTCGTGATACACAAGCAGCTGAACACCA

ACCGCTCAGTTGCAGACAGGGACGTCCAGTTCCTGCAGGACATCACCCGCCT

AAGCTCGCCCAGCCGCCTGGCTGTAACACCCTGGGGAGTGCGCAAGCTCCTT

GCGTGGATCCGGAGGAACTACAGAGACAGGGATATCTACATCACAGCCAATG

GCATCGATGACCTGGCTCTAGAGGATGATCAGATCCGAAAGTACTACTTGGA

GAAGTATGTCCAGGAGGCTCTGAAAGCATATCTCATTGACAAGGTCAAAATC

AAAGGCTACTATGCATTCAAACTGACTGAAGAGAAATCTAAGCCTAGATTTG

GATTTTTCACCTCTGACTTCAGAGCTAAGTCCTCTGTCCAGTTTTACAGCAAG

CTGATCAGCAGCAGTGGCCTCCCCGCTGAGAACAGAAGTCCTGCGTGTGGTC

AGCCTGCGGAAGACACAGACTGCACCATTTGCTCATTTCTCGTGGAGAAGAA

ACCACTCATCTTCTTCGGTTGCTGCTTCATCTCCACTCTGGCTGTACTGCTATC

CATCACCGTTTTTC'ATCATCAAAAGAGAAGAAAATTCCAGAAAGCAAGGAAC

TTACAAAATATACCATTGAAGAAAGGCCACAGCAGAGTTTTCAGCTAA (SEQ ID NO:469)

The amino add sequence of full length murine β-Klotho (GenBank Accession Number NP_112457) is provided:

MKTGCAAGSPGNEWJFFSSDERNTRSRKTMSNRALQRSAVLSAFVLLRA

VTGFSGDGKAIWDKKQYVSPVNPSQLFLYDTFPKNFSWGVGTGAFQVEG

SWKTDGRGPSIWDRYVYSHLRGVNGTDRSTDSYIFLEKDLLALDFLGVSF

YQFSISWPRLFPNGTVAAVNAQGLRYYRALLDSLVLRNIEP1VTLYHWDL

PLTLQEEYGGWKNATMIDLFNDYATYCFQTFGDRVKYWIT1HNPYLVAW

HGFGTGMHAPGEKGNLTAVYTVGHNLIKAHSKVWHNYDKNFRPHQKG

WLSITLGSHWIEPNRTDNMEDVINCQHSMSSVLGWFANPIHGDGDYPEF

MKTGAMIPEFSEAEKEEVRGTADFFAFSFGPNNFRPSNTVVKMGQNVSLN

LRQVLNWIKLEYDDPQILISENGWFTDSYIKTEDTTAIYMMKNFLNQVLQ

AIKFDEIRVFGYTAWTLLDGFEWQDAYTTRRGLFYVDFNSEQKERKPKSS

AHYYKQIIQDNGFPLKESTPDMKGRFPCDFSWGVTESVLRPEFTVSSPQFT

D P H L Y V WNV'PGN R L LY R V EG V RL KTRPSQC'TD Y VS IKKR V EM L AICM K V

THYQFALDWTSILPTGNLSKVNRQVLRYYRCVVSEGLKLGVFPMVTLYH

PTHSI-ILGLPLPLLSSGGWLNMNTAKAFQDYAELCFRELGDLVKLWITINE

PNRLSDMYNRTSNDTYRAAHNLMIAHAQVWHLYDRQYRPVQHGAVSLS

LHCDWAEPANPFVDSHWKAAERFLQFEIAWFADPLFKTGDYPSVMKEYI

AS KN QRGLSSS VLPRFTAKESRL VKGT VD F Y ALN H FTTRF V1H KQLNTN R

SVADRDVQFLQDITRLSSPSRLAVTPWGVRKXLAW1RRNYRDRDIY1TAN

GIDDLALEDDQIRKYYLEKYVQEALKAYLIDKVKJKGYYAFKLTEEKSKP

RFGFFTSDFRAKSSVQFYSKLISSSGLPAENRSPACGQPAEDTDCTICSFLV

EKKPLIFFGCCF1STLAVLLSITVFHHQKRRKFQKARNLQNIPLKKGHSRVF S(SEQJDNO:468)

As described herein, β-KIotho proteins can also include fragments. As used herein, the terms are used interchangeably to mean a co-receptor, in particular and unless otherwise specified, a human co-receptor, that upon association with FGFRlc and FGF21 induces FGF21-like signaling activity.

The term β-Klotho also includes post-translational modifications of the β-KIotho amino acid sequence, for example, possible N-linkcd glycosylation sites. Thus, the antigen binding proteins can bind to or be generated from proteins glycosylated at one or more of the positions.

Antiecn Binding Proteins that Specifically Bind One or iMore of β-Klotho. FGFRlc. FGFR2c. FGFR3c. FGFR4c A variety of selective binding agents useful for modulating FGF21-like signaling arc provided. These agents include, for instance, antigen binding proteins that contain an antigen binding domain (e.g., single chain antibodies, domain antibodies, hcmibodics, immunoadhesions, and polypeptides with an antigen binding region) and specifically bind to FGFRlc, β-KIotho or both FGFRlc and β-Klotho, in particular human FGFRlc and human β-Klotho. Some of the agents, for example, are useful in mimicking the signaling effect generated in vivo by the association of FGFRlc with β-Klotho and with FGF21, and can thus be used to enhance or modulate one or more activities associated with FGF21 -like signaling.

In general, the antigen binding proteins that arc provided typically comprise one or more CDRs as described herein (e.g., 1, 2, 3, 4, 5 or 6 CDRs). In some embodiments the antigen binding proteins are naturally expressed by clones, while in other embodiments, the antigen binding protein can comprise (a) a polypeptide framework structure and (b) one or more CDRs that arc inserted into and/or joined to the polypeptide framework structure. In some of these embodiments a CDR forms a component of a heavy or light chains expressed by the clones described herein; in other embodiments a CDR can be inserted into a framework in which the CDR is not naturally expressed. A polypeptide framework structure can take a variety of different forms. For example, a polypeptide framework structure can be, or comprise, the framework of a naturally occurring antibody, or fragment or variant thereof, or it can be completely synthetic in nature. Examples of various antigen binding protein structures are further described below.

In some embodiments in which the antigen binding protein comprises (a) a polypeptide framework structure and (b) one or more CDRs that arc inserted into and/or joined to the polypeptide framework structure, the polypeptide framework structure of an antigen binding protein is an antibody or is derived from an antibody, including, but not limited to, monoclonal antibodies, bispecific antibodies, minibodics, domain antibodies, synthetic antibodies (sometimes referred to herein as “antibody mimetics”), chimeric antibodies, humanized antibodies, antibody fusions (sometimes referred to as “antibody conjugates”), and portions or fragments of each, respectively. In some instances, the antigen binding protein is an immunological fragment of an antibody (<?.g., a Fab, a Fab’, a F(ab’h, or a scFv).

Certain of the antigen binding proteins as provided herein specifically bind to (i) β-Kiotho; (it) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4, including the human forms of these proteins . In one embodiment, an antigen binding protein specifically binds to both human FGFRlc comprising the amino acid sequence of SEQ ID NO:5, and human β-KIotho comprising the amino acid sequence of SEQ ID NO:8, and in another embodiment an antigen binding protein specifically binds to both human FGFRlc comprising the amino acid sequence of SEQ ID NO:5 and human β-Klotho having the amino acid sequence of SEQ ID NO:8 and induces FGF21-like signaling. Thus, an antigen binding protein can, but need not, induce FGF21-like signaling.

Antigen Binding Protein Structure

Some of the antigen binding proteins that specifically bind (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4, including the human forms of these proteins that are provided herein have a structure typically associated with naturally occurring antibodies. The structural units of these antibodies typically comprise one or more tetramers, each composed of two identical couplets of polypeptide chains, though some species of mammals also produce antibodies having only a single heavy chain. In a typical antibody, each pair or couplet includes one full-length “light” chain (in certain embodiments, about 25 kDa) and one full-length “heavy” chain (in certain embodiments, about 50-70 kDa). Each individual immunoglobulin chain is composed of several “immunoglobulin domains,” each consisting of roughly 90 to 110 amino acids and expressing a characteristic folding pattern. These domains are the basic units of which antibody polypeptides are composed. The amino-terminal portion of each chain typically includes a variable domain that is responsible for antigen recognition. The carboxy-tcrmina! portion is more conserved evolutionarily than the other end of the chain and is referred to as the “constant region” or "C region”. Human light chains generally arc classified as kappa (“tc”) and lambda (“λ”) light chains, and each of these contains one variable domain and one constant domain. Heavy chains arc typically classified as mu, delta, gamma, alpha, or epsilon chains, and these define the antibody’s isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subtypes, including, but not limited to, IgG!, lgG2, lgG3, and IgG4. IgM subtypes include IgM, and IgM2. IgA subtypes include IgAl and lgA2. In humans, the IgA and IgD isotypes contain four heavy chains and four light chains; the IgG and IgE isotypes contain two heavy chains and two light chains; and the IgM isotype contains five heavy chains and five light chains. The heavy chain C region typically comprises one or more domains that can be responsible for effector function. The number of heavy chain constant region domains will depend on the isotype. IgG heavy chains, for example, each contain three C region domains known as Cnl, Cn2 and Cn3. The antibodies that arc provided can have any of these isotypes and subtypes, in certain embodiments, an antigen binding protein that specifically binds one or more of (i) β-Klotho; (ii) FGFRIc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRIc, FGFR2c, FGFR3c, and FGFR4 is an antibody of the IgG 1, IgG2, or IgG4 subtype.

In full-length light and heavy chains, the variable and constant regions are joined by a “J” region of about twelve or more amino acids, with the heavy chain also including a “D” region of about ten more amino acids. See, e.g., Fundamental Immunology, 2nd cd., Ch. 7 (Paul, W„ ed.) 1989, New York: Raven Press (hereby incorporated by reference in its entirety for all purposes). The variable regions of each tight/hcavy chain pair typically form the antigen binding site.

One example of an IgG2 heavy constant domain of an exemplary monoclonal antibody that specifically binds (i) β-Klotho; (ii) FGFRIc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRIc, FGFR2c, FGFR3c, and FGFR4 has the amino acid sequence:

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV

HTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCMVDHKPSNTKVDKTVER

KCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE

VQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYK

CKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKG

FYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 9).

One example of a kappa light constant domain of an exemplary monoclonal antibody that binds (i) β-Klotho: (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFR Ic, FGFR2c, FGFR3c, and FGFR4 has the amino acid sequence: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC (SEQ ID NO: 10).

One example of a lambda light constant domain of an exemplary monoclonal antibody that binds (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2e, FGFR3c, and FGFR4 has the amino acid sequence: GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVK AGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKT VAPTECS (SEQ ID NO: 11)

Variable regions of immunoglobulin chains generally exhibit the same overall structure, comprising relatively conserved framework regions (FR) joined by three hypervariable regions, more often called “complementarity determining regions” or CDRs. The CDRs from the two chains of each heavy chain/light chain pair mentioned above typically are aligned by the framework regions to form a structure that binds specifically with a specific epitope on the target protein (e.g„ (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4). From N-terminal to C-tcrminal, naturally-occurring light and heavy chain variable regions both typically conform with the following order of these elements: FRI, CDR1, FR2, CDR2, FR3, CDR3 and FR4. A numbering system has been devised for assigning numbers to amino acids that occupy positions in each of these domains. This numbering system is defined in Rabat Sequences of Proteins of Immunological Interest (1987 and 1991, N1H, Bcthcsda. MD). As desired, the CDRs can also be redefined according an alternative nomenclature scheme, such as that of Chothia (see Chothia &amp; Lcsk, 1987, J. Mol. Biol. 196:901-917; Chothia ei ol., 1989, Nature 342:878-883 or Honegger &amp; Pluckthun, 2001,./. Mol. Biol. 309:657-670.

The various heavy chain and light chain variable regions of antigen binding proteins provided herein are depicted in Table 2. Each of these variable regions can be attached to the above heavy and light chain constant regions to form a complete antibody heavy and light chain, respectively. Further, each of the so-generated heavy and light chain sequences can be combined to form a complete antibody structure, it should be understood that the heavy chain and light chain variable regions provided herein can also be attached to other constant domains having different sequences than the exemplary sequences listed above.

Specific examples of some of the full length light and heavy chains of the antibodies that are provided and their corresponding amino acid sequences are summarized in Tables 1A and IB. Table IA shows exemplary light chain sequences, and Table IB shows exemplary heavy chain sequences.

Table 1A - Exemplary Antibody Light Chain Sequences

Table IB - Exemplary Antibody Heavy Chain Sequences

Again, each of the exemplary heavy chains (HI, H2, H3 etc.) listed in Table IB and 6A, infra, can be combined with any of the exemplary light chains shown in Table 1A and 6A, in fra, to form an antibody. Examples of such combinations include HI combined with any of LI through LI8; H2 combined with any of LI through LI8: H3 combined with any of LI through LI8, and so on. In some instances, the antibodies include at least one heavy chain and one light chain from those listed in Tables 1A and IB and 6A, infra: particular examples pairings of light chains and heavy chains include LI with 111, L2 with H2, L3 with H3, L4 with H4, L5 with H5, L6 with H6, L7 with H7, L8 with H8, L9 with H9, L10 with H10. LI 1 with HI 1, L12 with HI2, L13 with HI3, L14 with HI4, LI5 with HI5, LI6 with HI6, LI7 with Ml7, and LI8 with H18. In addition to antigen binding proteins comprising a heavy and a light chain from the same clone, a heavy chain from a first clone can be paired with a light chain from a second clone (e.g., a heavy chain from 46D11 paired with a light chain from 16H7 or a heavy chain from 16H7 paired with a light chain from 46D11). Generally, such pairings can include VL with 90% or greater homology can be paired with the heavy chain of the naturally occurring clone. In some instances, the antibodies comprise two different heavy chains and two different light chains listed in Tables 1A and IB and 6A, infra. In other instances, the antibodies contain two identical light chains and two identical heavy chains. As an example, an antibody or immunologically functional fragment can include two HI heavy chains and two LI light chains, or two H2 heavy chains and two L2 light chains, or two H3 heavy chains and two L3 light chains and other similar combinations of pairs of light chains and pairs of heavy chains as listed in Tables 1A and IB and 6A, infra.

In another aspect of the instant disclosure, “hemibodies” arc provided. A hemibody is a monovalent antigen binding protein comprising (i) an intact light chain, and (ii) a heavy chain fused to an Fc region (e.g, an IgG2 Fc region of SEQ ID NO:441), optionally via a tinker, The linker can be a (G_jS)x linker where “x” is a non-zero integer (e.g., (GjST; SEQ ID NO:440). Hemibodies can be constructed using the provided heavy and light chain components. Specific examples of hemibodies are disclosed in Example 14.

Other antigen binding proteins that are provided are variants of antibodies formed by combination of the heavy and light chains shown in Tables 1A and IB and 6A, infra and comprise light and/or heavy chains that each have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequences of these chains. In some instances, such antibodies include at least one heavy chain and one light chain, whereas in other instances the variant forms contain two identical light chains and two identical heavy chains.

Variable Domains of Antigen. Binding Proteins

Also provided are antigen binding proteins that contain an antibody heavy chain variable region selected from the group consisting of VH1, VH2, VH3, Vh4, Vh5, Vh6, Vh7, Vh8, Vh9, Vh 10, Vn 11, V„ 12, V„ 3 3, V„ 14, V„ 15, V„ 16, V„ 17 and V„ 18 as shown in Table 2B and/or an antibody light chain variable region selected from the group consisting of VlI, V|,2, Vl3, Vl4, V[,5, V[„6, V[7, V[_8, Vl9, Vi.10, VL11, VLI2, V,J3, V,14, V,.15, V, 16, V,.17 and V,J8 as shown in Table 2A, and immunologically functional fragments, derivatives, mutcins and variants of these light chain and heavy chain variable regions.

Table 2A - Exemplary Antibody Variable Light fVi.f Chains

Table 2B - Exemplary Antibody Variable Heavy (Vnl Chains

Table 2C - Coding Sequence for Antibody Variable Light (Vi,) Chains

Table 2D - Coding Sequence for Antibody Variable Heavy (VM) Chains

Eacli of the heav-y chain variable regions listed in Table 2B can be combined with any of the light chain variable regions shown in Table 2A to form an antigen binding protein. Examples of such combinations include Vnl combined with any of V| 1, Vj 2, Vi.3, V|4, V|5, V[,6, Vi.7, V18, Vl9, Vi JO, Vi 11, Vtj2, Vi 13, V[J4, VL15, VrJ6, VL17 or VjJ8; Vn2 combined with any of Vt.l, V,.2, V, 3, V,,4, Vi.5, V,6, V,.7, V,.8, V,.9, V,J0, V,J 1, V,J2, V, 13, V,J4, V, 15, V,J6, Vj 17 or Vi. 18; Vh3 combined with any of VL1, Vi.2, V| 3, VL4, Vt 5, VL6, V| 7, VjB, Vt,9, Vj.10, VL11, Vl12, Vl13, Vj 14, VL15, VL16, VtJ7 or VL18; and so on.

In some instances, the antigen binding protein includes at least one heavy chain variable region and/or one light chain variable region from those listed in Tables 2A and 2B. In some instances, the antigen binding protein includes at least two different heavy chain variable regions and/or light chain variable regions from those listed in Table 2B. An example of such an antigen binding protein comprises (a) one Vtih and (b) one of Vh2, Vh3, Vh4, Vu5, V|[6, Vh7, Vh8, Vh9, V„I0. V„] ], Vh12, VH|?, VH14, VH,5, VH,6, VH,7 or VH1S. Another example comprises (a) one V„2, and (b) one of VhU V„3, V,.,4, Vh5, V„6, V„7, V„8, V„9, V„10, V„ll, Vh12, Vh13, Vn14, Vnl5, VE1I6, Vnl7 or VH18. Again another example comprises (a) one Vtt3, and (b) one of VHI, Vh2, V„4, V„5, Vh6, V„7, V„8, Vh9, V„10, V„11, V„12, V„13, V„14, V„15 V„16, V„17orVn.l8, etc.

Again another example of such an antigen binding protein comprises (a) one Vj.I, and (b) one of Vl2, VL3, VL4, VL5, VL(\ V,7, V,„8, V,9, VL10, V, Π, Vl12, VL13, V,.14, Vt15, VL16, Vi 17, or V| 18. Again another example of such an antigen binding protein comprises (a) one V,2, and (b) one of V,.l, Vs3, V,4, V,5, Vr6, V, 7, VL8, VL9. Vi.10, Vf 11 or V,.32. Again another example of such an antigen binding protein comprises (a) one VE 3, and (b) one of Vi.l, Vt 2, Vj4, V[,5, V] 6, Vi 7, V,.8, Vr.9, V, 10, VL1I, V,12, Vt.13, V, 14, Vj 15, Vt.16, V, 17, or Vi, 18, etc.

The various combinations of heavy chain variable regions can be combined with any of the various combinations of light chain variable regions.

In other embodiments, an antigen binding protein comprises two identical light chain variable regions and/or two identical heavy chain variable regions. As an example, the antigen binding protein can be an antibody or immunologically iimctional fragment thereof that includes two light chain variable regions and two heavy chain variable regions in combinations of pairs of light chain variable regions and pairs of heavy chain variable regions as listed in Tables 2A and 2B.

Some antigen binding proteins that are provided comprise a heavy chain variable domain comprising a sequence of amino acids that differs from the sequence of a heavy chain variable domain selected from Vj|l, Vh2, Vn3, Vn4, Vn5, Vh6, Vh7, Vh8, Vu9, Vnl0, V»!!, Vnl2, VH13, VhI4, Vh15, Vii16, Vn17and V,il8atonly 1,2,3,4, 5,6,7,8, 9, 10, II, 12, 13, 14 or 15 amino acid residues, wherein each such sequence difference is independently either a deletion, insertion or substitution of one amino acid, with the deletions, insertions and/or substitutions resulting in no more than 15 amino acid changes relative to the foregoing variable domain sequences. The heavy chain variable region in some antigen binding proteins comprises a sequence of amino acids that has at least 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% sequence identity to the amino acid sequences of the heavy chain variable region of VH1, Vh2, V]]3, Vj|4, Vj.]5, V„6, Vh7, Vir8, Vn9, VnlO, V„] 1, Vn12, V„13, V„14, Vnl5, V„16, Vn]7 and V„18.

Certain antigen binding proteins comprise a iight chain variable domain comprising a sequence of amino acids that differs from the sequence of a light chain variable domain selected from V,.l, V,2, VL3, Vt4, V[.5, V,6, V,7, V,.8, VL9, V,.I0, VL11, V,.12, V,J3, Vr14, VL15, Vi.16, Vt.17 and Vj.18 at only I, 2, 3,4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14 or 15 amino acid residues, wherein each such sequence difference is independently either a deletion, insertion or substitution of one amino acid, with the deletions, insertions and/or substitutions resulting in no more than 15 amino acid changes relative to the foregoing variable domain sequences. The light chain variable region in some antigen binding proteins comprises a sequence of amino acids that has at least 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% sequence identity to the amino acid sequences of the light chain variable region of Vt 1, Vt 2, V] 3, V| 4, Vi,5, Vj 6, V( 7, V| 8, V] 9, Vi 10, Vt 11, V,.12, V,.13, V, 14, V,J5, V, 16, V,.17 or VL18.

In additional instances, antigen binding proteins comprise the following pairings of light chain and heavy chain variable domains: V|J with Vul, Vi,2 with Vh2, Vi2 with Vu3, Vi.3 with Vn4, Vi,4 with Vn5, Vi.5 with Vn6, Vi.6 with Vti7, VL7 with Vu8, VL8 with Vn8, Vt.9 with V(!9, V|.9 with VnlO, VL10 with Vn 11, V». 11 with Vnll, Vj,l2 with Vul2, V|,13 with Vnl3, V],14 with Vh14, Vtj15 with Vjil5, Vj 16 with Vh16, Vl17 with Vh17 and VL18 with Vh18. In some instances, the antigen binding proteins in the above pairings can comprise amino acid sequences that have 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with the specified variable domains.

Still other antigen binding proteins, e.g,, antibodies or immunologically functional fragments, include variant forms of a variant heavy chain and a variant light chain as just described.

Antiuen Binding Protein CDRs

In various embodiments, the antigen binding proteins disclosed herein can comprise polypeptides into which one or more CDRs are grafted, inserted and/or joined. An antigen binding protein can have .1, 2, 3, 4, 5 or 6 CDRs. An antigen binding protein thus can have, for example, one heavy chain CDRl (“CDRHl’j, and/or one heavy chain CDR2 (“CDRH2”), and/or one heavy chain CDR3 (‘‘CDRH3”), and/or one light chain CDRI (‘'CDRLl”), and/or one light chain CDR2 (“CDRL2”), and/or one light chain CDR3 (“CDRL3”). Some antigen binding proteins include both a CDRH3 and a CDRL3. Specific heavy and light chain CDRs are identified in Tables 3A and 3B, respectively and in Table 6C, infra.

Complementarity determining legions (C'DRs) and framework regions (FR) of a given antibody can be identified using the system described by Rabat et al„ in Sequences of Proteins of Immunological Interest, 5th Ed., US Dept, of Health and Human Services, PHS, NIH, NIH Publication no. 91-3242, 1991. As desired, the CDRs can also be redefined according an alternative nomenclature scheme, such as that of C'hothia (see Chothia &amp; Lesk, 1987,./. Mol. Biol. 196:901-917; Chothia et aL. 1989, Nature 342:878-883 or Honegger &amp; Pluckthun, 2001,./. Mol. Biot. 309:657-670). Certain antibodies that are disclosed herein comprise one or more amino acid sequences that arc identical or have substantial sequence identity to the amino acid sequences of one or more of the CDRs presented in Table 3A (CDRHs) and Table 3B (CDRLs) and Table 6C, infra.

Table 3A - Exemplary CDRH Sequences

Tabic 3B - Exemplary CDRL Sequences

Table 3C - Coding Setiuences for CDRHs

Table 3D - Coding Sequences for CDRLs

The structure and properties of CDRs within a naturally occurring antibody has been described, supra. Briefly, in a traditional antibody, the CDRs are embedded within a framework in the heavy and light chain variable region where they constitute the regions responsible for antigen binding and recognition, A variable region comprises at least three heavy or light chain CDRs, see, supra (Rabat et a!., 1991, Sequences of Proteins of Immunological Interest, Public Health Service Bethesda, MD; see also Chothia and Lcsk, 1987,,/. Mol. Biol. 196:901- 917; Chothia et al,, 1989, Nature 342: 877-883), within a framework region (designated framework regions 1-4, FRI, FR2, FR3, and FR4, by Rabat et al., 1991; see also Chothia and Lesk, 1987, supra). The CDRs provided herein, however, can not only be used to define the antigen binding domain of a traditional antibody structure, but can be embedded in a variety of other polypeptide structures, as described herein.

In one aspect, the CDRs provided are (a) a C'DRH selected from the group consisting of (i) a CDRHI selected from the group consisting of SEQ ID NO: 121-131; (ii) a CDRH2 selected from the group consisting of SEQ ID NO: 132-144; (iii) a CDRH3 selected from the group consisting of SEQ ID NO: 145-157; and (iv) a CDRH of (i), (ii) and (iii) that contains one or more amino acid substitutions, deletions or insertions of no more than five, four, three, two, or one amino acids; (B) a CDRL selected from the group consisting of (i) a CDRL1 selected from the group consisting of SEQ ID NO:!58-I70; (ii) a CDRL2 selected from the group consisting of SEQ ID NO: 171-179; (iii) a CDRL3 selected from the group consisting of SEQ ID NO: 180-194; and (iv) a CDRL of (i), (ii) and (iii) that contains one or more amino acid substitutions, deletions or insertions of no more than 1, 2,3,4, or 5 amino acids amino acids.

In another aspect, an antigen binding protein comprises 1,2,3,4, 5, or 6 variant forms of the CDRs listed in Tables 3A and 3B and Table 6C, infra, each having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a CDR sequence listed in Tables 3A and 3B and Table 6C, infra. Some antigen binding proteins comprise I, 2, 3, 4, 5, or 6 of the CDRs listed in Tables 3A and 3B and Table 6C, infra, each differing by no more than 1, 2, 3, 4 or 5 amino acids from the CDRs listed in these tables.

In still another aspect, an antigen binding protein includes the following associations of CDRLJ, CDRL2 and CDRL3: SEQ ID NOs:167, 176, and 190; SEQ ID NOs: 167, 176, and 189, SEQ ID NOs:166, 176, and 188; SEQ ID NOs:166, 176, and 188; SEQ ID NOs:161, 174, and 183; SEQ ID NOs;I62, 173, and 184; SEQ ID NOs: 162, 173, and 186; SEQ ID NOs:164, 173, and 186; SEQ ID NOs:!60, 173, and 182; SEQ ID NOs: 163, 173, and 185; SEQ ID NOs:l63, 173, and 185; SEQ ID NOs:159, 172, and 181; SEQ ID NOs: 165, 175, and 187; SEQ ID NOs: 158, 171, and 180; SEQ ID NOs:!68, 171, and 191; SEQ ID NOs:!69, 177 and 192; SEQ ID NOs:170, 378, and 193; SEQ ID NOs:163, 173, and 194; SEQ ID NOs:163, 173 and 194; and SEQ ID NOs:l63, 179, and 194. in an additional aspect, an antigen binding protein includes the following associations of CDRH1, CDRH2 and CDRH3: SEQ ID NOs:122, 133, and 146; SEQ ID NOs:122, 133, and 147; SEQ ID NOs:I22, 133, and 148; SEQ ID NOs:122, 134, and 148; SEQ ID NOs:124, 136, and 150; SEQ ID NOs:124, 138, and 152; SEQ ID NOs:124, 139, and 152; SEQ ID NOs:124, 137, and 151; SEQ ID NOs:124, 137, and 151; SEQ ID NOs:131. 140, and 153; SEQ ID NOs:125, 140, and 153; SEQ ID NOs:123, 135, and 149; SEQ ID NOs:123, 135, and 149; SEQ ID NOs:121, 132, and 145; SEQ ID NOs:126, 133, and 154; SEQ ID NOs:130, 144, and 157; SEQ ID NOs:127, 135, and 155; SEQ ID NOs:129, 142, and 156; SEQ ID NOs:128, 141, and 156; and SEQ ID NOs: 128, 143, and 156.

In another aspect, an antigen binding protein includes the following associations of CDRLI, CDRL2 and CDRL3 with CDRH1, CDRH2 and CDRH3: SEQ ID NOs:l67, 176, and 190; SEQ ID NOs:167, 176, and 189, SEQ ID NOs:166, 176, and 188; SEQ ID NOs:166, 176, and 188; SEQ ID NOs:161, 174, and 183; SEQ ID NOs:162, 173, and 184: SEQ ID NOs:162, 173, and 186; SEQ ID NOs:164, 173, and 186; SEQ ID NOs:160, 173, and 182; SEQ ID NOs:163, 173, and 185; SEQ ID NOs:163, 173, and 185; SEQ ID NOs: 159, 172, and 181; SEQ ID NOs: 165, 175, and 187; SEQ ID NOs:158, 171, and 180; SEQ ID NOs:168, 171, and 191; SEQ ID NOs: 169, 177 and 192; SEQ ID NOs: 170, 178, and 193; SEQ ID NOs: 163, 173, and 194; SEQ ID NOs: 163, 173 and 194; SEQ ID NOs: 163, 179, and 194 with SEQ ID NOs: 122, 133, and 146; SEQ ID NOs:322, 133, and 147; SEQ ID NOs:122, 133, and 148; SEQ ID NOs: 122, 134, and!48; SEQ ID NOs: 124, 136, and 150; SEQ ID NOs: 124, 138, and 152; SEQ ID NOs:124, 139, and 152; SEQ ID NOs:I24, 137, and 151; SEQ ID NOs:324, 137, and 151; SEQ ID N0s:131, 140, and 153; SEQ ID NOs:125, 140, and 153; SEQ ID NOs:123, 135, and 149: SEQ ID NOs:123, 135, and 149; SEQ ID NOs:121, 132, and 145; SEQ ID NOs:l26, 133, and 154; SEQ ID NOs:130, 144, and 157; SEQ ID NOs:127, 135, and 155; SEQ ID NOs:129, 142, and 156; SEQ IDNOs:I28, 141, and 156; and SEQ ID NOs:128, 143, and 156.

Consensus Sequences

In yet another aspect, the CDRs disclosed herein include consensus sequences derived from groups of related monoclonal antibodies. As described herein, a “consensus sequence” refers to amino acid sequences having conserved amino acids common among a number of sequences and variable amino acids that vary within a given amino acid sequences. The CDR consensus sequences provided include CDRs corresponding to each of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3.

Consensus sequences were determined using standard analyses of the CDRs corresponding to the VSI and VL of the disclosed antibodies, some of which specifically bind (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4. The consensus sequences were determined by keeping the CDRs contiguous within the same sequence corresponding to a Vh or V],.

Light Chain CDR3 Group 1 LQFINSYPLT (SEQ ID NO: 267)

Group 2 MQSLQTPFT (SEQ ID NO: 268) QQYNNWPPT (SEQ ID NO: 269)

Group 4 MQS1QLPRT (SEQ ID NO: 270)

Group 5 QQANDFPIT (SEQ ID NO: 271)

Group 6 MQALQTPCS (SEQ ID NO: 272)

Group 7 QVWD G N SDHVV (SEQ ID NO: 273) QVWD N T SDHVV (SEQ ID NO: 274) QVWD S S SDHVV (SEQ ID NO: 275) QVWD X, X2 SDHVV (SEQ ID NO: 276) wherein X| is G, S or N and X2 is S, T or N,

Group 8 QQ C G S S P L T (SEQ ID NO: 277) QQ Y G G S P L T (SEQ ID NO: 278) QQ Y G S A P L T (SEQ ID NO: 279) QQ Y G S S F T (SEQ ID NO: 280) QQ Y G S S P L T (SEQ ID NO: 281) QQ S G S S P L T (SEQ ID NO: 282) QQ X3 G X4 X5 Xo X7 T (SEQ ID NO: 283) wherein X3 is C, Y or S, X4 is S or G, Xs is S or A, X6 is P or F and X7 is L or absent.

Light Chain CDR2 Grour> 1 AASSLQS (SEQ ID NO: 284)

Group 2 GVSTRAT (SEQ ID NO: 285)

Group 3 DDSDRPS (SEQ ID NO: 286)

Group 4 EVSNRFS (SEQ ID NO: 287) L G S N R A S (SEQ ID NO: 288) L G S D R A S (SEQ ID NO: 289) L G S X27 R A S (SEQ ID NO: 290) wherein X2i is N or D.

Group 6 GASS RAT (SEQ ID NO: 291) G T S S RAT (SEQ ID NO: 292) GASP RAT (SEQ ID NO: 293) G X8 S X2R RAT (SEQ ID NO: 294) wherein Xs is A or T and X2s is S or F.

Light Chain CDRI RASQSVNSNLA (SEQ ID NO: 295)

Group 2 RASQDIRYDLG (SEQ ID NO: 296)

Group 3 RASQG1SIWLA (SEQ ID NO: 297)

Group 4 KSSQSLLQSDGKTYLY (SEQ ID NO: 298)

Group 5 RASQN F D S S S LA (SEQ ID NO: 299) RASQN F D S S Y LA (SEQ ID NO: 300) RASQS V S G N Y LA (SEQ ID NO: 301) RASQS V S G T Y LA (SEQ ID NO: 302) RASQN F D S N Y LA (SEQ ID NO: 303) RASQX, Xw X„ X12 Xu X|4 LA (SEQ ID NO: 304) wherein X<> is A or S, Xt<> is V or F, Χι i is D or S, Xu is G or S, Xn is S, N or T, and Xm is S or Y.

Group 6 GGNNIGS E SVH (SEQ ID NO: 305) GGNNIGS Q SVH (SEQ ID NO: 306) GGNNIGS X!S SVH (SEQ ID NO: 307) wherein Xu is E or Q.

Group 7 RSSQSLL Y Y NG F T Y LD (SEQ ID NO: 308) RSSQSLL H S NG Y N F LD (SEQ ID NO: 309) RSSQSLL X29 Xjo NG X.„ XS2 XM LD (SEQ ID NO: 310) wherein X2<> is Y or H, X30 is Y or S, X3i is F or Y, X.;2 is T or N and X33 is Y or F. HEAVY CDR3

Group 1 IVVVPAAIQSYYYYYGMGV (SEQ ID NO: 311)

Group 2 DPDGDYYYYGMDV (SEQ ID NO: 312)

Group 3 TYSSGWYVWDYYGMDV (SEQ ID NO: 313)

Group 4 DRVLSYYAMAV (SEQ ID NO: 314)

Group 5 VR1AGDYYYYYGMDV (SEQ ID NO: 315)

Group 6 EN1VVIPAA1FAGWFDP (SEQ ID NO: 316)

Group 7 DRAAAGLHYYYGMDV (SEQ ID NO: 317)

Group 8 I L L L G A YYY Y GMDV (SEQ ID NO: 318) I L L V G A YYY C GMDV (SEQ ID NO: 319) V V T G G YYY D GMDV (SEQ ID NO: 320) S V V T G G YYY D GMDV (SEQ ID NO: 321) X34 Xi6 Xi7 Xis G Xu YYY X20 GMDV (SEQ ID NO: 322)

Wherein is I, V or S, XJ6 is L or V, Xp is L, T or V, X]S is L, V, G or T, X19 is A, G or absent and X20 is Y, C or D.

Group 9 SLIVV 1 VY A LD H (SEQ ID NO: 323) SLIVV I VY A LD Y (SEQ ID NO: 324) SLIVV M VY V LD Y (SEQ ID NO: 325) SLIVV X2, VY X22 LD X» (SEQ ID NO: 326) Wherein X2) is I or M, X22 is A or V and X22 is H or Y, HEAVY CDR2 Group 1 GFDPEDGETIYAQKFQG (SEQ ID NO: 327)

Groun 2 RIKSK T DGGTTDYAAPVKG (SEQ ID NO: 328) RIKSK DGGTTDYAAPVKG (SEQ ID NO: 330) RIKSK X42 DGGTTDYAAPVKG (SEQ ID NO:483) wherein X^2 is T or absent,

Groun 3 HIFSNDEKSYSTSLK S (SEQ ID NO: 331) HIFSNDEKSYSTSLK N (SEQ ID NO: 332) HIFSNDEKSYSTSLK XM (SEQ ID NO: 333) wherein X24 is S or N.

Groun 4 G ISGSGVST H YADSVKG (SEQ ID NO: 334) G ISGSGVST Y YADSVKG (SEQ ID NO: 335) A ISGSGVST Y YADSVKG (SEQ ID NO: 336) A ISGSGVST N YADSVKG (SEQ ID NO: 337) X25 ISGSGVST X26 YADSVKG (SEQ ID NO: 338) wherein X25 is G or A and X2<-, is Η, Y or N.

Group 5 VIWYDGS D KYY A DSVKG (SEQ ID NO: 339) VIWYDGS I KYY G DSVKG (SEQ ID NO: 340) VIWYDGS X3S KYY X36 DSVKG (SEQ ID NO: 341) wherein X?s is D or I and Χΐύ is A or G.

Group 6 N iY Y SGST Y YNPSLKS (SEQ ID NO: 342) R IY T SGST Y YNPSLKS (SEQ ID NO: 343) R 1Y T SGST N YNPSLKS (SEQ ID NO: 329) X37 IY x.rs SGST X4, YNPSLKS (SEQ ID NO: 344) wherein X37 is N or R, X?s is Y or T and X4I is Y or N. HEAVY CPR1

Group 1 DLSMH (SEQ ID NO: 345)

Group 2 DAWMS (SEQ ID NO: 346)

Group 3 TYAMS (SEQ ID NO: 347)

Group 4 SYFWS (SEQ ID NO: 348)

Group 5 SGGYNWS (SEQ JD NO: 349)

Group 6 NARMGV S (SEQ ID NO: 350) NARMGV N (SEQ ID NO: 351) NARMGV X39 (SEQ ID NO: 352) wherein X39 is S or N.

Group 7 S YGIH (SEQ ID NO: 353) N YGIH (SEQ ID NO: 354) X<e YGIH (SEQ ID NO: 355) wherein XLio is S or N.

In some cases an antigen binding protein comprises at least one heavy chain CDRI, CDR2, or CDR3 having one of the above consensus sequences. In some cases, an antigen binding protein comprises at least one light chain CDRI, CDR2, or CDR3 having one of the above consensus sequences. In other cases, the antigen binding protein comprises at least two heavy chain C’DRs according to the above consensus sequences, and/or at least two light chain CDRs according to the above consensus sequences, in still other cases, the antigen binding protein comprises at least three heavy chain CDRs according to the above consensus sequences, and/or at least three light chain CDRs according to the above consensus sequences.

Exemplary Antigen Binding Proteins

According to one aspect, an isolated antigen binding protein comprising (a) otic or more heavy chain complementary determining regions (CDRHs) selected from the group consisting of: (i) a CDRH1 selected from the group consisting of SEQ ID NO:121-131; (ii) a CDRH2 selected from the group consisting of SEQ ID NO: 132-144; (iii) a CDRH3 selected from the group consisting of SEQ ID NO: 145-157; and (iv) a CDRH of (i), (ii) and (iii) that contains one or more amino acid substitutions, deletions or insertions of no more than 1, 2, 3, 4, or 5 amino acids; (b) one or more light chain complementary determining regions (CDRLs) selected from the group consisting of: (i) a CDRL1 selected from the group consisting of SEQ ID NO: 158-170; (ii) a CDRL2 selected from the group consisting of SEQ ID NO: 171-179; (iii) a CDRL3 selected from the group consisting of SEQ ID NO:180-194; and (iv) a CDRL of (i), (ii) and (iii) that contains one or more amino acid substitutions, deletions or insertions of no more than five, four, three, four, two or one amino acids; or (c) one or more heavy chain CDRHs of (a) and one or more light chain CDRLs of (b).

In another embodiment, the CDRHs have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with an amino acid sequence selected from the group consisting ofSEQ ID N0:121-1S7, and/or the CDRLs have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 158-194. In a further embodiment, the VH is selected from the group consisting ofSEQ ID NO: 121 -157, and/or the VL is selected from the group consisting of SEQ ID NO: 158-194.

According to one aspect, an isolated antigen binding protein comprising (a) one or more variable heavy chains (VHs) selected from the group consisting of: (i) SEQ ID NO: 121-157; and (ii) a VH of (i) that contains one or more amino acid substitutions, deletions or insertions of no more than five, four, three, four, two or one amino acids; (b) one or more variable light chains (VLs) selected from the group consisting of: (i) SEQ ID NO: 158-194, and (ii) a VL of (i) that contains one or more amino acid substitutions, deletions or insertions of no more than five, four, three, four, two or one amino acids; or (c) one or more variable heavy chains of (a) and one or more variable light chains of (b).

In another embodiment, the variable heavy chain (VH) has at least 70%, 75%, 80%, 85%, 90%, 95%i, 96%, 97%, 98% or 99%> sequence identity with an amino acid sequence selected from the group consisting ofSEQ ID NO: 121-157, and/or the variable light chain (VL) has at least 70%), 75%), 80%, 85%, 901½. 95%), 96%, 97%). 98%> or 99% sequence identity with an amino acid sequence selected from the group consisting ofSEQ ID NO: 158-194.

In one aspect, also provided is an antigen binding protein that specifically binds to an epitope comprising one or more amino acid residues from FGFRlc, FGRF2c, FGFR3c, and FGFR4.

In one aspect, also provided is an antigen binding protein that specifically binds to an epitope comprising one or more amino acid residues from β-Klotho.

In another aspect, also provided is an isolated antigen binding protein that specifically binds to an epitope comprising one or more amino acid residues from both β-Klotho and one or more amino acid residues from FGFRlc, FGFR2c, FGFR3c, or FGFR4.

In yet another embodiment, the isolated antigen binding protein described hereinabove comprises a first amino acid sequence comprising at least one of the CDRH consensus sequences disclosed herein, and a second amino acid sequence comprising at least one of the CDRL consensus sequences disclosed herein.

In one aspect, the first amino acid sequence comprises at least two of the CDRH consensus sequences, and/or the second amino acid sequence comprises at least two of the CDRL consensus sequences. In certain embodiments, the first and the second amino acid sequence arc covalently bonded to each other.

In a further embodiment, the first amino acid sequence of the isolated antigen binding protein comprises the CDRH3 of SEQ ID NO: 146, the CDRH2 of SEQ ID NO: 133. and the CDRH1 of SEQ 3D NO:122, and/or the second amino acid sequence of the isolated antigen binding protein comprises the CDRL3 of SEQ ID NO:190, the CDRL2 of SEQ ID NO: 176, and the CDRL 1 of SEQ ID NO: 167.

In a further embodiment, the first amino acid sequence of the isolated antigen binding protein comprises the CDRH3 of SEQ ID NO:147, the CDRH2 of SEQ ID NO: 133, and the CDRH I of SEQ ID NO: 122, and/or the second amino acid sequence of the isolated antigen binding protein comprises the CDRL3 of SEQ ID NO: 189, the CDRL2 of SEQ ID NO: 176, and the CDRL 1 of SEQ ID NO: 167.

In a further embodiment, the first amino acid sequence of the isolated antigen binding protein comprises the CDRH3 of SEQ ID NO: 148, the CDRH2 of SEQ ID NO: 133, and the CDRH 1 of SEQ ID NO: 122, and/or the second amino acid sequence of the isolated antigen binding protein comprises the CDRL3 of SEQ ID NO: 188, the CDRL2 of SEQ ID NO: 176, and the CDRL1 of SEQ ID NO: 166.

In a further embodiment, the first amino acid sequence of the isolated antigen binding protein comprises the CDRH3 of SEQ ID NO: 148, the CDRH2 of SEQ ID NO: 134, and the CDRH1 of SEQ ID NO: 122, and/or the second amino acid sequence of the isolated antigen binding protein comprises the CDRL3 of SEQ ID NO: 188, the CDRL2 of SEQ ID NO: 176, and the CDRL! of SEQ ID NO: 166. in a further embodiment, the first amino acid sequence of the isolated antigen binding protein comprises the CDRH3 of SEQ ID NO: 150, the CDRH2 of SEQ ID NO: 136, and the CDRH1 of SEQ ID NO: 124, and/or the second amino acid sequence of the isolated antigen binding protein comprises the CDRL3 of SEQ ID NO: 183, the CDRL2 of SEQ ID NO: 174, and the CDRL1 of SEQ ID NO: 161.

In a further embodiment, the first amino acid sequence of the isolated antigen binding protein comprises the CDRH3 of SEQ ID NO:152, the CDRH2 of SEQ ID NO:138, and the CDRH3 of SEQ ID NO: 124, and/or the second amino acid sequence of the isolated antigen binding protein comprises the CDRL3 of SEQ ID NO: 184, the CDRL2 of SEQ ID NO: 173, and the CDRL1 of SEQ ID NO: 162.

In a further embodiment, the first amino acid sequence of the isolated antigen binding protein comprises the CDRH3 of SEQ ID NO: 152, the CDRH2 of SEQ ID NO: 139, and the CDRH1 of SEQ ID NO: 124, and/or the second amino acid sequence of the isolated antigen binding protein comprises the CDRL3 of SEQ ID NO: 186, the CDRL2 of SEQ ID NO: 173, and the CDR.L1 of SEQ ID NO: 162. in a further embodiment, the first amino acid sequence of the isolated antigen binding protein comprises the CDRH3 of SEQ ID NO: 151, the CDRH2 of SEQ ID NO: 137, and the CDRH1 of SEQ ID NO: 124, and/or the second amino acid sequence of the isolated antigen binding protein comprises the CDRL3 of SEQ ID NO: 186, the CDRL2 of SEQ ID NO: 173, and the CDRL1 of SEQ ID NO: 164.

In a further embodiment, the first amino acid sequence of the isolated antigen binding protein comprises the CDRH3 of SEQ ID NO: 151, the CDRH2 of SEQ ID NO: 137, and the CDRH1 of SEQ ID NO: 124, and/or the second amino acid sequence of the isolated antigen binding protein comprises the CDRL3 of SEQ ID NO: 182, the CDRL2 of SEQ ID NO: 173, and thcCDRLl of SEQ ID NO: 3 60, in a further embodiment, the first amino acid sequence of the isolated antigen binding protein comprises the CDRH3 of SEQ ID NO: 153, the CDRH2 of SEQ ID NO: 140, and the CDRH1 of SEQ ID NO: 131, and/or the second amino acid sequence of the isolated antigen binding protein comprises the CDRL3 of SEQ ID NO: 185, the CDRL2 of SEQ ID NO: 173, and the CDRL1 of SEQ ID NO: 163.

In a further embodiment, the first amino acid sequence of the isolated antigen binding protein comprises the CDRH3 of SEQ ID NO: 153, the CDRH2 of SEQ ID NO: 140, and the CDRH3 of SEQ ID NO: 125, and/or the second amino acid sequence of the isolated antigen binding protein comprises the CDRL3 of SEQ ID NO: 185, the CDRL2 of SEQ ID NO: 173, and the CDRL1 of SEQ ID NO: 163.

In a further embodiment, the first amino acid sequence of the isolated antigen binding protein comprises the CDRH3 of SEQ ID NO:149, the CDRH2 of SEQ ID NO:135, and the CDRH1 of SEQ ID NO: 123, and/or the second amino acid sequence of the isolated antigen binding protein comprises the CDRL3 of SEQ ID NO: 18.1, the CDRL2 of SEQ ID NO: 172, and the CDRL1 of SEQ ID NO: 159.

In a further embodiment, the first amino acid sequence of the isolated antigen binding protein comprises the CDRH3 of SEQ ID NO: 149, the CDRH2 of SEQ ID NO:135, and the CDRH1 of SEQ ID NO: 123, and/or the second amino acid sequence of the isolated antigen binding protein comprises the CDRL3 of SEQ ID NO: 187, the CDRL2 of SEQ ID NO: 175, and the CDRL1 of SEQ ID NO: 165.

In a further embodiment, the first amino acid sequence of the isolated antigen binding protein comprises the CDRH3 of SEQ ID NO: 145, the CDRH2 of SEQ ID NO: 132, and the C'DRHl of SEQ ID NO: 121, and/or the second amino acid sequence of the isolated antigen binding protein comprises the CDRL3 of SEQ ID NO: 180, the CDRL2 of SEQ ID NO: 171, and the CDRL1 of SEQ ID NO: 158.

In a further embodiment, the first amino acid sequence of the isolated antigen binding protein comprises the CDRH3 of SEQ ID NO: 154, the CDRH2 of SEQ ID NO: 133, and the CDRH1 of SEQ ID NO: 126, and/or the second amino acid sequence of the isolated antigen binding protein comprises the CDRL3 of SEQ ID NO: 191, the CDRL2 of SEQ ID NO: 171, and the CDRL1 of SEQ ID NO: 168.

In a further embodiment, the first amino acid sequence of the isolated antigen binding protein comprises the CDRH3 of SEQ ID NO: 157, the CDRH2 of SEQ ID NO: 144, and the CDRH1 of SEQ ID NO: 130, and/or the second amino acid sequence of the isolated antigen binding protein comprises the CDRL3 of SEQ ID NO: 192, the CDRL2 of SEQ ID NO: 177, and the CDRL1 of SEQ ID NO: 169.

In a further embodiment, the first amino acid sequence of the isolated antigen binding protein comprises the CDRH3 of SEQ ID NO: 155, the CDRH2 of SEQ ID NO: 135, and the CDRH1 of SEQ ID NO: 127, and/or the second amino acid sequence of the isolated antigen binding protein comprises the CDRL3 of SEQ ID NO: 193, the CDRL2 of SEQ ID NO: 178, and the CDRL1 of SEQ ID NO: 170.

In a further embodiment, the first amino acid sequence of the isolated antigen binding protein comprises the CDRH3 of SEQ ID NO: 156, the CDR.H2 of SEQ ID NO: 142, and the CDRHI of SEQ ID NO: 129, and/or the second amino acid sequence of the isolated antigen binding protein comprises the CDRL3 of SEQ ID NO: 194, the CDRL2 of SEQ ID NO: 173, and the CDRL1 of SEQ ID NO: 163.

In a further embodiment, the first amino acid sequence of the isolated antigen binding protein comprises the CDRH3 of SEQ ID NO:!56, the CDRH2 of SEQ ID NO: 141, and the CDRH1 of SEQ ID NO: 128, and/or the second amino acid sequence of the isolated antigen binding protein comprises the CDRL3 of SEQ ID NO: 194, the CDRL2 of SEQ ID NO: 173, and the CDRL1 of SEQ ID NO: 163.

In a further embodiment, the first amino acid sequence of the isolated antigen binding protein comprises the CDRH3 of SEQ ID NO: 156, the CDRH2 of SEQ ID NO: 143, and the CDRH1 of SEQ JD NO: 128, and/or the second amino acid sequence of the isolated antigen binding protein comprises the CDRL3 of SEQ ID NO: 194, the CDRL2 of SEQ ID NO: 179, and the CDRL1 of SEQ ID NO: 163.

In a further embodiment, the antigen binding protein comprises at least two CDRH sequences of heavy chain sequences HI, H2, H3, H4, H5, H6, H7, H8, H9, H10, HI 1, HI 2, HI 3, HI4, H15, HI6, H17 or HI8, as shown in Tabic 4A. In again a further embodiment, the antigen binding protein comprises at least two CDRL sequences of light chain sequences LI, L2, L3, L4, L5, L6, L7, L8, L9, L10, LI 1, LI2, L13, L14, L15, LI6, L17or L18, as shown in Table 4B. In still a further embodiment, the antigen binding protein comprises at least two CDRH sequences of heavy chain sequences HI, H2, H3, H4, H5, H6, H7, H8, H9, H10, HU, HI2, H13, H14, HI5, HI6, HI 7 or H18 as shown in Table 4A, and at least two CDRLs of light chain sequences LI, L2, L3, L4, L5, L6, L7, L8, L9, LI0, LI 1, LI2, LI3, LI4, LI5, LI6, LI7 or LI8 as shown in Table 4B.

In again another embodiment, the antigen binding protein comprises the CDRH1, CDRH2, and CDRH3 sequences of heavy chain sequences HI, H2, H3, H4, H5, H6, H7, H8, H9, HIO, Hll, H12, H13, HI4, HIS, HI6, HI7 or H18 as shown in Table 4A. In yet another embodiment, the antigen binding protein comprises the CDRL1, CDRL2, and CDRL3 sequences of light chain sequences LI, L2, L3, L4, L5, L6, L7, L8, L9, L10, LI 1, LI 2, LI 3, LI 4, LIS, LI 6, LI 7 or L18 as shown in Table 4B.

In yet another embodiment, the antigen binding protein comprises all six CDRs of LI and HI, or L2 and H2, or L3 and H3, or L3 and H4, or L4 and H5, or L5 and H6, or L6 and H7, or L7 and H8, or L8 and H7, or L9 and H9, or L9 and H10, or L10 and HI 1, or LI I and HI 1, or L]2 and HI2, or LI3 and HI3, or L14 and H14, or LI5 and HI5, or LI6 and HI6. or L17 and Η17, or L18 and Η18, as shown in Tables 4A and 4B.

Table 4A - Heavy Chain Sequences

Table 4B - Light Chain Sequences

In one aspect, the isolated antigen binding proteins that specifically bind (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc. FGFR2c, FGFR3c, and FGFR4providcd herein can be a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, a chimeric antibody, a multispecific antibody, or an antibody fragment thereof. in another embodiment, the antibody fragment of the isolated antigen-binding proteins provided herein can be a Fab fragment, a Fab’ fragment, an F(ab’)2 fragment, an Fv fragment, a diabody, or a single chain antibody molecule.

In a further embodiment, an isolated antigen binding protein that specifically (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4 provided herein is a human antibody and can be of the IgG 1 -, igG2- lgG3- or lgG4-typc. in another embodiment, an isolated antigen binding protein that specifically binds (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4: or (in) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4 comprises a light or a heavy chain polypeptide as set forth in Tables 1A-IB. In some embodiments, an antigen binding protein that specifically binds (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4 comprises a variable light or variable heavy domain such as those listed in Tables 2A-2B. In still other embodiments, an antigen binding protein that specifically binds (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4 comprises one, two or three CDRHs or one, two or three CDRLs as set forth in Tables 3A-3B, 4A-4B and Table 6C, infra. Such antigen binding proteins, and indeed any of the antigen binding proteins disclosed herein, can be PEGylatcd with one or more PEG molecules, for examples PEG molecules having a molecular weight selected from the group consisting of 5K, 10K, 20K, 40K, 50K, 60K, 80K, 100K or greater than 100K.

In yet another aspect, any antigen binding protein that specifically binds (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4 provided herein can be coupled to a labeling group and can compete for binding to the extracellular portion of (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRIc, FGFR2c, FGFR3c, and FGFR4 with an antigen binding protein of one of the isolated antigen binding proteins provided herein. In one embodiment, the isolated antigen binding protein provided herein can reduce blood glucose levels, decrease triglyceride and cholesterol levels or improve other glycemic parameters and cardiovascular risk factors when administered to a patient.

As will be appreciated, for any antigen binding protein comprising more than one CDR provided in Tables 3A-3B, and 4A-4B, any combination of CDRs independently selected from the depicted sequences may be useful. Thus, antigen binding proteins with one, two, three, four, five or six of independently selected CDRs can be generated. However, as will be appreciated by those in the art, specific embodiments generally utilize combinations of CDRs that are non-repetitive, e.g., antigen binding proteins are generally not made with two CDRH2 regions, etc.

Some of the antigen binding proteins that specifically bind (i) β-Klotho; (ii) FGFRIc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRIc, FGFR2c, FGFR3c, and FGFR4 that are provided herein are discussed in more detail below.

Antigen Binding Proteins and Binding Epitopes and Binding Domains

When an antigen binding protein is said to bind an epitope on (i) β-Klotho; (ii) FGFRIc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRIc, FGFR2c, FGFR3c, and FGFR4, or the extracellular domain of β-Klotho, FGFRIc, FGFR2c, FGFR3c or FGFR4, for example, what is meant is that the antigen binding protein specifically binds to a specified portion of (i) β-Klotho; (ii) FGFRIc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRIc, FGFR2c, FGFR3c, and FGFR4. In some embodiments, e.g., in certain cases where the antigen binding protein binds only FGFRIc or β-Klotho, the antigen binding protein can specifically bind to a polypeptide consisting of specified residues (e.g., a specified segment of β-KIotho, FGFRIc, FGFR2c, FGFR3c or FGFR4, such as those residues disclosed in Example 14). In other embodiments, e.g., in certain cases where an antigen binding protein interacts with both β-Klotho and one or more of FGFRIc, FGFR2c, FGFR3c and FGFR4, the antigen binding protein can bind residues, sequences of residues, or regions in both β-Klotho and FGFRIc, FGFR2c, FGFR3c or FGFR4, depending on which receptor the antigen binding protein recognizes. In still other embodiments the antigen binding protein will bind residues, sequence or residues or regions of a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4, for example FGFRlc,

In any of the foregoing embodiments, such an antigen binding protein does not need to contact every residue of (i) β-Klotho: (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (Hi) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4, or the extracellular domain of the recited proteins or complexes. Nor does every single amino acid substitution or deletion within (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4, or the extracellular domain of the recited proteins or complexes, necessarily significantly affect binding affinity.

Epitope specificity and the binding domain(s) of an antigen binding protein can be determined by a variety of methods. Some methods, for example, can use truncated portions of an antigen. Other methods utilize antigen mutated at one or more specific residues, such as by employing an alanine scanning or arginine scanning-type approach or by the generation and study of chimeric proteins in which various domains, regions or amino acids are swapped between two proteins (e.g.. mouse and human forms of one or more of the antigens or target proteins), or by protease protection assays.

Competing Antigen Binding Proteins

In another aspect, antigen binding proteins are provided that compete with one of the exemplified antibodies or functional fragments for binding to (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4. Such antigen binding proteins can also bind to the same epitope as one of the herein exemplified antigen binding proteins, or an overlapping epitope. Antigen binding proteins and fragments that compete with or bind to the same epitope as the exemplified antigen binding proteins are expected to show similar functional properties. The exemplified antigen binding proteins and fragments include those with the heavy and light chains HI-HI 8 and L1-L18, variable region domains V|J- Vj,18 and Vnl- Vnl8, and CDRs provided herein, including those in Tables 1, 2, 3, and 4. Thus, as a specific example, the antigen binding proteins that are provided include those that compete with an antibody comprising: (a) 1, 2, 3. 4, 5 or all 6 of the CDRs listed for an antibody listed in Tables 3A and 3B, and 4A and 4B and Table 6C, infra; (b) a Vh and a Vf selected from Vj.l- V[ 18 and Vnl- Vh18 and listed for an antibody listed in Tables 2A and 2B; or (c) two light chains and two heavy chains as specified for an antibody listed in Tables 1A and 12B and Table 6A, infra.

Thus, in one embodiment, the present disclosure provides antigen binding proteins that competes for binding to (i) β-KIotho; (ii) FGFRic, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRic, FGFR2c, FGFR3c, and FGFR4 with a reference antibody, wherein the reference antibody comprises a combination of light chain and heavy chain variable domain sequences selected from the group consisting of LI HI, L2H2, L3H3, L3H4, L4H5, L5H6, L6H7, L7H8, L8H8, L9H9, L9H10, L10H11, LI1H11, L12H12, L13H13, L14H14, L15H15, L16H16, L17H17 or LI8H18. in another embodiment, the present disclosure provides human antibodies that compete for binding to (i) β-Klotho; (ii) FGFRic, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRic, FGFR2c, FGFR3c, and FGFR4 with a reference antibody, wherein the reference antibody is 17C3, 22H5, 16H7, 24H11, 18G1, 17D8, 26H11, 12E4, 12CI I, 21H2, 21B4, 18B1I.1, 18BI1.2, 20D4, 46D11,40D2, 37D3, 39F7, 39F1 or 39G5.

In a further embodiment, an isolated human antibody is provided that binds to (i) β-Klotho; (ii) FGFRic, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRic, FGFR2c, FGFR3c, and FGFR4 with substantially the same Kd as a reference antibody; initiates FGF21-like signaling in an in vitro ELK-Luciferasc assay to the same degree as a reference antibody; lowers blood glucose; lowers serum lipid levels; and/or competes for binding with said reference antibody to (i) β-Klotho; (ii) FGFRic, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRic, FGFR2c, FGFR3c, and FGFR4, wherein the reference antibody is selected from the group consisting of I7C3, 22H5, 16H7, 24H11, 18G1, 17D8, 26H11, 12E4, 12C11, 21H2, 21B4, 18B11.1, 18B11.2, 20D4, 46D11, 40D2, 37D3,39F7,39F1 or 39G5.

The ability to compete with an antibody can be determined using any suitable assay, such as that described in Example 8, in w'hich antigen binding proteins 17C3, 22H5, 16H7, 24H11, 18GJ, 17D8, 26HI1, 12E4, 32C11, 21H2, 21B4, I8B1I.1, 18B11.2, 20D4, 46D11, 40D2, 37D3, 39F7, 39F3 or 39G5 can be used as the reference antibody.

Monoclonal Antibodies

The antigen binding proteins that are provided include monoclonal antibodies that bind to (i) β-KIotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4, and induce FGF21-!ike signaling to various degrees. Monoclonal antibodies can be produced using any technique known in the art, e.g., by immortalizing spleen cells harvested from the transgenic animal after completion of the immunization .schedule. The spleen cells can be immortalized using any technique known in the art, e.g., by fusing them with myeloma cells to produce hybridomas. Myeloma cells for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render them incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas). Examples of suitable cell lines for use in mouse fusions include Sp-20, P3-X63/Ag8, P3-X63-Ag8.653, NSl/I.Ag 4 1, Sp210-Ag!4, FO, NSO/U, MPC-ll, MPC11-X45-GTG 1.7 and S194/5XXO Bui; examples of cell lines used in rat fusions include R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210. Other cell lines useful for cell fusions arc U-266, GM1500-GRG2, L1CR-LON-HMy2 and UC729-6.

In some instances, a hybridoma cell line is produced by immunizing an animat (e.g., a transgenic animal having human immunoglobulin sequences) with a FGFRlc, β-Klotho or FGFRlc and/or β-Klotho immunogen (e.g., a soluble complex comprising the extracellular domains of FGFRlc, FGFR2c, FGFRSc or FGFR4 and/or β-Klotho as shown in Examples 2, and 3; membranes on which the extracellular domains of FGFRlc, FGFR2c, FGFR3c or FGFR4 and/or β-Klotho arc expressed, as shown in Examples I and 3; or whole cells expressing FGFRlc and/or β-Klotho, as shown in Examples 1 and 3); harvesting spleen cells from the immunized animal; fusing the harvested spleen cells to a myeloma cell line, thereby generating hybridoma cells; establishing hybridoma cell lines from the hybridoma cells, and identifying a hybridoma cell line that produces an antibody that binds to (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4 (e.g., as described in the Example 4) and can induce FGF21-like signaling (e.g., as described in Examples 5-7). Such hybridoma cell lines, and the monoclonal antibodies produced by them, form aspects of the present disclosure.

Monoclonal antibodies secreted by a hybridoma cell line can be purified using any technique known in the art. Hybridomas or mAbs can be further screened to identify mAbs with particular properties, such as the ability to induce FGF21 -like signaling. Examples of such screens arc provided herein.

Chimeric and Humanized Antibodies

Chimeric and humanized antibodies based upon the foregoing sequences can readily be generated. One example is a chimeric antibody, which is an antibody composed of protein segments from different antibodies that are covalently joined to produce functional immunoglobulin light or heavy chains or immunologically functional portions thereof. Generally, a portion of the heavy chain and/or light chain is identical with or homologous to a corresponding sequence in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is/are identical with or homologous to a corresponding sequence in antibodies derived from another species or belonging to another antibody class or subclass. For methods relating to chimeric antibodies, see, for example, United States Patent No. 4,816,567; and Morrison et al., 1985, Proc. Natl. Acad. Sci. USA 81:6851-6855, which are hereby incorporated by reference. CDR grafting is described, for example, in United States Patent No. 6,180,370, No. 5,693,762, No. 5,693,761, No. 5,585,089, and No. 5,530,101.

Generally, the goal of making a chimeric antibody is to create a chimera in which the number of amino acids from the intended patient/rccipient species is maximized. One example is the “CDR-graflcd” antibody, in which the antibody comprises one or more complementarity determining regions (CDRs) from a particular species or belonging to a particular antibody class or subclass, while the remainder of the antibody chain(s) is/arc identical with or homologous to a corresponding sequence in antibodies derived from another species or belonging to another antibody class or subclass. For use in humans, the variable region or selected CDRs from a rodent antibody often are grafted into a human antibody, replacing the naturally-occurring variable regions or CDRs of the human antibody.

One useful type of chimeric antibody is a “humanized” antibody. Generally, a humanized antibody is produced from a monoclonal antibody raised initially in a non-human animal. Certain amino acid residues in this monoclonal antibody, typically from non-antigen recognizing portions of the antibody, arc modified to be homologous to corresponding residues in a human antibody of corresponding isotype. Humanization can be performed, for example, using various methods by substituting at least a portion of a rodent variable region for the corresponding regions of a human antibody (see, e.g., United States Patent No. 5,585,089, and No, 5,693,762; Jones et a/.. 1986, Nature 321:522-525: Riechmann et al., 1988, Nature 332:323-27; Verhoeyen et al... 1988, Science 239:1534-1536).

In one aspect, the CDRs of the light and heavy chain variable regions of the antibodies provided herein (e.g., in Tables 3 and 4) are grafted to framework regions (FRs) from antibodies from the same, or a different, phylogenetic species. For example, the CDRs of the heavy and light chain variable regions V^l, VH2, Vrt3, Vn4, VH5, VH6, Vn7, Vn8, Vn9, VH10, νΜ11, Val2, V„13, V]]14, V„15. Vn16, Vn17 or V„18 and/or V,.I, Vi.2, V,3, V,4, V, 5, V,6, V,.7, V, 8, V,9, Vi lO, VJ !, Vh!2, Vl!3, Vi,14. VJ5, VJ6, V|17 or Vj 18 can be grafted to consensus human FRs. To create consensus human FRs, FRs from several human heavy chain or light chain amino acid sequences can be aligned to identify a consensus amino acid sequence. In other embodiments, the FRs of a heavy chain or light chain disclosed herein arc replaced with the FRs from a different heavy chain or light chain. In one aspect, rare amino acids in the FRs of the heavy and light chains of an antigen binding protein (e.g., an antibody) that specifically binds (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4 are not replaced, while the rest of the FR amino acids are replaced. A “rare amino acid” is a specific amino acid that is in a position in which this particular amino acid is not usually found in an FR. Alternatively, the grafted variable regions from the one heavy or light chain can be used with a constant region that is different from the constant region of that particular heavy or light chain as disclosed herein. In other embodiments, the grafted variable regions are part of a single chain Fv antibody.

In certain embodiments, constant regions from species other than human can be used along with the human variable region(s) to produce hybrid antibodies.

Fully Human Antibodies

Fully human antibodies are also provided by the instant disclosure. Methods are available for making fully human antibodies specific for a given antigen without exposing human beings to the antigen (“fully human antibodies”). One specific means provided for implementing the production of fully human antibodies is the “humanization” of the mouse humoral immune system. Introduction of human immunoglobulin (Ig) loci into mice in which the endogenous Ig genes have been inactivated is one means of producing fully human monoclonal antibodies (mAbs) in mouse, an animal that can be immunized with any desirable antigen. Using fully human antibodies can minimize the immunogenic and allergic responses that can sometimes be caused by administering mouse or mouse-derived mAbs to humans as therapeutic agents.

Fully human antibodies can be produced by immunizing transgenic animals (typically mice) that are capable of producing a repertoire of human antibodies in the absence of endogenous immunoglobulin production. Antigens for this purpose typically have six or more contiguous amino acids, and optionally are conjugated to a carrier, such as a hapten. See, e.g., Jakobovits et a!., (1993) Proc. Natl. Acad. Sci. USA 90:2551-2555; Jakobovits et £//.,(1993) Nature 362:255-258; and Bruggermann et a/., (1993) Year in Immunol. 7:33. In one example of such a method, transgenic animals are produced by incapacitating the endogenous mouse immunoglobulin loci encoding the mouse heavy and light immunoglobulin chains therein, and inserting into the mouse genome large fragments of human genome DNA containing loci that encode human heavy and light chain proteins. Partially modified animals, which have less than the full complement of human immunoglobulin loci, are then cross-bred to obtain an animal having ail of the desired immune system modifications. When administered an immunogen, these transgenic animals produce antibodies that arc immunospecific for the immunogen but have human rather than murine amino acid sequences, including the variable regions. For further details of such methods, see, e.g., W096/33735 and W094/02602. Additional methods relating to transgenic mice for making human antibodies arc described in United States Patent No. 5,545,807; No. 6,713,610; No. 6,673,986; No. 6,162,963; No. 5,545,807; No. 6,300,129; No. 6,255,458; No. 5,877,397; No. 5,874,299 and No. 5,545,806; in PCT publications WO91/I0741, W090/04036, and in EP 546073B1 and EP 546073A1.

The transgenic mice described above, referred to herein as “HuMab" mice, contain a human immunoglobulin gene minilocus that encodes unrearranged human heavy ([μ, mu] and [γ, gamma]) and [k, kappa] light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous μ [mu] and tc [kappa] chain loci (Lonberg et al., 1994, Nature 368:856-859). Accordingly, the mice exhibit reduced expression of mouse IgM or [k, kappa] and in response to immunization, and the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high affinity human IgG [k, kappa] monoclonal antibodies (Lonberg et a 1., supra.; Lonberg and Huszar, (1995) Intern. Rev. Immunol. J_3: 65-93; Harding and Lonberg, (1995) Ann. N.Y Acad. Sci. 764:536-546). The preparation of HuMab mice is described in detail in Taylor et al., (1992) Nucleic Acids Research 20:6287-6295; C'hcn et at., (1993) International Immunology 5:647-656; Tuaillon et al., (1994) ,/. Immunol. 152:2912-2920; Lonberg et al., (1994) Nature 368:856-859; Lonberg, (1994) Handbook of Exp. Pharmacology 113:49-101; Taylor et al., (1994) Internationa! Immunology 6:579-591; Lonberg and Huszar, (1995) Intern. Rev. Immunol. j_3:65-93; Harding and Lonberg, (1995) Ann. N.Y Acad. Sci. 764:536-546; Fishwild et al., (1996) Nature Biotechnology 14:845-851; the foregoing references are hereby incorporated by reference in their entirety for all purposes, See, further United States Patent No. 5,545,806; No. 5,569,825; No. 5,625,126; No. 5,633,425; No. 5,789,650; No. 5,877,397; No. 5,661,016; No. 5,814,318; No. 5,874,299; and No. 5,770,429; as well as United States Patent No. 5,545,807; International Publication Nos. WO 93/1227; WO 92/22646; and WO 92/03918, the disclosures of all of which are hereby incorporated by reference in their entirety for all purposes. Technologies utilized for producing human antibodies in these transgenic mice are disclosed also in WO 98/24893, and Mendez et «/.,(1997) Nature Genetics 15:146-156, which are hereby incorporated by reference. For example, the HCo7 and HCol2 transgenic mice strains can be used to generate antigen binding proteins (e.g., antibodies) that bind to (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4 and may induce FGF21-likc signaling. Further details regarding the production of human antibodies using transgenic mice are provided in the examples below.

Using hybridoma technology, antigen-specific human mAbs with the desired specificity can be produced and selected from the transgenic mice such as those described above. Such antibodies can be cloned and expressed using a suitable vector and host cell, or the antibodies can be harvested from cultured hybridoma cells.

Fully human antibodies can also be derived from phage-display libraries (as disclosed in Hoogcnboom et al., (1991) ./ Mol. Biol. 227:38k and Marks et al., (1991) ,/. Mol. Biol. 222:581), Phage display techniques mimic immune selection through the display of antibody repertoires on the surface of filamentous bacteriophage, and subsequent selection of phage by their binding to an antigen of choice. One such technique is described in PCT Publication No. WO 99/10494 (hereby incorporated by reference), which describes the isolation of high affinity and functional agonistic antibodies for MPL- and msk-receptors using such an approach.

Bispecific or Bifunctional Antigen Binding Proteins

Also provided are bispccific and bifunctional antibodies that include one or more CDRs or one or more variable regions as described above. A bispccific or bifunclional antibody in some instances can be an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods including, but not limited to, fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai and Lachmann, 1990, Clin. Exp. Immunol. 79:315-321; Kostclny et al., 1992, ,/. Immunol. 148:1547-1553. When an antigen binding protein of the instant disclosure binds (i) both β-Klotho and one or more of FGFRlc, FGFR2c, FGFR3c or FGFR4; or (ii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4, the binding may lead to the activation of FGF21-like activity as measured by the FGF21-like functional and signaling assays described in Examples 5-7; when such an antigen binding protein is an antibody it is referred to as an agonistic antibody.

Various Other Forms

Some of the antigen binding proteins that specifically bind (i) β-Kiotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4 that are provided in the present disclosure include variant forms of the antigen binding proteins disclosed herein (e.g., those having the sequences listed in Tables 1-4).

In various embodiments, the antigen binding proteins disclosed herein can comprise one or more non-naturally occurring amino acids. For instance, some of the antigen binding proteins have one or more non-naturally occurring amino acid substitutions in one or more of the heavy or light chains, variable regions or C’DRs listed in Tables 1-4. Examples of non-naturally amino acids (which can be substituted for any naturally-occurring amino acid found in any sequence disclosed herein, as desired) include: 4-hydroxyprolinc, y-carboxyglutamatc, ε-Ν,Ν,Ν-trimethyllysine, ε-Ν-acetyllysine, O-phosphoscrine, N-acctylserine, N-formyimethionine, 3-methyihistidine, 5-hydroxyiysinc, σ-Ν-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxyl-terminal direction, in accordance with standard usage and convention. A non-limiting lists of examples of non-naturally occurring amino acids that can be inserted into an antigen binding protein sequence or substituted for a wild-type residue in an antigen binding sequence include β-amino acids, homoamino acids, cyclic amino acids and amino acids with derivatized side chains. Examples include (in the L-form or D-form; abbreviated as in parentheses): citruHinc (Cit), homocitrullinc (hCit), Να-methylcitrullinc (NMeCit), Na-mcthylhomocitrulline (Na-McHoCit), ornithine (Om), Na-Mcthylornithine (Na-McOrn or NMcOrn), sarcosine (Sar), homolysine (hLys or hK), homoarginine (hArg or hR), homoglutaminc (hQ), Να-mcthyiarginine (NMeR), Na-mcthyllcucinc (Na-MeL or NMcL), N-methylhomolysinc (NMcHoK), Na-mcthylglutamine ("NMeQ), norleucine (Nle), norvaline (Nva), l,2,3,4-tetrahydroisoquinoline (Tic), Octahydroindole-2-carboxylic acid (Oic), 3-(I-naphthyl)alanine (1-NaI), 3-(2-naphthyl)alanine (2-Nal), 1,2,3,4-tetrahydroisoquinoline (Tic), 2-indanylglycine (Igl), para-iodophenylalanine (pl-Phe), para-aminophenylalanine (4AmP or 4-Amino-Phe), 4-guanidino phenylalanine (Guf), glycyllysine (abbreviated “K(Ns-glycyl)” or “K(giycyl)” or “K(gly)”), nitrophcnylalaninc (nitrophe), aminophenylalaninc (aminophe or Amino-Phc), bcnzylphcnylalanine (bcnzylphc), y-carboxyg!utamic acid (γ-carbo.xyglu), hydroxyproline (hydroxypro), p-carboxyl-phcnylaianine (Cpa), α-aminoadipic acid (Aad), Να-methyl valine (NMcVal), N-u-mcthyl leucine (NMcLeu), Na-mcthylnorleucine (NMeNle), cyclopcntylglycinc (Cpg), cyclohcxylglycine (Chg), acetylargininc (acetylarg), a, β-diaminopropionoic acid (Dpr), a, y-diaminobutyric acid (Dab), diaminopropionic acid (Dap), cyclohexylalanine (Cha), 4-methyl-phenylalanine (MePhe), β, β-diphenyl-alanine (BiPhA), aminobutyric acid (Abu), 4-phcnyl-phenylalanine (or biphcnylaiantne; 4Bip), α-amino-isobutyric acid (Aib), beta-alanine, beta-aminopropionic acid, piperidinic acid, aminocaprioic acid, aminoheptanoic acid, aminopimclic acid, desmosine, diaminopimclic acid, N-cthylglycine. N-cthylasparginc, hydroxylysinc, allo-hydroxylysinc, isodcsmosine, allo-isolcucine, N-mcthylglycinc, N-methylisoIcucinc, N-mcthylvalinc, 4-hydroxyprolinc (Hyp), γ-carboxyghitamate, ε-Ν,Ν,Ν-trimcthylIysine, ε-Ν-acetyllysine, O-phosphoscrinc, N-acctylserine, N-formylmcthioninc, 3-mcthylhistidinc, 5-hydroxylysinc, co-methylarginine, 4-Amino-O-Phthalic Acid (4APA), and other similar amino acids, and derivatizcd forms of any of those specifically fisted.

Additionally, the antigen binding proteins can have one or more conservative amino acid substitutions in one or more of the heavy or light chains, variable regions or CDRs listed in Tables 1-4. Naturally-occurring amino acids can be divided into classes based on common side chain properties: 1) hydrophobic: norlcucinc, Met, Ala, Val, Leu, lie; 2) neutral hydrophilic: Cys, Scr, Thr, Asn, Gin; 3) acidic: Asp, Glu; 4) basic: His, Lys, Arg; 5) residues that influence chain orientation: Gly, Pro; and 6) aromatic: Tip, Tyr, Phc.

Conservative amino acid substitutions can involve exchange of a member of one of these classes with another member of the same class. Conservative amino acid substitutions can encompass non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. See Table 5, infra. These include pcptidomimetics and other reversed or inverted forms of amino acid moieties.

Non-conservative substitutions can involve the exchange of a member of one of the above classes for a member from another class. Such substituted residues can be introduced into regions of the antibody that are homologous with human antibodies, or into the non-homologous regions of the molecule.

In making such changes, according to certain embodiments, the hydropathic index of amino acids can be considered. The hydropathic profile of a protein is calculated by assigning each amino acid a numerical value (“hydropathy index”) and then repetitively averaging these values along the peptide chain. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. They are: isolcucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8): cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0,7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); prolific (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

The importance of the hydropathic profile in conferring interactive biological function on a protein is understood in the art (see, e.g., Kyte et al., 1982,,/. Mol. Biol. 157:105-131). It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, in certain embodiments, the substitution of amino acids whose hydropathic indices arc within ±2 is included. In some aspects, those which arc within ±1 arc included, and in other aspects, those within ±0.5 arc included.

It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity, particularly where the biologically functional protein or peptide thereby created is intended for use in immunological embodiments, as in the present case. In certain embodiments, the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigen-binding or immunogenicity, that is, with a biological property of the protein.

The following hydrophilicity values have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0+1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±1); alanine (-0.5); histidine (-0.5); cysteine (-3.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isolcucine (-3.8); tyrosine (-2.3); phenylalanine (-2.5) and tryptophan (-3.4), In making changes based upon similar hydrophilicity values, in certain embodiments, the substitution of amino acids whose hydrophilicity values arc within ±2 is included, in other embodiments, those which arc within ±1 are included, and in still other embodiments, those within ±0.5 are included. In some instances, one can also identify epitopes from primary amino acid sequences on the basis of hydrophilicity. These regions are also referred to as “epitopic core regions.”

Exemplary conservative amino acid substitutions are set forth in Table 5.

Table 5

Conservative Amino Acid Substitutions

A skilled artisan will be able to determine suitable variants of polypeptides as set forth herein using well-known techniques coupled with the information provided herein. One skilled in the art can identify suitable areas of the molecule that can be changed without destroying activity by targeting regions not believed to be important for activity. The skilled artisan also will be able to identify residues and portions of the molecules that are conserved among similar polypeptides. In further embodiments, even areas that can be important for biological activity or for structure can be subject to conservative amino acid substitutions without destroying the biological activity or without adversely affecting the polypeptide structure.

Additionally, one skilled in the art can review structure-function studies identifying residues in similar polypeptides that arc important for activity or structure. In view of such a comparison, one can predict the importance of amino acid residues in a protein that correspond to amino acid residues important for activity or structure in similar proteins. One skilled in the art can opt for chemically similar amino acid substitutions for such predicted important amino acid residues.

One skilled in the art can also analyze the 3-dimensional structure and amino acid sequence in relation to that structure in similar polypeptides. In view of such information, one skilled in the art can predict the alignment of amino acid residues of an antibody with respect to its three dimensional structure. One skilled in the art can choose not to make radical changes to amino acid residues predicted to be on the surface of the protein, since such residues can be involved in important interactions with other molecules. Moreover, one skilled in die art can generate test variants containing a single amino acid substitution at each desired amino acid residue. These variants can then be screened using assays for FGF21-likc signaling, (see the Examples provided herein) thus yielding information regarding which amino acids can be changed and which must not be changed, in other words, based on information gathered from such routine experiments, one skilled in the art can readily determine the amino acid positions where further substitutions should be avoided either alone or in combination with other mutations. A number of scientific publications have been devoted to the prediction of secondary structure. See, Moult, (1996) Chit, Op. in Biotech. 2:422-427; Chou et a/., (1974)

Biochem, 13:222-245; Chou et ai, (1974) Biochemistry Π3:211-222; Chou et al., (1978) Adv. Enzymol. Relat. Areas Mol. Biol. 47:45-148; Chou et ai, (1979) Ann. Rev, Biochem. 47:251-276; and Chou et ai, (1979) Biophys. ,/, 26:367-384. Moreover, computer programs arc currently available to assist with predicting secondary structure. One method of predicting secondary structure is based upon homology modeling. For example, two polypeptides or proteins that have a sequence identity of greater than 30%, or similarity greater than 40% can have similar structural topologies. The growth of the protein structural database (PDB) has provided enhanced predictability of secondary structure, including the potential number of folds within a polypeptide’s or protein’s structure. See, Holm et ai, (1999) Nad Add. Res. 27:244-247. It has been suggested (Brenner et ai, (1997) Carr, Op, Struct. Biol. 7:369-376) that there are a limited number of folds in a given polypeptide or protein and that once a critical number of structures have been resolved, structural prediction will become dramatically more accurate.

Additional methods of predicting secondary structure include “threading” (Jones, (1997) Cun·. Opin. Struct. Biol. 7:377-387; Sippl et a!.. (1996) Structure 4:15-19), “profile analysis” (Bowie et a/., (1991) Science 253:164-170; Gribskov et a!,, (1990) Meth. Enzym. 183:146-159; Gribskov et al., (1987) Proc. Nat. Acad. Sci. 84:4355-4358), and “evolutionary linkage” (See, Holm, (1999) supra; and Brenner, (1997) supra).

In some embodiments, amino acid substitutions arc made that: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter ltgand or antigen binding affinities, and/or (4) confer or modify other physicochemical or functional properties on such polypeptides. For example, single or multiple amino acid substitutions (in certain embodiments, conservative amino acid substitutions) can be made in the naturally-occurring sequence. Substitutions can be made in that portion of the antibody that lies outside the domain(s) forming intcrmoiecular contacts). In such embodiments, conservative amino acid substitutions can be used that do not substantially change the structural characteristics of the parent sequence (e.g., one or more replacement amino acids that do not disrupt the secondary structure that characterizes the parent or native antigen binding protein). Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed.), 1984, W. H. New York: Freeman and Company; Introduction to Protein Structure (Branden and Tooze, eds.), 1991, New York: Garland Publishing; and Thornton et at., (1991) Nature 354:105. which are each incorporated herein by reference.

Additional preferred antibody variants include cysteine variants wherein one or more cysteine residues in the parent or native amino acid sequence arc deleted from or substituted with another amino acid (e.g., serine). Cysteine variants are useful, inter alia when antibodies must be refolded into a biologically active conformation. Cysteine variants can have fewer cysteine residues than the native antibody, and typically have an even number to minimize interactions resulting from unpaired cysteines.

The heavy and light chains, variable regions domains and CDRs that are disclosed can be used to prepare polypeptides that contain an antigen binding region that can specifically bind (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4 and may induce FGF21-like signaling. For example, one or more of the CDRs listed in Tables 3 and 4 can be incorporated into a molecule (e.g., a polypeptide) covalently or noncovalently to make an immunoadhesion. An tmmunoadhesion can incorporate the C'DR(s) as part of a larger polypeptide chain, can covalently link the CDR(s) to another polypeptide chain, or can incorporate the C’DR(s) noncovalently. The CDR(s) enable the immunoadhesion to bind specifically to a particular antigen of interest (e.g., (i) β-Kiotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4 or an epitope thereon).

The heavy and light chains, variable regions domains and CDRs that are disclosed can be used to prepare polypeptides that contain an antigen binding region that can specifically bind (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4 and may induce FGF21-like signaling. For example, one or more of the CDRs listed in Tables 3 and 4 can be incorporated into a molecule (e.g., a polypeptide) that is structurally similar to a “half’ antibody comprising the heavy chain, the light chain of an antigen binding protein paired with a Fc fragment so that the antigen binding region is monovalent (like a Fab fragment) but with a dimeric Fc moiety.

Mimetics (e.g.. "peptide mimetics” or “peptidomimetics”) based upon the variable region domains and CDRs that are described herein are also provided. These analogs can be peptides, non-peptides or combinations of peptide and non-peptide regions. Fauchcrc, 1986, Adv. Drug Res. 15:29; Vcber and Freidingcr, 1985, TINS p. 392: and Evans et ai. 1987, J. Med. Chew. 30:1229, which arc incorporated herein by reference for any purpose. Peptide mimetics that arc structurally similar to therapeutically useful peptides can be used to produce a similar therapeutic or prophylactic effect. Such compounds arc often developed with the aid of computerized molecular modeling. Generally, peptidomimetics are proteins that are structurally similar to an antibody displaying a desired biological activity, such as here the ability to specifically bind (i) β-Klolho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4, but have one or more peptide linkages optionally replaced by a linkage selected from: -CH2NH-, -CH2S-, -CH2-CH2-, -CH-CH-(cis and trans), -COCH2-, -CH(OH)CH2-, and -CH2SO-, by methods well known in the art. Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type (e.g., D-lysine in place of L-lysine) can be used in certain embodiments to generate more stable proteins, in addition, constrained peptides comprising a consensus sequence or a substantially identical consensus sequence variation can be generated by methods known in the art (Rizo and Gicrasch, 1992, Aim. Rev. Biochem. 6 S :387), incorporated herein by reference), for example, by adding internal cysteine residues capable of forming intramolecular disulfide bridges which cyclizc the peptide.

Derivatives of the antigen binding proteins that specifically bind (i) β-Klotho; (it) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4 that arc described herein arc also provided. The derivatized antigen binding proteins can comprise any molecule or substance that imparts a desired property to the antibody or fragment, such as increased half-life in a particular use. The derivatized antigen binding protein can comprise, for example, a detectable (or labeling) moiety (e.g., a radioactive, colorimetric, antigenic or enzymatic molecule, a detectable bead (such as a magnetic or clectrodcnsc (e.g., gold) bead), or a molecule that binds to another molecule (e.g., biotin or streptavidin)), a therapeutic or diagnostic moiety (e.g., a radioactive, cytotoxic, or pharmaceutically active moiety), or a molecule that increases the suitability of the antigen binding protein for a particular use (e.g., administration to a subject, such as a human subject, or other in vivo or in vitro uses). Examples of molecules that can be used to dcrivatize an antigen binding protein include albumin (e.g,, human serum albumin) and polyethylene glycol (PEG). Albumin-linked and PEGylated derivatives of antigen binding proteins can be prepared using techniques well known in the art. Certain antigen binding proteins include a PEGylated single chain polypeptide as described herein. In one embodiment, the antigen binding protein is conjugated or otherwise linked to transthyretin (TTR) or a TTR variant. The TTR or TTR variant can be chemically modified with, for example, a chemical selected from the group consisting of dextran, poly(n-vinyl pyrrolidone), polyethylene glycols, propropylene glycol homopolymers, polypropylene oxide/cthylene oxide co-polymers, polyoxyethylated polyols and polyvinyl alcohols.

Other derivatives include covalent or aggregative conjugates of the antigen binding proteins that specifically bind (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4 that are disclosed herein with other proteins or polypeptides, such as by expression of recombinant fusion proteins comprising heterologous polypeptides fused to the N-terminus or C-termtnus of an antigen binding protein that induces FGF21-like signaling. For example, the conjugated peptide can be a heterologous signal {or leader) polypeptide, e.g., the yeast alpha-factor leader, or a peptide such as an epitope tag. An antigen binding protein-containing fusion protein of the present disclosure can comprise peptides added to facilitate purification or identification of an antigen binding protein that specifically binds (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4 (e.g, a poly-His tag) and that can induce FGF21-like signaling. An antigen binding protein that specifically binds (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4 also can be linked to the FLAG peptide as described in Flopp et ai., 1988, Bio/Technology 6:1204; and United States Patent No. 5,011,912. The FLAG peptide is highly antigenic and provides an epitope reversibly bound by a specific monoclonal antibody (mAb), enabling rapid assay and facile purification of expressed recombinant protein. Reagents useful for preparing fusion proteins in which the FLAG peptide is fused to a given polypeptide are commercially available (Sigma, St. Louis, MO).

Multimers that comprise one or more antigen binding proteins that specifically bind (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4 form another aspect of the present disclosure. Multimers can take the form of covalently-linked or non-covalently-linkcd dimers, trimers, or higher multimers. Multimers comprising two or more antigen binding proteins that bind (t) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4 and which may induce FGF21-like signaling are contemplated for use as therapeutics, diagnostics and for other uses as well, with one example of such a muitimer being a homodimer. Other exemplary multimers include heterodimers, homotrimers, hctcrotrimers, homotetramers, heterotetramers, etc.

One embodiment is directed to multimers comprising multiple antigen binding proteins that specifically bind (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4 joined via covalent or non-covalcnt interactions between peptide moieties fused to an antigen binding protein that specifically binds (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4. Such peptides can be peptide linkers (spacers), or peptides that have the property of promoting multimerization. Leucine zippers and certain polypeptides derived from antibodies are among the peptides that can promote multimerization of antigen binding proteins attached thereto, as described in more detail herein.

In particular embodiments, the multimcrs comprise from two to four antigen binding proteins that bind (i) β-KIotho; (ii) FGFRlc, FGFR2c, FGFRSc or FGFR4; or (iii) a complex comprising β-KIotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4. The antigen binding protein moieties of the multimer can be in any of the forms described above, e.g., variants or fragments. Preferably, the multimcrs comprise antigen binding proteins that have the ability to specifically bind (i) β-KIotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-KIotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4.

In one embodiment, an oligomer is prepared using polypeptides derived from immunoglobulins. Preparation of fusion proteins comprising certain heterologous polypeptides fused to various portions of antibody-derived polypeptides (including the Fc domain) has been described, e.g., by Ashkenazi et al., (1991) Proc. Natl. Acad. Sci. USA 88:10535: Bym et a!., (1990) Nature 344:677: and Hoilcnbaugh et al., 1992 "Construction of Immunoglobulin Fusion Proteins", in Current Protocols in Immunology, Suppl. 4, pages 10.19.1-10.19.1!.

One embodiment comprises a dimer comprising two fusion proteins created by fusing an antigen binding protein that specifically binds (i) β-KIotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-KIotho and one of FGFRlc, FGFR2c, FGFRSc, and FGFR4 to the Fc region of an antibody. The dimer can be made by, for example, inserting a gene fusion encoding the fusion protein into an appropriate expression vector, expressing the gene fusion in host cells transformed with the recombinant expression vector, and allowing the expressed fusion protein to assemble much like antibody molecules, whereupon interchain disulfide bonds form between the Fc moieties to yield the dimer.

The term “Fc polypeptide” as used herein includes native and mutcin forms of polypeptides derived from the Fc region of an antibody. Truncated forms of such polypeptides containing the hinge region that promotes dimerization also arc included. Fusion proteins comprising Fc moieties (and oligomers formed therefrom) offer the advantage of facile purification by affinity chromatography over Protein A or Protein G columns.

One suitable Fc polypeptide, described in PCT application WO 93/10151 and United States Patent. No. 5,426,048 and No. 5,262,522, is a single chain polypeptide extending from the N-terminal hinge region to the native C-tcrminus of the Fc region of a human IgGl antibody. Another useful Fc polypeptide is the Fc mutein described in United States Patent No. 5,457,035, and in Baum et al., (1994) EMBOJ. 13:3992-4001. The amino acid sequence of this mutein is identical to that of the native Fc sequence presented in WO 93/10151, except that amino acid 19 has been changed from Leu to Ala, amino acid 20 has been changed from Leu to Gtu, and amino acid 22 has been changed from Gly to Ala. The mutein exhibits reduced affinity for Fc receptors.

In other embodiments, the variable portion of the heavy and/or light chains of a antigen binding protein such as disclosed herein can be substituted for the variable portion of an antibody heavy and/or light chain.

Alternatively, the oligomer is a fusion protein comprising multiple antigen binding proteins that specifically bind (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4, with or without peptide linkers (spacer peptides). Among the suitable peptide linkers are those described in United States Patent. No. 4,751,180 and No. 4,935,233.

Another method for preparing oligomeric derivatives comprising that antigen binding proteins that specifically bind (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4 involves use of a leucine zipper. Leucine zipper domains arc peptides that promote oligomerization of the proteins in which they are found. Leucine zippers were originally identified in several DNA-binding proteins (Landschulz et al., (1988) Science 240:1759). and have since been found in a variety of different proteins. Among the known leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize. Examples of leucine zipper domains suitable for producing soluble oligomeric proteins are described in PCT application WO 94/10308, and the leucine zipper derived from lung surfactant protein D (SPD) described in Hoppe et a!., (1994) FEBS Letters 344:191. hereby incorporated by reference. The use of a modified leucine zipper that allows for stable trimerization of a heterologous protein fused thereto is described in

Fanslow et a/., (1994) Semin. Immunol 6:267-278. In one approach, recombinant fusion proteins comprising an antigen binding protein fragment or derivative that specifically binds (i) β-Klotho; (ϋ) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4 is fused to a leucine zipper peptide are expressed in suitable host cells, and the soluble oligomeric antigen binding protein fragments or derivatives that form arc recovered from the culture supernatant.

In certain embodiments, the antigen binding protein has a K[> (equilibrium binding affinity) of less than 1 pM, 10 pM, 100 pM, 1 nM, 2 nM, 5 nM, 10 nM, 25 nM or 50 nM.

In another aspect the instant disclosure provides an antigen binding protein having a half-life of at least one day in vitro or in vivo (e.g.. when administered to a human subject). In one embodiment, the antigen binding protein has a half-life of at least three days. In another embodiment, the antibody or portion thereof has a half-life of four days or longer. In another embodiment, the antibody or portion thereof has a half-life of eight days or longer. In another embodiment, the antibody or portion thereof has a half-life of ten days or longer. In another embodiment, the antibody or portion thereof has a half-life of eleven days or longer. In another embodiment, the antibody or portion thereof has a half-life of fifteen days or longer. In another embodiment, the antibody or antigen-binding portion thereof is derivatized or modified such that it has a longer half-life as compared to the underivatized or unmodified antibody. In another embodiment, an antigen binding protein that specifically binds (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-KIotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4 contains point mutations to increase serum half life, such as described in WO 00/09560, published Feb. 24, 2000, incorporated by reference.

Givcosvlation

An antigen binding protein that specifically binds (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-KJotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4 can have a glycosylation pattern that is different or altered from that found in the native species. As is known in the art, glycosylation patterns can depend on both the sequence of the protein (e.g., the presence or absence of particular glycosylation amino acid residues, discussed below), or the host cell or organism in which the protein is produced. Particular expression systems are discussed below.

Glycosylation of polypeptides is typically either N-Iinked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tri-peptide sequences asparagine-X-scrine and asparagine-X-thrconinc, where X is any amino acid except praline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of cither of these tri-peptide sequences in a polypeptide creates a potential glycosylation site. O-linkcd glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used.

Addition of glycosylation sites to the antigen binding protein is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tri-peptide sequences (for N-linkcd glycosylation sites). The alteration can also be made by the addition of, or substitution by, one or more serine or threonine residues to the starting sequence (for O-linkcd glycosylation sites). For ease, the antigen binding protein amino acid sequence can be altered through changes at the DNA level, particularly by mutating the DNA encoding the target polypeptide at preselected bases such that codons are generated that will translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on the antigen binding protein is by chemical or enzymatic coupling of glycosides to the protein. These procedures arc advantageous in that they do not require production of the protein in a host cell that has glycosylation capabilities for N- and O-iinked glycosylation. Depending on the coupling mode used, the sugar(s) can be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine, or hydroxyprolinc, (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine. These methods arc described in WO 87/05330 and in Aplin and Wriston, (1981) CRC Crit. Rev. Biochem., pp. 259-306.

Removal of carbohydrate moieties present on the starting antigen binding protein can be accomplished chemically or enzymatically. Chemical deglycosylation requires exposure of the protein to the compound trifluoromethanesulfonic acid, or an equivalent compound. This treatment results in the cleavage of most or all sugars except the linking sugar (N-acctylglucosamine or N-acetylgalactosamine), while leaving the polypeptide intact. Chemical deglycosylation is described by Hakimuddin et ai, (1987) Arch. Biochem. Biophys. 259:52 and by Edge et £//., (1981) Anal. Biochem. 118:131. Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and cxo-glycosidascs as described by Thotakura et a/., (1987) Meth. Enzymot. 138:350. Glycosylation at potential glycosylation sites can be prevented by the use of the compound tunicamycin as described by Duskin et a/., (1982) ./. Biol. Chem. 257:3105. Tunicamycin blocks the formation of protein-N-glycoside linkages.

Hence, aspects of the present disclosure include glycosylation variants of antigen binding proteins that specifically bind (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4 wherein the number and/or type of glycosylation site(s) has been altered compared to the amino acid sequences of the parent polypeptide. In certain embodiments, antibody protein variants comprise a greater or a lesser number of N-linked glycosylation sites than the native antibody. An N-linkcd glycosylation site is characterized by the sequence: Asn-X-Ser or Asn-X-Thr, wherein the amino acid residue designated as X can be any amino acid residue except proline. The substitution of amino acid residues to create this sequence provides a potential new site for the addition of an N-linkcd carbohydrate chain. Alternatively, substitutions that eliminate or alter this sequence will prevent addition of an N-linked carbohydrate chain present in the native polypeptide. For example, the glycosylation can be reduced by the deletion of an Asn or by substituting the Asn with a different amino acid. In other embodiments, one or more new N-linked sites are created. Antibodies typically have a N-linkcd glycosylation site in the Fc region.

Labels and Effector Groups

In some embodiments, an antigen binding protein that specifically binds (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4 comprises one or more labels. The term “labeling group” or “label” means any detectable label. Examples of suitable labeling groups include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3H, l4C, 15N, 35S,90Y, 99Tc, 11 'in, l25l, l31l), fluorescent groups (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic groups (e.g., horseradish peroxidase, β-galactosidase, lucifcrasc, alkaline phosphatase), chemiluminescent groups, biotinyl groups, or predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, nictal binding domains, epitope tags), in some embodiments, the labeling group is coupled to the antigen binding protein via spacer arms of various lengths to reduce potential steric hindrance. Various methods for labeling proteins arc known in the art and can be used as is seen Fit.

The term “effector group” means any group coupled to an antigen binding protein that specifically binds one (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-KIotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4 that acts as a cytotoxic agent. Examples for suitable effector groups arc radioisotopes or radionuclides (e.g., Ή, MC, l5N, ?5S, 90Y, "Tc, ntln, I25I, 1λ1ϊ). Other suitable groups include toxins, therapeutic groups, or chemotherapeutic groups. Examples of suitable groups include calicheamicin, auristalins, geldanamycin and cantansine. In some embodiments, the effector group is coupled to the antigen binding protein via spacer arms of various lengths to reduce potential steric hindrance.

In general, labels fall into a variety of classes, depending on the assay in which they arc to be detected: a) isotopic labels, which can be radioactive or heavy isotopes; b) magnetic labels (e.g., magnetic particles); c) redox active moieties; d) optical dyes; enzymatic groups (e.g. horseradish peroxidase, β-galactosidase, lucifcrasc, alkaline phosphatase); c) biotinylated groups; and f) predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags, etc.). In some embodiments, the labeling group is coupled to the antigen binding protein via spacer arms of various lengths to reduce potential steric hindrance. Various methods for labeling proteins are known in the art.

Specific labels include optical dyes, including, but not limited to, chromophores, phosphors and fluorophores, with the latter being specific in many instances. Fluorophores can be either “small molecule” fluores, or proteinaceous fluores,

By "fluorescent label” is meant any molecule that can be detected via its inherent fluorescent properties. Suitable fluorescent labels include, but are not limited to, fluorescein, rhodaminc, tetramethylrhodamine, cosin, crythrosin, coumarin, methyl-coumarins, pyrene, Malacitc green, stilbenc, Lucifer Yellow, Cascade Blue, Texas Red, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy 5, Cy 5.5, LC Red 705, Oregon green, the Aiexa-Fluor dyes (Alcxa Fluor 350, Alcxa Fluor 430, Alcxa Fluor 488, Alcxa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680), Cascade Blue,

Cascade Yellow and R-phycoerythrin (PE) (Molecular Probes, Eugene, OR), FITC, Rhodamine, and Texas Red (Pierce, Rockford, IL), Cy5, Cy5.5, Cy7 (Amersham Life Science, Pittsburgh, PA). Suitable optical dyes, including fhiorophorcs, are described in Molecular Probes Handbook, by Richard Ρ. Haugland, hereby expressly incorporated by reference.

Suitable proteinaceous fluorescent labels also include, but arc not limited to, green fluorescent protein, including a Rend la, Ptihsarcus, or Aequorea species of GFP (Chalfie et a/., (1994) Science 263:802-805). EGFP (Clontech Labs., Inc., Genbank Accession Number U55762), blue fluorescent protein (BFP, Quantum Biotechnologies, Inc., Quebec, Canada; Stauber, (1998) Biotechniques 24:462-471; Heim et al„ ( .1996) Cnrr. Biol. 6:178-182), enhanced yellow fluorescent protein (EYFP, Clontech Labs., Inc.), lucifcrase (Ichiki et at.,(1993) J. Immunol. 150:5408-5417). β galactosidasc (Nolan et al., (1988) Proc. Natl. Acad. Sci, U.S.A. 85:2603-2607) and Rcnilla (W092/15673, WO95/07463, WO98/14605, W098/26277, WO99/49019, United States Patents No. 5292658, No. 5418155, No. 5683888, No. 5741668, No. 5777079, No. 5804387, No. 5874304, No. 5876995, No. 5925558).

Preparing Of Antigen Binding Proteins

Non-human antibodies that are provided can be, for example, derived from any antibody-producing animal, such as mouse, rat, rabbit, goat, donkey, or non-human primate (such as monkey (e.g., cynomolgus or rhesus monkey) or ape (e.g., chimpanzee)). Non-human antibodies can be used, for instance, in in vitro cell culture and cell-culture based applications, or any other application where an immune response to the antibody does not occur or is insignificant, can be prevented, is not a concern, or is desired. In certain embodiments, the antibodies can be produced by immunizing with full-length β-Klotho, FGFRlc, FGFR2c, FGFR3c or FGFR4 (Example 1), with the extracellular domain of β-Klotho, FGFRlc, FGFR2c, FGFR3c or FGFR4 (Example 2), or two of β-Klotho, FGFRlc, FGFR2c, FGFR3c and FGFR4 (Example 1), with whole cells expressing FGFRlc, β-Klotho or both FGFRlc and β-Klotho (Example I and 3), with membranes prepared from cells expressing FGFRlc, β-Klotho or both FGFRlc and β-Klotho (Example 1 and 3), with fusion proteins, e.g., Fc fusions comprising FGFRlc, β-Klotho or FGFRlc and β-Klotho (or extracellular domains thereof) fused to Fc (Example 2 and 3), and other methods known in the art, e.g., as described in the Examples presented herein. Alternatively, the certain non-human antibodies can be raised by immunizing with amino acids which are segments of one or more of β-Klotho, FGFRlc, FGFR2c, FGFR3c or FGFR4 that form part of the epitope to which certain antibodies provided herein bind. The antibodies can be polyclonal, monoclonal, or can be synthesized in host cells by expressing recombinant DNA.

Fully human antibodies can be prepared as described above by immunizing transgenic animals containing human immunoglobulin loci or by selecting a phage display library that is expressing a repertoire of human antibodies.

The monoclonal antibodies (mAbs) can be produced by a variety of techniques, including conventional monoclonal antibody methodology, e.g., the standard somatic cell hybridization technique of Kohler and Milstcin, (1975) Nature 256:495. Alternatively, other techniques for producing monoclonal antibodies can be employed, for example, the viral or oncogenic transformation of B-lymphocytes. One suitable animal system for preparing hybridomas is the murine system, which is a very well established procedure. Immunization protocols and techniques for isolation of immunized splenocytes for fusion are known in the art. For such procedures, B ceils from immunized mice arc fused with a suitable immortalized fusion partner, such as a murine myeloma cell line. If desired, rats or other mammals besides can be immunized instead of mice and B cells from such animals can be fused with the murine myeloma cell line to form hybridomas. Alternatively, a myeloma cell line from a source other than mouse can be used. Fusion procedures for making hybridomas also arc well known. SLAM technology can also be employed in the production of antibodies.

The single chain antibodies that are provided can be formed by linking heavy and light chain variable domain (Fv region) fragments via an amino acid bridge (short peptide linker), resulting in a single polypeptide chain. Such single-chain Fvs (scFvs) can be prepared by fusing DNA encoding a peptide linker between DNAs encoding the two variable domain polypeptides (V[. and Vh). The resulting polypeptides can fold back on themselves to form antigen-binding monomers, or they can form multimers (e.g., dimers, trimers, or tetramers), depending on the length of a flexible linker between the two variable domains (Kortt et a/., (1997) Prot. Eng. 10:423; Kortt et a/., (2001) Biomoi. Eng. 18:95-108). By combining different Vi, and Vh-comprising polypeptides, one can form multimeric scFvs that bind to different epitopes (Kriangkum et al, (2001) Biomoi. Eng. 18:31-40). Techniques developed for the production of single chain antibodies include those described in U.S. Pat. No. 4,946,778; Bird, (1988) Science 242:423; Huston et al., (1988) Proc. Natl. Acad. Sci. U.S.A. 85:5879; Ward et a/., (1989) Nature 334:544. de Graaf et a/., (2002) Methods Mol Biol. 178:379-387. Single chain antibodies derived from antibodies provided herein include, but are not limited to scFvs comprising the variable domain combinations of the heavy and light chain variable regions depicted in Table 2, or combinations of light and heavy chain variable domains which include CDRs depicted in Tables 3 and 4.

Antibodies provided herein that arc of one subclass can be changed to antibodies from a different subclass using subclass switching methods. Thus, IgG antibodies can be derived from an IgM antibody, for example, and vice versa. Such techniques allow the preparation of new antibodies that possess the antigen binding properties of a given antibody (the parent antibody), but also exhibit biological properties associated with an antibody isotype or subclass different from that of the parent antibody. Recombinant DNA techniques can be employed. Cloned DNA encoding particular antibody polypeptides can be employed in such procedures, e.g., DNA encoding the constant domain of an antibody of the desired isotype. See, e.g., Lantto et al., (2002) Methods Mol. Biot. 178:303-316.

Accordingly, the antibodies that are provided include those comprising, for example, the variable domain combinations described, supra., having a desired isotype (for example, IgA, IgG 1, IgG2, EgG3, IgG4, IgE, and IgD) as well as Fab or F(ab'h fragments thereof. Moreover, if an IgG4 is desired, it can also be desired to introduce a point mutation (CPSCP->CPPCP (SEQ ID NOS 380-381, respectively, in order of appearance)) in the hinge region as described in Bloom et al., (1997) Protein Science 6:407, incorporated by reference herein) to alleviate a tendency to form intra-H chain disulfide bonds that can lead to heterogeneity in the lgG4 antibodies.

Moreover, techniques for deriving antibodies having different properties (/. e., varying affinities for the antigen to which they bind) arc also known. One such technique, referred to as chain shuffling, involves displaying immunoglobulin variable domain gene repertoires on the surface of filamentous bacteriophage, often referred to as phage display. Chain shuffling has been used to prepare high affinity antibodies to the hapten 2-phcnyioxazol-5-onc, as described by Marks et al., (1992) BioTechnolosv 10:779.

Conservative modifications can be made to the heavy and light chain variable regions described in Table 2, or the CDRs described in Tables 3A and 3B. 4A and 4B, and Table 6C, infra (and corresponding modifications to the encoding nucleic acids) to produce an antigen binding protein having functional and biochemical characteristics. Methods for achieving such modifications are described above.

Antigen binding proteins that specifically bind one or more of (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (tit) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFRSc, and FGFR4 can be further modified in various ways. For example, if they arc to be used for therapeutic purposes, they can be conjugated with polyethylene glycol (PEGylatcd) to prolong the scrum half-life or to enhance protein dclivciy. Alternatively, the V region of the subject antibodies or fragments thereof can be fused with the Fc region of a different antibody molecule. The Fc region used for this purpose can be modified so that it docs not bind complement, thus reducing the likelihood of inducing cell lysis in the patient when the fusion protein is used as a therapeutic agent. In addition, the subject antibodies or functional fragments thereof can be conjugated with human serum albumin to enhance the serum half-life of the antibody or fragment thereof. Another useful fusion partner for the antigen binding proteins or fragments thereof is transthyretin (TTR). TTR has the capacity to form a tetramcr, thus an untibody-TTR fusion protein can form a multivalent antibody which can increase its binding avidity.

Alternatively, substantial modifications in the functional and/or biochemical characteristics of the antigen binding proteins described herein can be achieved by creating substitutions in the amino acid sequence of the heavy and light chains that differ significantly in their effect on maintaining (a) the structure of the molecular backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulkiness of the side chain. A “conservative amino acid substitution” can involve a substitution of a native amino acid residue with a nonnative residue that has little or no effect on the polarity or charge of the amino acid residue at that position. See, Table 5, supra. Furthermore, any native residue in the polypeptide can also be substituted with alanine, as has been previously described for alanine scanning mutagenesis.

Amino acid substitutions (whether conservative or non-conservative) of the subject antibodies can be implemented by those skilled in the art by applying routine techniques. Amino acid substitutions can be used to identify important residues of the antibodies provided herein, or to increase or decrease the affinity of these antibodies for one or more of (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4 or for modifying the binding affinity of other antigenbinding proteins described herein.

Methods of Expressing Antigen Binding Proteins

Expression systems and constructs in the form of plasmids, expression vectors, transcription or expression cassettes that comprise at least one polynucleotide as described above are also provided herein, as well host cells comprising such expression systems or constructs.

The antigen binding proteins provided herein can be prepared by any of a number of conventional techniques. For example, antigen binding proteins that specifically bind (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4 can be produced by recombinant expression systems, using any technique known in the art. See, e.g., Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Kennel et al. (eds.) Plenum Press, New York (1980): and Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988).

Antigen binding proteins can be expressed in hybridoma cell lines (e.g., in particular antibodies can be expressed in hybridomas) or in cell lines other than hybridomas. Expression constructs encoding the antibodies can be used to transform a mammalian, insect or microbial host cell. Transformation can be performed using any known method for introducing polynucleotides into a host cell, including, for example packaging the polynucleotide in a virus or bacteriophage and transducing a host cell with the construct by transfection procedures known in the art, as exemplified by United States Patent No. 4,399,216: No. 4,912,040; No. 4,740,461; No. 4,959,455. The optimal transformation procedure used will depend upon which type of host cell is being transformed. Methods for introduction of heterologous polynucleotides into mammalian cells arc well known in the art and include, but are not limited to, dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, mixing nucleic acid with positively-charged lipids, and direct microinjcction of the DNA into nuclei.

Recombinant expression constructs typically comprise a nucleic acid molecule encoding a polypeptide comprising one or more of the following: one or more CDRs provided herein; a light chain constant region; a light chain variable region; a heavy chain constant region (e.g., C||I, Cjj2 and/or Cn3); and/or another scaffold portion of an antigen binding protein. These nucleic acid sequences are inserted into an appropriate expression vector using standard ligation techniques. In one embodiment, the heavy or light chain constant region is appended to the C-terminus of the anti-p-Klotho, -FGFRlc, -FGFR2c, -FGFR3c, -FGFR4, or β-Klotho and FGFR1 c-specific heavy or light chain variable region and is ligated into an expression vector. The vector is typically selected to be functional in the particular host cell employed (/.<?., the vector is compatible with the host cell machinery, permitting amplification and/or expression of the gene can occur). In some embodiments, vectors arc used that employ protein-fragment complementation assays using protein reporters, such as dihydrofolatc reductase (see, for example, U.S. Pat. No. 6,270,964, which is hereby incorporated by reference). Suitable expression vectors can be purchased, for example, from Invitrogen Life Technologies or BD Biosciences (formerly "Clontech”). Other useful vectors for cloning and expressing the antibodies and fragments include those described in Bianchi and McGrew, (2003) Biotech. Biotechnol. Bioeng. 84:439-44. which is hereby incorporated by reference. Additional suitable expression vectors arc discussed, for example, in Methods Enzymol., vol. 185 (D. V. Goeddcl, cd.), 1990, New York: Academic Press.

Typically, expression vectors used in any of the host cells will contain sequences for plasmid maintenance and for cloning and expression of exogenous nucleotide sequences. Such sequences, collectively referred to as “flanking sequences” in certain embodiments will typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcriptional termination sequence, a complete intron sequence containing a donor and acceptor splice site, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, and a selectable marker clement. Each of these sequences is discussed below.

Optionally, the vector can contain a “tag”-cncoding sequence, /.<?., an oligonucleotide molecule located at the 5’ or 3’ end of an antigen binding protein coding sequence; the oligonucleotide sequence encodes polyHis (such as hexaHis (SEQ ID NO: 382)), or another “tag” such as FLAG, HA (hemagiutinin influenza virus), or myc, for which commercially available antibodies exist. This tag is typically fused to the polypeptide upon expression of the polypeptide, and can serve as a means for affinity purification or detection of the antigen binding protein from the host cell. Affinity purification can be accomplished, for example, by column chromatography using antibodies against the tag as an affinity matrix. Optionally, the tag can subsequently be removed from the purified antigen binding protein by various means such as using certain peptidases for cleavage.

Flanking sequences can be homologous (/.¢., from the same species and/or strain as the host cell), heterologous (/.<?., from a species other than the host cell species or strain), hybrid (/'.<?., a combination of flanking sequences from more than one source), synthetic or native. As such, the source of a flanking sequence can be any prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, provided that the flanking sequence is functional in, and can be activated by, the host cell machinery.

Flanking sequences useful in the vectors can be obtained by any of several methods well known in the art. Typically, flanking sequences useful herein will have been previously identified by mapping and/or by restriction endonuclease digestion and can thus be isolated from the proper tissue source using the appropriate restriction endonucleases. In some cases, the full nucleotide sequence of a flanking sequence can be known. Here, the flanking sequence can be synthesized using the methods described herein for nucleic acid synthesis or cloning.

Whether all or only a portion of the flanking sequence is known, it can be obtained using polymerase chain reaction (PCR) and/or by screening a genomic library with a suitable probe such as an oligonucleotide and/or flanking sequence fragment from the same or another species. Where the flanking sequence is not known, a fragment of DNA containing a flanking sequence can be isolated from a larger piece of DNA that can contain, for example, a coding sequence or even another gene or genes. Isolation can be accomplished by restriction endonuclease digestion to produce the proper DNA fragment followed by isolation using agarose gel purification, QiagenJl column chromatography (Chatsworth, CA), or other methods known to the skilled artisan. The selection of suitable enzymes to accomplish this purpose will be readily apparent to one of ordinary skill in the art.

An origin of replication is typically a part of those prokaryotic expression vectors purchased commercially, and the origin aids in the amplification of the vector in a host cell. If the vector of choice docs not contain an origin of replication site, one can be chemically synthesized based on a known sequence, and ligated into the vector. For example, the origin of replication from the plasmid pBR322 (New England Biolabs, Beverly, MA) is suitable for most gram-negative bacteria, and various viral origins (e.g., SV40, polyoma, adenovirus, vesicular stomatitus virus (VSV), or papillomaviruses such as HPV or BPV) are useful for cloning vectors in mammalian cells. Generally, the origin of replication component is not needed for mammalian expression vectors (for example, the SV40 origin is often used only because it also contains the virus early promoter). A transcription termination sequence is typically located 3' to the end of a polypeptide coding region and serves to terminate transcription. Usually, a transcription termination sequence in prokaryotic cells is a G-C rich fragment followed by a poly-T sequence. While the sequence is easily cloned from a library or even purchased commercially as pail of a vector, it can also be readily synthesized using methods for nucleic acid synthesis such as those described herein. A selectable marker gene encodes a protein necessary for the survival and growth of a host cell grown in a selective culture medium. Typical selection marker genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, tetracycline, or kanamycin for prokaryotic host cells; (b) complement auxotrophic deficiencies of the cell; or (c) supply critical nutrients not available from complex or defined media. Specific selectable markers are the kanamycin resistance gene, the ampicillin resistance gene, and the tetracycline resistance gene. Advantageously, a neomycin resistance gene can also be used for selection in both prokaryotic and eukaryotic host cells.

Other selectable genes can be used to amplify the gene that will be expressed. Amplification is the process wherein genes that are required for production of a protein critical for growth or cell survival are reiterated in tandem within the chromosomes of successive generations of recombinant cells. Examples of suitable selectable markers for mammalian cells include dihydrofolate reductase (DHFR) and promoterlcss thymidine kinase genes. Mammalian ceil transformants are placed under selection pressure wherein only the transformants are uniquely adapted to survive by virtue of the selectable gene present in the vector. Selection pressure is imposed by culturing the transformed cells under conditions in which the concentration of selection agent in the medium is successively increased, thereby leading to the amplification of both the selectable gene and the DNA that encodes another gene, such as an antigen binding protein that binds (i) β-KLlotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4. As a result, increased quantities of a polypeptide such as an antigen binding protein are synthesized from the amplified DNA. A ribosome-binding site is usually necessary for translation initiation of mRNA and is characterized by a Shinc-Dalgarno sequence (prokaryotes) or a Kozak sequence (eukaryotes). The element is typically located 3’ to the promoter and 5' to the coding sequence of the polypeptide to be expressed.

In some cases, such as where glycosylation is desired in a eukaryotic host cell expression system, one can manipulate the various pre- or pro-sequences to improve glycosylation or yield. For example, one can alter the peptidase cleavage site of a particular signal peptide, or add prosequences, which also can affect glycosylation. The final protein product can have, in the -1 position (relative to the first amino acid of the mature protein), one or more additional amino acids incident to expression, which may not have been totally removed. For example, the final protein product can have one or two amino acid residues found in the peptidase cleavage site, attached to the amino-terminus. Alternatively, use of some enzyme cleavage sites can result in a slightly truncated form of the desired polypeptide, if the enzyme cuts at such area within the mature polypeptide.

Expression and cloning will typically contain a promoter that is recognized by the host organism and operably linked to the molecule encoding an antigen binding protein that specifically binds (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4. Promoters are untranscribcd sequences located upstream (/.<?., 5’) to the start codon of a structural gene (generally within about 100 to 1000 bp) that control transcription of the structural gene. Promoters are conventionally grouped into one of two classes: inducible promoters and constitutive promoters. Inducible promoters initiate increased levels of transcription from DNA under their control in response to some change in culture conditions, such as the presence or absence of a nutrient or a change in temperature. Constitutive promoters, on tiic other hand, uniformly transcribe a gene to which they arc operably linked, that is, with little or no control over gene expression. A large number of promoters, recognized by a variety of potential host cells, arc well known. A suitable promoter is operably linked to the DNA encoding heavy chain or light chain comprising an antigen binding protein by removing the promoter from the source DNA by restriction enzyme digestion and inserting the desired promoter sequence into the vector.

Suitable promoters for use with yeast hosts arc also well known in the art. Yeast enhancers arc advantageously used with yeast promoters. Suitable promoters for use with mammalian host cells arc well known and include, but are not limited to, those obtained from the genomes of viruses such as polyoma virus, fowl pox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma vims, cytomegalovirus, retroviruses, hepatitis-B virus, and Simian Virus 40 (SV40). Other suitable mammalian promoters include heterologous mammalian promoters, for example, heat-shock promoters and the actin promoter.

Additional promoters which can be of interest include, but are not limited to: SV40 early promoter (Benoist and Chambon, (1981) Nature 290:304-310): CMV promoter (Thomsen et a/., (1984) Proc. Natl. Acad, U.S.A. 81:659-663); the promoter contained in the 3’ long terminal repeat of Rous sarcoma virus (Yamamoto et a/., (1980) Cell 22:787-797): herpes thymidine kinase promoter (Wagner et a!., (1981) Proc. Natl. Acad. Sci. U.S.A. 78:1444-1445); promoter and regulatory sequences from the metallothionine gene (Prinster et al., (1982) Nature 296:39-42); and prokaryotic promoters such as the beta-lactamase promoter (Villa-Kamaroff et al., (1978) Proc. Natl. Acad. Set. U.S.A. 75:3727-3731); or the tac promoter (DeBoer et al., (1983) Proc. Natl. Acad. Sci. U.S.A. 80:21-25). Also of interest are the following animal transcriptional control regions, which exhibit tissue specificity and have been utilized in transgenic animals: the elastase I gene control region that is active in pancreatic acinar cells (Swift et al., (1984) Cell 38:639-646; Ornitz et al., (1986) Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, (1987) Hepatology 7:425-515); the insulin gene control region that is active in pancreatic beta cells (Hanahan, (1985) Nature 315:115-122); the immunoglobulin gene control region that is active in lymphoid cells (Grosschcdl et a!., (1984) Cel! 38:647-658; Adames et al., (1985) Nature 318:533-538; Alexander et al., (1987) Mol. Cell. Biol. 2:1436-1444); the mouse mammary tumor virus control region that is active in testicular, breast, lymphoid and mast cells (Lcdcr et al., (1986) Cell 45:485-495); the albumin gene control region that is active in liver (Pinkert et al., (1987) Genes and Devel. \ :268-276); the aipha-feto-protein gene control region that is active in liver (Krumlauf et al., (1985) Mol. Cell. Biol. 5:1639-1648; Hammer et a/., (1987) Science 253:53-58); the alpha 1-antitrypsin gene control region that is active in liver (Kelsey et a!., (1987) Genes and Devel. 1:161-171); the bcta-globin gene control region that is active in myeloid ceils (Mogram et al., (1985) Nature 315:338-340: Kollias et al., (1986) Cell 46:89-94); the myelin basic protein gene control region that is active in oligodendrocyte cells in the brain (Readhcad et #/.,(1987) Cell 48:703-712); the myosin light chain-2 gene control region that is active in skeletal muscle (Sani, (1985) Nature 314:283-286); and the gonadotropic releasing hormone gene control region that is active in the hypothalamus (Mason et al., (1986) Science 234:1372-1378).

An enhancer sequence can be inserted into the vector to increase transcription of DNA encoding light chain or heavy chain comprising an antigen binding protein that specifically binds (i) β-Klotho; (ii) FGFRlc, FGFR2c. FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4 by higher eukaryotes, e.g., a human antigen binding protein that specifically binds (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4. Enhancers are cis-acting elements of DNA, usually about 10-300 bp in length, that act on the promoter to increase transcription. Enhancers are relatively orientation and position independent, having been found at positions both 5’ and 3' to the transcription unit. Several enhancer sequences available from mammalian genes arc known (e.g., globin, clastase, albumin, alpha-feto-protein and insulin). Typically, however, an enhancer from a virus is used. The SV40 enhancer, the cytomegalovirus early promoter enhancer, the polyoma enhancer, and adenovirus enhancers known in the art are exemplary enhancing elements for the activation of eukaryotic promoters. While an enhancer can be positioned in the vector cither 5' or 3’ to a coding sequence, it is typically located at a site 5' from the promoter. A sequence encoding an appropriate native or heterologous signal sequence (leader sequence or signal peptide) can be incorporated into an expression vector, to promote extracellular secretion of the antibody. The choice of signal peptide or leader depends on the type of host cells in which the antibody is to be produced, and a heterologous signal sequence can replace the native signal sequence. Examples of signal peptides that are functional in mammalian host cells include the following: the signal sequence for interleukin-7 (1L-7) described in US Patent No. 4,965,195; the signal sequence for interleukin-2 receptor described in Cosman et al., (1984) Nature 312:768: the interlcukin-4 receptor signal peptide described in EP Patent No. 0367 566; the type I interleukin-1 receptor signal peptide described in U.S. Patent No. 4,968,607; the type II interleukin-1 receptor signal peptide described in EP Patent No, 0 460 846.

The expression vectors that are provided can be constructed from a starting vector such as a commercially available vector. Such vectors can but need not contain all of the desired flanking sequences. Where one or more of the flanking sequences described herein are not already present in the vector, they can be individually obtained and ligated into the vector. Methods used for obtaining each of the flanking sequences arc well known to one skilled in the art.

After the vector has been constructed and a nucleic acid molecule encoding light chain, a heavy chain, or a light chain and a heavy chain comprising an antigen binding protein that specifically binds (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-KJotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4 has been inserted into the proper site of the vector, the completed vector can be inserted into a suitable host cell for amplification and/or polypeptide expression. The transformation of an expression vector for an antigen binding protein into a selected host cell can be accomplished by well known methods including transfection, infection, calcium phosphate co-precipitation, electroporation, microinjcction, lipofcction, DEAE-dextran mediated transfection, or other known techniques. The method selected will in part be a function of the type of host cell to be used. These methods and other suitable methods arc well known to the skilled artisan, and arc set forth, for example, in Sambrook et a!., (2001), supra. A host cell, when cultured under appropriate conditions, synthesizes an antigen binding protein that can subsequently be collected from the culture medium (if the host cell secretes it into the medium) or directly from the host cell producing it (if it is not secreted). The selection of an appropriate host cell will depend upon various factors, such as desired expression levels, polypeptide modifications that are desirable or necessary for activity (such as glycosylation or phosphorylation) and ease of folding into a biologically active molecule.

Mammalian cell lines available as hosts for expression are well known in the art and include, but are not limited to, immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) ceils, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and a number of other cell lines. In certain embodiments, cell lines can be selected through determining which cell lines have high expression levels and constitutivcly produce antigen binding proteins with desirable binding properties (e.g., the ability to bind (i) β-

Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3e, and FGFR4). In another embodiment, a cell line from the B cell lineage that docs not make its own antibody but has a capacity to make and secrete a heterologous antibody can be selected. The ability to induce FGF21-like signaling can also form a selection criterion.

Uses of Antigen Binding Proteins for Diagnostic and Therapeutic Purposes

The antigen binding proteins disclosed herein arc useful for detecting (i) β-KIotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-KIotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4 in biological samples and identification of cells or tissues that produce one or more of (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4. For instance, the antigen binding proteins disclosed herein can be used in diagnostic assays, e.g., binding assays to detect and/or quantify (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-KIotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4 expressed in a tissue or cell. Antigen binding proteins that specifically bind to (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4 can be used in treatment of diseases related to FGF21-like signaling in a patient in need thereof, such as type 2 diabetes, obesity, dyslipidemia, NASH, cardiovascular disease, and metabolic syndrome. By forming a signaling complex comprising an antigen binding protein, and (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4, the natural in vivo activity of FGF21, which associates with FGFRlc, FGFR2c, FGFR3c, FGFR4 and β-Klotho in vivo to initiate signaling, can be mimicked and/or enchanced, leading to therapeutic effects.

Indications A disease or condition associated with human FGF21 includes any disease or condition whose onset in a patient is caused by, at least in part, the induction of FGF21-like signaling, which is initiated in vivo by the formation of a complex comprising FGFRlc, FGFR2c, FGFR3c or FGFR4 and β-KIotho and FGF21. The severity of the disease or condition can also be decreased by the induction of FGF21-likc signaling. Examples of diseases and conditions that can be treated with the antigen binding proteins include type 2 diabetes, obesity, dyslipidemia, NASH, cardiovascular disease, and metabolic syndrome.

The antigen binding proteins described herein can be used to treat type 2 diabetes, obesity, dyslipidemia, NASH, cardiovascular disease, and metabolic syndrome, or can be employed as a prophylactic treatment administered, e.g., daily, weekly, biweekly, monthly, bimonthly, biannuaily, etc to prevent or reduce the frequency and/or severity of symptoms, e.g., elevated plasma glucose levels, elevated triglycerides and cholesterol levels, thereby providing an improved glycemic and cardiovascular risk factor profile.

Diagnostic Methods

The antigen binding proteins described herein can be used for diagnostic purposes to detect, diagnose, or monitor diseases and/or conditions associated with FGFRIc, FGFR2c, FGFR3c, FGFR4, β-Klotho, FGF21 or combinations thereof. Also provided are methods for the detection of the presence of (i) β-Klotho; (ii) FGFRIc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-KIotho and one of FGFRIc, FGFR2c, FGFR3c, and FGFR4 in a sample using classical immunohistological methods known to those of skill in the art (e.g., Tijssen, 1993, Practice and Theory of Enzyme Immunoassays, Voi 15 (Eds R.H. Burdon and P.H. van Knippcnbcrg, Elsevier, Amsterdam); Zola, (1987) Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc.): Jalkancn et a!., (1985)./. Cell. Biol. 101:976-985: Jalkancn et al., (1987)./. Cell Biol 105:3087-3096). The detection of (i) β-Klotho; (ii) FGFRIc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRIc, FGFR2c, FGFR3c, and FGFR4 can be performed in vivo or in vitro.

Diagnostic applications provided herein include use of the antigen binding proteins to detect expression of (i) β-Klotho; (ii) FGFRIc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRIc, FGFR2c, FGFR3c, and FGFR4 and/or binding to (i) β-Klotho; (ii) FGFRIc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRIc, FGFR2c, FGFR3c, and FGFR4. Examples of methods useful in the detection of the presence of (i) β-Klotho; (ii) FGFRIc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRIc, FGFR2c, FGFR3c, and FGFR4 include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).

For diagnostic applications, the antigen binding protein typically will be labeled with a detectable labeling group. Suitable labeling groups include, but are not limited to, the following; radioisotopes or radionuclides (e.g., 'Ή, ‘‘‘C, l5N, '5S, 90Y, 99Tc, 11‘in, |2ί1, !λ1Ι), fluorescent groups (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic groups horseradish peroxidase, β-galactosidasc, lucifcrase, alkaline phosphatase), chemiluminescent groups, biotinyl groups, or predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, the labeling group is coupled to the antigen binding protein via spacer arms of various lengths to reduce potential steric hindrance. Various methods for labeling proteins are known in the art and can be used.

In another aspect, an antigen binding protein can be used to identify a cell or cells that express (i) β-Klotho; (ii) FGFRIc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Kiotho and one of FGFRIc, FGFR2c, FGFR3c, and FGFR4. In a specific embodiment, the antigen binding protein is labeled with a labeling group and the binding of the labeled antigen binding protein to (i) β-Klotho: (ii) FGFRIc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRIc, FGFR2c, FGFR3c, and FGFR4 is detected. In a further specific embodiment, the binding of the antigen binding protein to (i) β-Klotho; (ii) FGFRIc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRIc, FGFR2c, FGFR3c, and FGFR4 detected in vivo. In a further specific embodiment, the antigen binding protein is isolated and measured using techniques known in the art. See, for example, Harlow ami Lane, (1988) Antibodies: A Laboratory Manual, New York: Cold Spring Harbor (ed. 1991 and periodic supplements); John E. Coligan, ed„ (1993) Current Protocols In Immunology New York: John Wiley &amp; Sons.

Another aspect provides for detecting the presence of a test molecule that competes for binding to (i) β-Klotho; (ii) FGFRIc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRIc, FGFR2c, FGFR3c, and FGFR4 with the antigen binding proteins provided, as disclosed herein. An example of one such assay could involve detecting the amount of free antigen binding protein in a solution containing an amount of one or more of (i) β-KIotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (Hi) a complex comprising β-KIotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4 in the presence or absence of the test molecule. An increase in the amount of free antigen binding protein (/.<?., the antigen binding protein not bound to (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-KIotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4) would indicate that the test molecule is capable of competing for binding to (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4: or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4 with the antigen binding protein. In one embodiment, the antigen binding protein is labeled with a labeling group. Alternatively, the test molecule is labeled and the amount of free test molecule is monitored in the presence and absence of an antigen binding protein.

Methods of Treatment: Pharmaceutical Formulations and Routes of Administration

Methods of using the antigen binding proteins arc also provided. In some methods, an antigen binding protein is provided to a patient. The antigen binding protein induces FGF21-like signaling.

Pharmaceutical compositions that comprise a therapeutically effective amount of one or a plurality of the antigen binding proteins and a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative, and/or adjuvant arc also provided. In addition, methods of treating a patient by administering such pharmaceutical composition are included. The term “patient” includes human patients.

Acceptable formulation materials arc nontoxic to recipients at the dosages and concentrations employed. In specific embodiments, pharmaceutical compositions comprising a therapeutically effective amount of human antigen binding proteins that specifically bind (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4 are provided.

In certain embodiments, acceptable formulation materials preferably arc nontoxic to recipients at the dosages and concentrations employed, In certain embodiments, the pharmaceutical composition can contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. In such embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as cthylenediamine tctraacetic acid (EDTA)); complcxing agents (such as caffeine, polyvinylpyrrolidone, bcta-cyclodcxtrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose ordextrins); proteins (such as scrum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as bcnzalkonium chloride, benzoic acid, salicylic acid, thimcrosal, phenethyl alcohol, mcthylparabcn, propylparaben, chlorhcxidinc, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as Pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and/or pharmaceutical adjuvants. See, Remington’s Pharmaceutical Sciences. 18th Edition, (A.R. Gcnnaro, cd.), 1990, Mack Publishing Company.

In certain embodiments, the optimal pharmaceutical composition will be determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format and desired dosage. See, for example, Remington’s Pharmaceutical Sciences, supra. In certain embodiments, such compositions can influence the physical state, stability, rate of in vivo release and rate of in vivo clearance of the antigen binding proteins disclosed. In certain embodiments, the primary vehicle or carrier in a pharmaceutical composition can be either aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier can be water for injection, physiological saline solution or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin arc further exemplary vehicles. Jn specific embodiments, pharmaceutical compositions comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5,5, and can further include sorbitol or a suitable substitute. In certain embodiments, compositions comprising antigen binding proteins that specifically bind (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4 can be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents (Remington's Pharmaceutical Sciences, supra) in the form of a lyophilized cake or an aqueous solution. Further, in certain embodiments, antigen binding protein that bind (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4 can be formulated as a lyophilizatc using appropriate excipients such as sucrose.

The pharmaceutical compositions can be selected for parenteral delivery. Alternatively, the compositions can be selected for inhalation or for delivery through the digestive tract, such as orally. Preparation of such pharmaceutically acceptable compositions is within the skill of the art.

The formulation components are present preferably in concentrations that are acceptable to the site of administration. In certain embodiments, buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8.

When parenteral administration is contemplated, the therapeutic compositions can be provided in the form of a pyrogen-frec, parenterally acceptable aqueous solution comprising the desired antigen binding protein in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water in which the antigen binding protein is formulated as a sterile, isotonic solution, properly preserved. In certain embodiments, the preparation can involve the formulation of the desired molecule with an agent, such as injectable microspheres, bio-erodiblc particles, polymeric compounds (such as polyiactic acid or polyglycolic acid), beads or liposomes, that can provide controlled or sustained release of the product which can be delivered via depot injection. In certain embodiments, hyaluronic acid can also be used, which can have the effect of promoting sustained duration in the circulation. In certain embodiments, implantable drug delivery devices can be used to introduce the desired antigen binding protein.

Certain pharmaceutical compositions are formulated for inhalation. In some embodiments, antigen binding proteins that bind to (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFRSc, and FGFR4 are formulated as a dry, inhalable powder, in specific embodiments, antigen binding protein inhalation solutions can also be formulated with a propellant for aerosol delivery. In certain embodiments, solutions can be nebulized. Pulmonary administration and formulation methods therefore arc further described in International Patent Application No. PCT/US94/001875, which is incorporated by reference and describes pulmonary delivery of chemically modified proteins. Some formulations can be administered orally. Antigen binding proteins that specifically bind (i) β-KIotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4 that arc administered in this fashion can be formulated with or without carriers customarily used in the compounding of solid dosage forms such as tablets and capsules. In certain embodiments, a capsule can be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized. Additional agents can be included to facilitate absorption of an antigen binding protein. Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders can also be employed.

Some pharmaceutical compositions comprise an effective quantity of one or a plurality of human antigen binding proteins that specifically bind (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4 in a mixture with non-toxic excipients that are suitable for the manufacture of tablets. By dissolving the tablets in sterile water, or another appropriate vehicle, solutions can be prepared in unit-dose form. Suitable excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc.

Additional pharmaceutical compositions will be evident to those skilled in the art, including formulations involving antigen binding proteins that specifically bind (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4 in sustained- or controllcd-dciivery formulations.

Techniques for formulating a variety of other sustained- or controlled-delivery means, such as liposome carriers, bio-crodible microparticles or porous beads and depot injections, arc also known to those skilled in the art. See, for example, International Patent Application No. PCT/US93/00829, which is incorporated by reference and describes controlled release of porous polymeric microparticles for delivery of pharmaceutical compositions. Sustained-release preparations can include scmipcrmeablc polymer matrices in the form of shaped articles, e.g., films, or microcapsules. Sustained release matrices can include polyesters, hydrogels, polylactides (as disclosed in U.S. Patent No. 3,773,919 and European Patent Application Publication No. EP 058481, each of which is incorporated by reference), copolymers of L-giutamic acid and gamma ethyl-L-glutamate (Sidman eta/., 1983, Biopolymers 2:547-556), poly (2-hydiOxycthyl-incthacrylatc) (Langcr et a/., 1981, ,/. Biomed. Mater. Res. J5 :.167-277 and Langcr, 1982, Chem. Tech. j_2:98-105), ethylene vinyl acetate (Langcr et ai, 1981, supra) or poiy-D(-)-3-hydroxybutyric acid (European Patent Application Publication No. EP 133,988). Sustained release compositions can also include liposomes that can be prepared by any of several methods known in the art. See, e.g., Eppstein et al., 1985, Proc. Natl. Acad. Sci. U.S.A. 82:3688-3692; European Patent Application Publication Nos. EP 036,676; EP 088,046 and EP 143,949, incorporated by reference.

Pharmaceutical compositions used for in vivo administration are typically provided as sterile preparations. Sterilization can be accomplished by filtration through sterile filtration membranes. When the composition is lyophiiized, sterilization using this method can be conducted either prior to or following lyophilization and reconstitution. Compositions for parenteral administration can be stored in lyophiiized form or in a solution. Parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceablc by a hypodermic injection needle.

In certain embodiments, cells expressing a recombinant antigen binding protein as disclosed herein is encapsulated for delivery (see, Invest. Ophthalmol Vis Sci (2002) 43:3292-3298 and Proc. Natl. Acad. Sciences USA (2006) 103:3896-3901).

In certain formulations, an antigen binding protein has a concentration of at least 10 mg/ml, 20 mg/ml, 30 mg/ml, 40 mg/ml, 50 mg/ml, 60 mg/ml, 70 mg/ml, 80 mg/ml, 90 mg/ml, 100 mg/ ml or 150 mg/ml. Some formulations contain a buffer, sucrose and polysorbate. An example of a formulation is one containing 50-100 mg/ml of antigen binding protein, 5-20 mM sodium acetate, 5-10% w/v sucrose, and 0.002 - 0.008% w/v polysorbate. Certain, formulations, for instance, contain 65-75 mg/ml of an antigen binding protein in 9-11 m.M sodium acetate buffer. 8-10% w/v sucrose, and 0.005-0.006% w/v polysorbate. The pH of certain such formulations is in the range of 4.5-6. Other formulations have a pH of 5.0-5,5 (<?.g., pH of 5.0, 5.2 01-5.4).

Once the pharmaceutical composition lias been formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, crystal, or as a dehydrated or lyophilized powder. Such formulations can be stored either in a ready-to-use form or in a form (e.g., lyophilized) that is reconstituted prior to administration. Kits for producing a single-dose administration unit are also provided. Certain kits contain a first container having a dried protein and a second container having an aqueous formulation. In certain embodiments, kits containing single and multi-chambered prc-fillcd syringes (e.g., liquid syringes and lyosyringcs) arc provided. The therapeutically effective amount of an antigen binding protein-containing pharmaceutical composition to be employed will depend, for example, upon the therapeutic context and objectives. One skilled in the art will appreciate that the appropriate dosage levels for treatment will vary depending, in part, upon the molecule delivered, the indication for which the antigen binding protein is being used, the route of administration, and the size (body weight, body surface or organ size) and/or condition (the age and general health) of the patient. In certain embodiments, the clinician can titer the dosage and modify the route of administration to obtain the optimal therapeutic effect. A typical dosage can range from about 1 pg/kg to up to about 30 mg/kg or more, depending on the factors mentioned above. In specific embodiments, the dosage can range from 10 pg/kg up to about 30 mg/kg, optionally from 0.1 mg/kg up to about 30 mg/kg, alternatively from 0.3 mg/kg up to about 20 mg/kg. In some applications, the dosage is from 0.5 mg/kg to 20 mg/kg. In some instances, an antigen binding protein is dosed at 0.3 mg/kg, 0.5mg/kg, 1 mg/kg, 3 mg/kg, 10 mg/kg, or 20 mg/kg. The dosage schedule in some treatment regimes is at a dose of 0.3 mg/kg qVV, 0.5mg/kg qW, 1 mg/kg qW, 3 mg/kg qW, 10 mg/kg qW, or 20 mg/kg qW.

Dosing frequency will depend upon the pharmacokinetic parameters of the particular antigen binding protein in the formulation used. Typically, a clinician administers the composition until a dosage is reached that achieves the desired effect. The composition can therefore be administered as a single dose, or as two or more doses (which can but need not contain the same amount of the desired moiecuie) over time, or as a continuous infusion via an implantation device or catheter. Appropriate dosages can be ascertained through use of appropriate dose-response data. In certain embodiments, the antigen binding proteins can be administered to patients throughout an extended time period. Chronic administration of an antigen binding protein minimizes the adverse immune or allergic response commonly associated with antigen binding proteins that are not fully human, for example an antibody raised against a human antigen in a non-human animal, for example, a non-fully human antibody or non-human antibody produced in a non-human species.

The route of administration of the pharmaceutical composition is in accord with known methods, e.g., orally, through injection by intravenous, intrapcritoncai, intracerebral (intra-parcnchymal), intraccrcbrovcntricular, intramuscular, intra-ocular, intraarterial, intraportal, or intralcsional routes; by sustained release systems or by implantation devices. In certain embodiments, the compositions can be administered by bolus injection or continuously by infusion, or by implantation device.

The composition also can be administered locally via implantation of a membrane, sponge or another appropriate material onto which the desired molecule has been absorbed or encapsulated, in certain embodiments, where an implantation device is used, the device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be via diffusion, timed-release bolus, or continuous administration.

It also can be desirable to use antigen binding protein pharmaceutical compositions ex vivo. In such instances, cells, tissues or organs that have been removed from the patient are exposed to antigen binding protein pharmaceutical compositions after which the ceils, tissues and/or organs arc subsequently implanted back into the patient. in particular, antigen binding proteins that specifically bind (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4 can be delivered by implanting certain cells that have been genetically engineered, using methods such as those described herein, to express and secrete the polypeptide. In certain embodiments, such cells can be animal or human cells, and can be autologous, heterologous, or xenogeneic. In certain embodiments, the cells can be immortalized. In other embodiments, in order to decrease the chance of an immunological response, the cells can be encapsulated to avoid infiltration of surrounding tissues. In further embodiments, the encapsulation materials arc typically biocompatible, semi-permeable polymeric enclosures or membranes that allow the release of the protein product(s) but prevent the destruction of the cells by the patient’s immune system or by other detrimental factors from the surrounding tissues.

Combination Theranics

In another aspect, the present disclosure provides a method of treating a subject for diabetes with a therapeutic antigen binding protein of the present disclosure, such as the fully human therapeutic antibodies described herein, together with one or more other treatments. In one embodiment, such a combination therapy achieves an additive or synergistic effect. The antigen binding proteins can be administered in combination with one or more of the type 2 diabetes or obesity treatments currently available. These treatments for diabetes include biguanide (metaformin), and sulfonylureas (such as glyburide, glipizide). Additional treatments directed at maintaining glucose homeostasis include PPAR gamma agonists (pioglitazonc, rosiglitazonc); glinidcs (mcglitinidc, rcpaglinidc, and natcglinidc); DPP-4 inhibitors (JanuviaOi) and Onglyza®) and alpha glucosidase inhibitors (acarbose, voglibose).

Additional combination treatments for diabetes include injectable treatments such as insulin and incrctin mimctics (Byctta©, Excnatidc®), other GLP-1 (glucagon-like peptide) analogs such as liraglutide, other GLP-1R agonists and Symlin® (pramlintide).

Additional combination treatments directed at weight loss include Mcridia® and XenicaKa).

EXAMPLES

The following examples, including the experiments conducted and the results achieved, are provided for illustrative purposes only and are not to be construed as limiting. EXAMPLE 1

PREPARATION OF FGFRlc OVER EXPRESSING CELLS FOR USE AS AN

ANTIGEN

Nucleic acid sequences encoding the full length human FGFRlc polypepetide (SEQ ID NO:4; Figures 1A-1B) and a separate sequence encoding the full length human β-Klotho polypeptide (SEQ ID NO:7; Figures 2A-2C) were subcloned into suitable mammalian cell expression vectors (e.g., pcDNA3.1 Zeo, pcDNA3.1 Hyg (Jnvitrogen, Carlsbad, CA) or pDSRa20. The pDSRa20 vector contains SV40 early promoter/enliancer for expressing the gene of interest and a mouse DHFR expression cassette for selection in CHO DHFR (-) host cells such as AM 1 CHO (a derivative of DG44, CHO DHFR (-))- AM-1 CHO cells were seeded at 1.5 xlO6 cells per 100mm dish. After 24 hours, the cells were co-transfected with linearized DNAs of pDSRoc20/huFGFRlc and pDSRa20/huP-Klotho with FuGenefi (Roche Applied Science). The transfected cells were trypsinized 2 days after transfection and seeded into CHO DHFR selective growth medium containing 10% dialyzed FBS and without hypoxanthinc/thymidine supplement. After 2 weeks, the resulting transfected colonies were trypsinized and pooled. HEK293T cells were transfected with the full length huFGFRic and hup-Klotho in pcDNA3.1 series or pTT14 (an expression vector developed by Durocher, NRCC, with CMV promoter and EBV ori, similar to pTT5 and a puromycin selection marker) based vector and selected with the corresponding drugs following similar procedure as for the CHO transfection and selection.

The FGF21R (/.<?., FGFRlc and βΚ-lotho) transfected AMI CHO or 293T cell pools were sorted repeatedly using Alcxa 647-labelcd FGF21. As a cell-surface staining reagent, FGF21 was labeled with Alexa 647-NHS followed the method recommended by the manufacturer (Molecular Probes, Inc. Cat A 2006). The Alexa 647-labelcd FGF21 showed specific staining of FGF21R receptor expressing cells and not the non-transfccted parental cells (Figure 3). High expressing cells were collected at the end of the final sorting, expanded and frozen into vials. The AM-l/huFGF2!R cells were prepared for immunization and the 293T/huFGF21R cells were used for titering mouse sera by FACS after immunization and in binding screens of the hybridoma supernatants by FMAT (sec Example 4). EXAMPLE 2

PREPARATION OF A SOLUBLE FGFRie/B-KLOTHO COMPLEX

FOR USE AS ANTIGEN

Soluble FGF21 receptor constructs were generated in pTT14 or pcDNA3.1 expression vectors. The FGFRlc ECD-Fc construct (SEQ ID NO:362, Figure 4) comprises the N-terminal extracelluar domain of FGFRlc (amino acid residues #1 - 374; SEQ ID NO:5) fused to Fc (SEQ ID NO:384). The β-Klotho ECD-Fc construct (SEQ ID NO:363, Figure 5) comprises the N-termina! extracellular domain of β-Klotho (amino acid residues #1-996; SEQ ID NO:8) fused to Fc (SEQ ID 190:384). HEK293 ceils (293F, Invitrogen) were transfected with huFGFRlc ECD-Fc/pTT5, hup-Klotho ECD-Fc/pTT14-puro and dGFP/pcDNA3.1-Nco and selected in the presence of the corresponding drugs followed by repeated FACS sorting based on dGFP expression. Cells were grown in scrum-free Dulbccco's Modified Eagle Medium (DMEM) supplemented with noncsscntial amino acids in HypcrFiasks (Corning) for 4 days and conditioned media (CM) harvested for purification.

The 293 CM was concentrated 6 fold and applied to Protein A FF equilibrated in PBS. The protein was eluted with Pierce Gentle Ag/Ab elution buffer. The Protein A pool was dialyzed against 20mM Tris-HCl, pH 7. IOmM NaCI and applied to SP HP at pH 7.0. The FGFRlc ECD-Fc was present in the flow-through (FT) and the heterodimer was eluted with linear gradient of 0- 0.4 M NaCI, 20 mM Tris-HCI pH 7.0. N-terminus amino acid sequencing verified the purified soluble FGF21R to be a heterodimer composed of (1:1) ratio of FGFRlc ECD-Fc and β-Klotho ECD-Fc. The purified soluble FGF21R-Fc (Figure 6) was used as the antigen for immunization. EXAMPLE 3

PREPARATION OF MONOCLONAL ANTIBODIES

Immunizations were conducted using one or more suitable forms of FGF21 receptor antigen, including: (1) cell bound receptor of CHO transfcctants expressing full length human FGFRlc and β-Klotho at the cell surface, obtained by transfecting CHO cells with cDNA encoding a human full length FGFRlc polypeptide of SEQ ID NO:4 (see also Figuresla-b) and cDNA encoding a human β-KIotho polypeptide of SEQ ID NO:7 (sec also Figures 2a-c); (2) membrane extract from the aforementioned cells expressing the FGF21R receptor complex; or (3) soluble FGF21R receptor obtainable by co-exprcssing the N-tcrminal extracellular domain (ECD) of FGFRlc (SEQ ID NO:5; see also Figure 4) and the N-terminal extracellular domain (ECD) of β-Klotho (SEQ ID NO:8; see also Figure 5) or (4) combinations thereof. A suitable amount of immunogen (/.<?., 10 pgs/mouse of soluble FGF21R or 3 - 4 x 106 celis/mouse of stably transfected CHO cells or 150 pgs/mouse of purified FGF21R membranes prepared from CHO cells stably expressing FGF21R) was used for initial immunization in XenoMouse™ according to the methods disclosed in International Patent Application Nos. WO 98/24893, and WO 00/76310, the disclosures of which are hereby incorporated by reference. Following the initial immunization, subsequent boost immunizations of immunogen (5 pg/mouse of soluble FGF21R or 1.7 x 106 FGF21R transfected cells/mouse or 75 pgs of purified FGF21R membranes) were administered on a schedule and for the duration necessary to induce a suitable anti-FGF21R titer in the mice. Titers were determined by a suitable method, for example, by enzyme immunoassay, fluorescence activated cell sorting (FACS), or by other methods (including combinations of enzyme immunoassays and FACS).

Animals exhibiting suitable titers were identified, and lymphocytes were obtained from draining lymph nodes and, if necessary, pooled for each cohort. Lymphocytes were dissociated from lymphoid tissue by grinding in a suitable medium (for example, Dulbecco’s Modified Eagle Medium; DMEM; obtainable from Invitrogen, Carlsbad, CA) to release the cells from the tissues, and suspended in DMEM. B cells were selected and/or expanded using standard methods, and fused with suitable fusion partner, for example, nonsecretory myeloma P3X63Ag8.653 cells (American Type Culture Collection CRL 1580; Kearney et al, J. Immunol. 123, 1979, 1548-1550), using techniques that were known in the art.

In one suitable fusion method, lymphocytes were mixed with fusion partner cells at a ratio of 1:4. The cell mixture was gently pelleted by centrifugation at 400 x g for 4 minutes, the supernatant decanted, and the cell mixture gently mixed (for example, by using a 1 ml pipette). Fusion was induced with PEG/DMSO (polyethylene glycol/dimethyl sulfoxide; obtained from Sigma-Aldrich, St. Louis MO; 1 ml per million of lymphocytes). PEG/DMSO was slowly added with gentle agitation over one minute followed, by one minute of mixing. IDMEM (DMEM without glutamine; 2 ml per million of B cells), was then added over 2 minutes with gentle agitation, followed by additional IDMEM (8 ml per million B-cells) which was added over 3 minutes.

The fused cells were pelleted (400 x g 6 minutes) and resuspended in 20 ml Selection media (for example, DMEM containing Azaserine and Hypoxanthine [HA] and other supplemental materials as necessary) per million B-cells. Cells were incubated for 20-30 _ minutes at 37°C and then resuspended in 200 m! selection media and cultured for three to four days in T175 flasks prior to 96 well plating.

Cells were distributed into 96-wcil plates using standard techniques to maximize clonality of the resulting colonies. After several days of culture, supernatants were collected and subjected to screening assays as detailed in the examples below, including confirmation of binding to human FGF21 receptor, specificity and/or cross-species reactivity. Positive cells were further selected and subjected to standard cloning and subcloning techniques. Clonal lines were expanded in vitro, and the secreted human antibodies obtained for analysis.

In this manner, mice were immunized with either cells or membranes expressing full length FGF21R cells, or soluble FGF21R extracellular domain, with a range of 11-17 immunizations over a period of approximately one to three and one-half months. Several cell lines secreting FGF21 R-spccific antibodies were obtained, and the antibodies were further characterized. The sequences thereof arc presented herein and in the Sequence Listing, and results of various tests using these antibodies arc provided. EXAMPLE 4

SELECTION OF BINDING ANTIBODIES BY FMAT

After 14 days of culture, hybridoma supernatants were screened for FGF21R-specific monoclonal antibodies by Fluoromctric Microvolumc Assay Technology (FMAT) by screening against cither the CHO AMl/huFGF21R cell line or recombinant HEK293 cells that were transfected with human FGF21R and counter-screening against parental CHO or HEK293 cells. Briefly the cells in Freestyle media (Invitrogcn) were seeded into 384-well FMAT plates in a volume of 50 gL/well at a density of 4,000 cells/wcll for the stable transfectants, and at a density of 16,000 cells/well for the parental cells, and cells were incubated overnight at 37°C. 10 uL/well of supernatant was then added, and the plates were incubated for approximately one hour at 4°C’, after which 10 μ L/wel 1 of anti-human lgG-Cy5 secondary antibody was added at a concentration of 2.8 ug/mi (400ng/ml final concentration). Plates were then incubated for one hour at 4°C, and fluorescence was read using an FMAT Cellular Detection System (Applied Biosystems).

In total, over 3,000 hybridoma supernatants were identified as binding to the FGF21 receptor expressing ceils but not to parental ceils by the FMAT method. These supernatants were then tested in the FGF21 functional assays as described below. EXAMPLE 5

SELECTION OF ANTIBODIES THAT INDUCE FGF21-LIKE SIGNALING

Experiments were performed to identify functional antibodies that mimic wild-type FGF21 activity (e.g., the ability to induce FGF21 -like signaling) using a suitable FGF21 reporter assay. The disclosed FGF21 reporter assay measures activation of FGFR signaling via a MARK pathway readout. β-Klotho is a co-receptor for FGF2I signaling, and although it is believed not to have any inherent signaling capability due to its very short cytoplasmic domain, it is required for FGF21 to induce signaling through FGFRs.

Example 5.1 ELK-Luciferase Reporter Assay ELK-lucifcrase assays were performed using a recombinant human 293T kidney cell or CHO ceil system. Specifically, the host cells were engineered to over-express β-KIotho and lucifcrase reporter constructs. The reporter constructs contain sequences encoding GAL4-ELK1 and 5xUAS-Luc, a lucifcrase reporter driven by a promoter containing five tandem copies of the GaI4 binding site. Activation of the FGF21 receptor complex in these recombinant reporter cell lines induces intracellular signal transduction, which in turn leads to ERK and ELK phosphorylation. Lucifcrase activity is regulated by the level of phosphorylatcd ELK, and is used to indirectly monitor and quantify FGF21 activity.

In one example, CHO cells were transfected sequentially using the Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer’s protocol with the receptor constructs expressing β-Klotho, FGFRlc and the reporter plasmids: 5x Gal4-Lucifcrasc (minimal TK promoter with 5xGal4 binding sites upstream of lucifcrase) and Gal4-ELK1. Gal4-ELKI binds to the Gal4 binding sites and activates transcription when it is phosphorylatcd by ERK. Lucifcrase transcription, and thereby the corresponding enzymatic activity in this context is regulated by the level of phosphorylatcd ELK1, and is used to indirectly monitor and quantify FGF2 I activity.

Clone 2E10 was selected as the FGF2] luciferase reporter cell line based on the optimal assay window of 10-20 fold with native FGF21 exhibiting an EC50 in the single nM range.

For the assay, the ELK-luciferase reporter ceils were plated in 96 well assay plates, and scrum starved overnight. FGF21 or test samples were added for 6 hours at 37 degrees.

The plates were then allowed to cool to room temperature and the luciferase activity in the cell lysates was measured with Bright-Glo (Promcga).

Example 5.2 ERK-Pliospliorvlation Assay

Alternative host cell lines specifically L6 (a rat myoblastic cell line) was developed and applied to identify antibodies with FGF21-like signaling activity. The rat L6 cell line is a desirable host cell line for the activity assay because it is known to express minimal levels of cndogcncous FGF receptors. The L6 cells do not respond to FGF21 even when transfected with β-Klotho expression vector and therefore provides a cleaner background. (Kurosu et ah, (2007) ./. Biol. Cheat. 282, 26687-26695). L6 cells were maintained in Dulbccco’s modified Eagle’s medium supplemented w'ith 10% fetal bovine serum and penicillin/streptomycin. Cells were transfected with plasmids expressing pKlotho and individual FGFR using the Lipofcctaminc 2000 transfection reagent (Invitrogcn) according to the manufacturer’s protocol.

Analysis of FGF signaling in L6 cells was performed as described in the literature (Kurosu et al., (2007),/. Biol. Chem. 282. 26687-26695). Cel! cultures were collected 10 min after the treatment of FGF21 or test molecules and snap frozen in liquid nitrogen, homogenized in the lysis buffer and subjected to western blot analysis using an anti-phospho-p44/42 MAP kinase (ERK1/2) antibody and an anti-ERK antibody (Cell Signaling). The percent of phosphorylated ERK versus total ERK protein was determined in this way.

In addition, the factor-dependent mouse BaF3 cell-based proliferation assay used frequently for cytokine receptors can also be developed and applied.

Among the hybridoma supernatants tested in the CHO cell (clone 2E10) based human FGF21 ELK-luciferase reporter assay, over 30 were identified as positive (> 5% of the activity of FGF21) when compared to 20nM FGF21 as the positive control. Antibodies were then purified from the conditioned media of the hybridoma cultures of these positives and tested again in the CHO cell based ELK-Iucifcrase reporter assay. {Figure 7) showed the representative antibodies in the dose-responsive potency assay with estimated EC50 less than lpg/ml (or 6.7nM). The activities were confirmed in the L6 cell based ERKl/2-phosphrylation assay (Figure 8) with EC50 less than 10 nM which is consistent to the ELK-luciferase assay in the CHO stable cell line 2EI0. EXAMPLE 6

INDUCTION OF FGF21-L1KE SIGNALING IS SPECIFIC TO THE FGFRIc/BKLOTHO COMPLEX FGF21 has been reported to signal through multiple receptor complexes including FGFRIc, 2c, 3c and 4 when paired with β-KJotho. The selectivity of the FGF2I agonistic antibodies was tested in the rat myoblastic L6 cells transfected with vectors expressing the respective FGFRs and βΚΙοίΙιο. The results shown in Figure 9 demonstrate that the activity was mediated selectively and exclusively through FGFRIc and not through FGFR2c. 3c or 4 when they were paired with β-KIofho because no activity was detected on the latter receptors up to 100 nM of the agonistic antibodies. This unique selectivity strongly suggests that the action of these antibodies is β-Klotho-dcpcndcnt yet it must also involve specifically the FGFRIc component of the signaling complex. EXAMPLE 7

ACTIVITY IN PRIMARY HUMAN ADIPOCYTES FGF21 stimulates glucose uptake and lipolysis in cultured adipocytes, and adipocytes are considered to be more physiologically relevant than the recombinant reporter cell system. A panel of the antibodies was shown to exhibit Erk-phosphorylation activity similar to FGF2I in the human adipocyte assay (Figure 10) with estimated EC50 less than 10 nM. EXAMPLE 8

COMPETITION BINDING AND EPITOPE BINNING

To compare the similarity of the binding sites of the antibodies on the FGF21 receptor, a series of competition binding experiments were performed and measured by Biacore. In one example (and as shown in Figure 11), two representative agonistic FGF2] receptor antibodies (24H11 and 17D8) and one non-functional FGF23 receptor binding antibodies (1A2.1) were immobilized on the sensor chip surface. Soluble human FGFRlc/p-Klotho ECD-Fc complex or β-Klotho was then captured on the immobilized antibody surfaces. Finally, several of the test FGF21 receptor antibodies were injected individually over the captured soluble human FGF21 receptor or β-Kfotho. If the injected antibody recognizes a distinct binding site relative to that recognized by the immobilized antibody, a second binding event will be observed. If the antibodies recognize very similar binding site, no more binding will be observed.

As shown in (Figure 11 A), there arc two distinct yet partially overlapping binding sites for the agonistic antibodies tested. One site is covered by 24H13, 21H2, 18B11.1 and 17C3 (Group A) and the other site covered by 17D8, 12E4 and 18G1 (Group B). The two nonfunctional antibodies 2G10 and 1A2, bind to different sites from each other and are distinct from the two sites covered by the agonistic antibodies in Group A and B. Other functional antibodies binding to Group A epitope included 20D4,22H5, 16H7,40D2 and 46D31. Two other functional antibodies 26H11 and 37D3 were shown by this method to bind the same site covered by the Group B antibodies. In addition, a third binding site for functional antibodies was identified for 39F11, 39F7 and 39G5 (group C) which appeared to be distinct from Group A and B binding sites (Figure 1 IB).

Another Biacore analysis was carried out with biotinylated-FGF21 immobilized on the sensor ship. 10 nM soluble β-Klotho was then passed over the chip alone or mixed with the individual test antibodies at ΙΟΟηΜ. (Figure 12) showed that several agonistic antibodies in group A (24H11, 18B11, 17C3) and antibody 12E4 (from group B) competed significantly with FGF2.1 in binding to soluble β-Klotho whereas the non-functional antibodies 2GI0 and 1A2 and several other functional antibodies did not show competition binding with FGF2I.

Figure 11C summarizes the binning results obtained. EXAMPLE 9

RECOGNITION OF NATIVE AND DENATURES STRUCTURES

The ability of disclosed antigen binding proteins to recognize denatured and native structures was investigated. The procedure and results were as follows.

Example 9.1 FGF21 Receptor Agonistic Antibodies do not Recognize Denatured Structures, as Shown

bv FACS

Cell lysates from CHO cells stably expressing FGF21 receptor (FGFRlc and β-Klotho) or CHO parental cells were diluted with sample buffer without beta-mercaptoethanoi (nonreducing conditions). 20μ1 of cell lysate was loaded per lane on adjacent lanes separated with a molecular weight marker lane on 4-20% SDS-PAGE gels. Following electrophoresis, the gels were blotted onto 0.2μ nitrocellulose filters. The blots were treated with Tris-buffered saline/Triton-X (TBST) plus 5% non-fat milk (blocking buffer) for 30 minutes. The blots were then cut along the molecular weight marker lanes. The strips were then probed with FGF21 receptor agonistic antibodies (12C3, 26H11, 12E4, 21H2, 18B11, or 20D4), and commercial goat anti-murine βΚΙοίΙιο or mouse anti-huFGFRl (R&amp;D Diagnostics) in TBST/5% milk. Blots were incubated with the antibodies for one hour at room temperature, followed by three washes with TBST + 1% milk. The blots were then probed with anti-human or anti-goat lgG-HRP secondary antibodies for 20 min. Blots were given three 15 min. washes with TBST followed by treatment with Pierce Supersignal West Dura developing reagent (1 min.) and exposure to Kodak Biomax X-ray film.

The commercial anti-p-Klotho and anti-FGFRI antibodies detected the corresponding receptor proteins in the SDS-PAGE indicating they bind to denatured receptor proteins. In contrast, none of the FGF21 receptor agonistic antibodies tested detected the corresponding protein species suggesting they bind to the native conformational epitope distinct from the commercial antibodies which bind to denatured sequences.

Example 9.2 FGF21 Receptor Agonistic Antibodies Bind To Native Receptor Structure,

As Shown Bv FACS A FACS binding assay was performed with several commercially available FGFRlc and β-Klotho antibodies, and several of the disclosed FGF21 receptor agonistic antibodies. The experiments were performed as follows. CHO cells stably expressing FGF21 receptor were treated with R&amp;D Systems mouse anti-huFGFRl, goat anti-mu β-Klotho, or FGF21 receptor antibodies 24H11, 17C3, 17D8, 18G1, or 2G10 (1μ§ per lxl0f’ cells in ΙΟΟμΙ PBS/0.5% BSA). Cells were incubated with the antibodies at 4°C followed by two washes with PBS/BSA. Cells were then treated with F1TC-labeled secondary antibodies at 4°C followed by two washes. The cells were resuspended in 1ml PBS/BSA and antibody binding was analyzed using a FACS Calibur instrument.

Consistent with western blot results, all of the FGF21 receptor agonistic antibodies tested bind well to cell surface FGF21 receptor in FACS whereas the commercial anti-p-Klotho or anti-FGFRl antibodies did not. This observation further confirmed that the FGF21 receptor agonistic antibodies recognize the native structure whereas the commercial antibodies to the receptor components do not.

EXAMPLE 10 ARGININE SCANNING

As described above, antigen binding proteins that bind human FGF21R, e.g., FGFRlc, β-Klotho or both FGFRlc and β-Klotho, were created and characterized. To determine the neutralizing determinants on human FGFRlc and/or β-Klotho that these various antigen binding proteins bound, a number of mutant FGFRlc and/or β-Klotho proteins can be constructed having arginine substitutions at select amino acid residues of human FGFRlc and/or β-Klotho. Arginine scanning is an art-recognized method of evaluating where antibodies, or other proteins, bind to another protein, sec, e.g., Nanevicz et al., (1995) J. Biol. Chem., 270:37. 2161.9-21625 and Zupnick et al., (2006) ./. Biot. Chem., 281:29, 20464-20473. In general, the arginine sidechain is positively charged and relatively bulky as compared to other amino acids, which can disrupt antibody binding to a region of the antigen where the mutation is introduced. Arginine scanning is a method that determines if a residue is part of a neutralizing determinant and/or an epitope.

Various amino acids distributed throughout the human FGFRlc and/or β-Klotho extracellular domains can be selected for mutation to arginine. The selection can be biased towards charged or polar amino acids to maximize the possibility of the residue being on the surface and reduce the likelihood of the mutation resulting in misfolded protein. Using standard techniques known in the art, sense and anti-sense oligonucleotides containing the mutated residues can be designed based on criteria provided by Stratagene Quickchangc* II protocol kit (Stratagene/Agilcnt, Santa Clara, CA). Mutagenesis of the wild-type (WT) FGFRlc and/or β-

Klotho sequences can be performed using a Quickchange* II kit (Stratagene). Chimeric constructs can be engineered to encode a FLAG-histidine tag (six histidines (SEQ ID NO: 382)) on the carboxy terminus of the extracellular domain to facilitate purification via the poly-His tag.

Multiplex analysis using the Bio-Plex Workstation and software (BioRad, Hercules, CA) can be performed to determine neutralizing determinants on human FGFRlc and/or β-Klotho by analyzing exemplary human FGFRlc and/or β-Klotho mAbs differential binding to arginine mutants versus wild-type FGFRlc and/or β-Klotho proteins. Any number of bead codes of pcntaHis-coatcd beads (“penta-His” disclosed as SEQ ID NO: 383) (Qiagen, Valencia, CA; see wwwl.qiagen.com) can be used to capture histidine-tagged protein. The bead codes can allow the multiplexing of FGFRlc and/or β-Klotho arginine mutants and wild-type human FGFRlc and/or β-Klotho.

To prepare the beads, lOOul of wild-type FGFRlc and/or β-Klotho and FGFRlc and/or β-Klotho arginine mutant supernatants from transient expression culture arc bound to penta-His-coatcd beads ("penta-His” disclosed as SEQ ID NO: 383) overnight at 4°C or 2 hours at room temperature with vigorous shaking. The beads are then washed as per the manufacturer’s protocol and the bead set pooled and aliquoted into 2 or 3 columns of a 96-weil filter plate (Miiliporc, BcIIcrica, MA, product #MSBVNI250) for duplicate or triplicate assay points, respectively. ΙΟΟμΙ anti-FGFRlc and/or anti-p-Klotho antibodies in 4-fold dilutions arc added to the wells, incubated for 1 hour at room temperature, and washed. ΙΟΟμΙ of a 1:100 dilution of PE-conjugatcd anti-human lgG Fc (Jackson Labs., Bar Harbor, ME, product #109-116-170) is added to each well, incubated for 1 hour at room temperature and washed. Beads arc resuspended in 1% BSA, shaken for 3 minutes, and read on the Bio-Plex workstation. Antibody binding to FGFRlc and/or β-Klotho arginine mutant protein is compared to antibody binding to the human FGFRlc and/or β-Klotho wild-type from the same pool. A titration of antibody over approximately a 5 log scale can be performed. Median Fluorescence Intensity (MF1) of FGFRlc and/or β-Klotho arginine mutant proteins can be graphed as a percent of maximum wild-type human FGFRlc and/or β-Klotho signal. Those mutants for which signal from all the antibodies are below a cut-off value, e.g., 30% of wild-type FGFRlc and/or β-Klotho can be deemed to be cither of too low a protein concentration on the bead due to poor expression in the transient culture or possibly misfoldcd and can be excluded from analysis. Mutations (/.<;., arginine substitutions) that increase the EC50 for the FGFRIc and/or β-Klotho mAb by a cut-off value, e.g., 3-fold or greater (as calculated by, e.g., GraphPad Prism*) can be considered to have negatively affected FGFRIc and/or β-KJotho mAb binding. Through these methods, neutralizing determinants and epitopes for various FGFRIc β-Klotho antibodies are elucidated. EXAMPLE 11

CONSTRUCTION OF CHIMERIC RECEPTORS

In another method of determining the activation determinants on human FGFRIc and/or β-Klotho that these various antigen binding proteins bind, specific chimeric FGFRIc and/or β-Klotho proteins between human and mouse species can be constructed, expressed in transient or stable 293 or CHO cells as described before and tested. For example, a chimeric FGF21 receptor can be constructed comprising native human FGFRIc, FGFR2C, FGFR3c or FGFR4, in one example FGFRIc, paired with chimeric human/mouse β-Klotho in which selected regions or sequences on the human β-Klotho are systematically replaced by the corresponding mouse-specific residues (see, e.g., Figure 2A-2C). Similarly, native human β-Klotho paired with chimeric human/mouse FGFRIc, FGFR2c, FGFR3c or FGFR4, in one example FGFRIc in which selected regions or sequences on the human FGFRIc arc systematically replaced by the corresponding mouse-specific residues (sec, e.g., the alignments of Figures! A-IB). The critical sequences involved in the binding and/or activity of the antigen binding proteins can be derived through binding assay or activity measurements described in previous Examples 4, 5, 6 and 7 based on the chimeric FGF21 receptors.

Example 11.1 Construction of Specific Chimeras Human-mouse β-Klotho chimeras were constructed using the methodology described in Example 14. A schematic of the chimeras constructed is presented in Figure 29; summarily, the chimeras generated comprised (from N to C terminus) a fusion of a human β-Klotho sequence fused to a murine β-Klotho sequence fused to a human β-Klotho sequence. Human β-Klotho (SEQ ID NO:8) was used as a framework into which regions of murine β-Klotho (full length sequence shown in SEQ ID NO:468) were inserted. The regions of murine β-KIotho that were inserted were as follows:

Murine Residues 82P-520P

PKNFSWGVGTGAFQVEGSWK.TDGRGPSIWDRYVYSHLRGVNGTDRSTDSYIFLEKDLL

ALDFLGVSFYQFSISWPRLFPNGTVAAVNAQGLRYYRALLDSLVLRNIEPIVTLYHWDLP

LTLQEEYGGWKNATMIDLFNDYATYCFQTFGDRVKYW1TIHNPYLVAWHGFGTGMHA

PGEKGNLTAVYTVGHNLIKAHSKVWHNYDKNFRPHQKGWLSITLGSHWIEPNRTDNM

EDVINCQHSMSSVLGWFANPIHGDGDYPEFMKTGAMIPEFSEAEfCEEVRGTADFFAFSF

GPNNFRPSNTVVKMGQNVSLNLRQVLNWIKLEYDDPQILISENGWFTDSYIKTEDTTAIY

MMKNFLNQVLQA3KFDE1RVFGYTAWTLLDGFEWQDAYTTRRGLFYVDFNSEQKERKP KSSAHYYKQIIQDNGFPLKESTPDMKGRFP (SEQ ID NO:470)

Murine Residues 506F-I043S

FPLKESTPDMKGRFPCDFSWGVTESVLKPEFTVSSPQFTDPHLYVWNVTGNRLLYRVEG

VRLKTRPSQCTDYVS1KKRVEMLAKMKVTHYQFALDWTS1LPTGNLSKVNRQVLRYYR

CVVSEGLKLGVFPMVTLYHPTHSHLGLPLPLLSSGGWLNMNTAKAFQDYAELCFRELG

DLVKLWITI1MEPNRLSDMYNRTSNDTYRAAHNLMIAHAQVWHLYDRQYRPVQHGAVS

LSLHCDWAEPANPFVDSHWKAAERFLQFE1AWFADPLFKTGDYPSVMKEY1ASKNQRG

LSSSVLPRFTAKESRLVKGTVDFYALNHFTTRFVTHKQLNTNRSVADRDVQFLQD1TRLS

SPSRLAVTPWGVRKLLAWIRRNYRDRDIYITANGIDDLALEDDQIRKYYLEKYVQEALK

AYLIDKVK1KGYYAFK.LTEEKSKPRFGFFTSDFRAKSSVQFYSK.L1SSSGLPAENRSPACG

QPAEDTDCTICSFLVEKKPUFFGCCFISTLAVLLSITVFHHQKRRKFQKARNLQNIPLKK GHSRVFS (SEQ ID NO:471)

Murine Residues 1M-393L MKTGCAAGSPGNEWIFFSSDERNTRSRKTMSNRALQRSAVLSAFVLLRAVTGFSGDGK AIWDKKQYVSPVNPSQLFLYDTFPKNFSWGVGTGAFQVEGSWKTDGRGPSIWDRYVYS HLRGVNGTDRSTDSYIFLEKDLLALDFLGVSFYQFSISWPRLFPNGTVAAVNAQGLRYY RALLDSLVLRNIEPIVTL (SEQ ID NO:472)

Murine Residues 82P-302S PKNFSWGVGTGAFQVEGSWKTDGRGPSIWDRYVYSHLRGVNGTDRSTDSYIFLEKDLL ALDFLGVSFYQFSISWPRLFPNGTVAAVNAQGLRYYRALLDSLVLRNIEPIVTLYHWDLP LTLQEEYGGWKNATMIDLFNDYATYCFQTFGDRVKYWITIHNPYLVAWHGFGTGMHA PGEKGNLTAVYTVGHNLIKAHSKVWUNYDKNFRPHQKGWLSITLGS (SEQ ID NO:473)

Murine Residues 194Y-416G

YHWDLPLTLQEEYGGWKNATMIDLFNDYATYCFQTFGDRVKYW1TIHNPYLVAWHGF

GTGMHAPGEKGNLTAVYTVGHNLIKAHSKVWHNYDKNFRPHQKGWLSITLGSHWIEP

NRTDNMEDVINCQHSMSSVLGWFANPIHGDGDYPEFMKTGAMIPEFSEAEKEEVRGTA DFFAFSFGPNNFRPSNTVVKMGQNVSLNLRQVLNWIKLEYDDPQILISENG {SEQ ID 140:474)

Murine Residues 302S-506F SHWIEPNRTDNMEDVINCQHSMSSVLGWFANP1HGDGDYPEFMKTGAMIPEFSEAEKEE VRGTADFFAFSFGPNNFRPSNTWKMGQNVSLNLRQVLNWIKLEYDDPQIL1SENGWFT DSYIKTEDTTAIYMMK.NFLNQVLQA1KFDEIRVFGYTAWTLLDGFEWQDAYTTRRGLFY VDFNSEQKERKPKSSAHYYKQIIQDNGF (SEQ ID NO:475)

Murine Residues 416G-519P GWFTDSYIKTEDTTAIYMMKNFLNQVLQA1KFDEIRVFGYTAWTLLDGFEWQDAYTTR RGLFYVDFNSEQKJERKPKSSAHYYKQIIQDNGFPLKESTPDMKGRF (SEQ ID NO:476)

Murine Residues 507P-632G PLKESTPDMKGRFPCDFSWGVTESVLKPEFTVSSPQFTDPHLYVWNVTGNRLLYRVEGV RLKTRPSQCTDYVSIKK.RVEMLAKMKVTHYQFALDWTSILPTGNLSKVNRQVLRYYRC VVSEGLKLG (SEQ ID NO:477)

Murine Residues 520P-735A PCDFSWGVTESVLKPEFTVSSPQFTDPHLYVWNVTGNRLLYRVEGVRLKTRPSQCTDY VSIKKRVEMLAKLMKVTHYQFALDWTSILPTGNLSKVNRQVLRYYRCVVSEGLKLGVFP MVTLYHPTHSHLGLPLPLLSSGGWLNMNTAKAFQDYAELCFRELGDLVKLWITINEPNR LSDMYNRTSNDTYRAAHNLMIAHAQVWHLYDRQYRPVQHGA (SEQ ID NO:478)

Murine Residues 632G-849Q GVFPMVTLYHPTHSHLGLPLPLLSSGGWLNMNTAKAFQDYAELCFRELGDLVKLWIT1N EPNRLSDMYNRTSNDTYRAAHNLMIAHAQVWHLYDRQYRPVQHGAVSLSLHCDWAE PANPFVDSHWKAAERFLQFEIAWFADPLFKTGDYPSVMKEYIASICNQRGLSSSVLPRFT AKESRLVKGTVDFYALNHFTTRFVIHKQLNTNRSVADRDVQFLQ (SEQ ID NO:479)

Murine Residues 735A-963S AVSLSLHCDWAEPANPFVDSHWKAAERFLQFEIAWFADPLFKTGDYPSVMKEYIASKN QRGLSSSVLPRFTAKESRLVKGTVDFYALNHFTTRFVIHKQLNTNRSVADRDVQFLQDIT RLSSPSRLAVTPWGVRKJLLAW1RRNYRDRDIYITANG1DDLALEDDQIRK.YYLEKYVQE ALKAYLIDKVK1KGYYAFKLTEEKSKPRFGFFTSDFRAKSSVQFYSKLISSS (SEQ ID NO:480)

Murine Residues 1M-81F

MKTGCAAGSPGNEWIFFSSDERNTRSRKTMSNRALQRSAVLSAFVLLRAVTGFSGDGK AIWDKKQYVSPVNPSQLFLYDTF (SEQ ID NO:481)

Murine Residues 82P-193L

PKNFSWGVGTGAFQVEGSWKTDGRGPSIWDRYVYSHLRGVNGTDRSTDSYIFLEKDLL ALDFLGVSFYQFSISWPRLFPNGTVAAVNAQGLRYYRALLDSLVLRNIEPIVTL (SEQ !D NO :482)

The chimeras generated using the murine β-Klotho sequences comprised the following components:

The generated chimeras comprised the following amino acid sequences: (i) huBeta_Klotho(l-81, 523-1044)(muBctaKLOTHO 82-520) MKPGCAAGSPGNEWIFFSTDEITTRYRNTMSNGGLQRSVILSALILLRAV TGFSGDGRAIWSKNPNFTPVNESQLFLYDTFPKNFSWGVGTGAFQVEGSW KTDGRGPSIWDRYVYSHLRGVNGTDRSTDSYIFLEKDLLALDFLGVSFYQ FSISWPRLFPNGTVAAVNAQGLRYYRALLDSLVLRNIEP1VTLYHWDLPL TLQEEYGGWKNATMIDLFNDYATYCFQTFGDRVKYW1TIHNPYLVAWHGF GTGMHAPGEKGNLTAVYTVGHNLIKAHSKVWHNYDKNFRPHQKGWLSITL GSHWIEPNRTDNMEDVIMCQHSMSSVLGWFANPIHGDGDYPEFMKTGAM1 PEFSEAEfCEEVRGTADFFAFSFGPNNFRPSNTVVKMGQNVSLNLRQVLNW IKLEYDDPQlLlSENGWFTDSYlKTEDTTAIYMMRNFLNQVLQAliCFDEl RVFGYTAWTLLDGFEWQDAYTTRRGLFYVDFNSEQKERKPKSSAHYYKQ1 IQDNGFPLKESTPDMKGRFPCDFSWGVTESVLKPESVASSPQFSDPHLYV WNATGNRLLHRVEGVRLKTRPAQCTDFVNIKKQLEMLARMKVTHYRFALD WASVLPTGNLSAVNRQALRYYRCVVSEGLKLG1SAMVTLYYPTHAHLGLP EPLLHADGWLNPSTAEAFQAYAGLCFQELGDLVKLWITINEPNRLSDIYN RSGNDTYGAAHNLLVAHALAWRLYDQQFRPSQRGAVSLSLHADWAEPANP YADSHWRAAERFLQFEIAWFAEPLFKTGDYPAAMREY1ASKHRRGLSSSA LPRLTEAERRLLKGTVDFCALNHFTTRFVMHEQLAGSRYDSDRDIQFLQD ITRLSSPTRLAVIPWGVRKLLRWVRRNYGDMDIYITASGIDDQALEDDRL RKYYLGKYLQEVLKAYL1DKVRIKGYYAFKLAEEKSKPRFGFFTSDFKAK SSIQFYNKV1SSRGFPFENSSSRCSQTQENTECTVCLFLVQKKPL1FLGC CFFSTLVLLLSIAIFQRQKRRKFWKAKNLQHIPLKKGKRWS (SEQ ID NO:455)

(ii) huBeta _KIotho( 1 -507)(muBetaKLOTHO 506F-1045S) MKPGCAAGSPGNEWIFFSTDEITTRYRNTMSNGGLQRSVILSALILLRAV TGFSGDGRAIWSKNPNFTPVNESQLFLYDTFPKNFFWGIGTGALQVEGSW KKDGKGPSIWDHFIHTHLKNVSSTNGSSDSYIFLEKDLSALDFIGVSFYQ FSISWPRLFPDGIVTVANAKGLQYYSTLLDALVLRNIEPIVTLYHWDLPL ALQEKYGGWKNDTHDIFNDYATYCFQMFGDRVKYW1TIHNPYLVAWHGY GTGMHAPGEKGNLAAVYTVGHNLIKAHSKVWHNYNTHFRPHQKGWLS1TL GSHWIEPNRSENTMD1FKCQQSMVSVLGWFANPIHGDGDYPEGMRK.KLFS VLPIFSEAERHEMRGTADFFAFSFGPNNFKPLNTMAKMGQNVSLNLREAL NWIKLEYNNPRILIAENGWFTDSRVKTEDTTAIYMMKNFLSQVLQAIRLD EIRVFGYTAWSLLDGFEWQDAYTIRRGLFYVDFNSKQKERKPKSSAHYYK

QIIRENGFPLKESTPDMKGRFPCDFSWGVTESVLKPEFTVSSPQFTDPHL

YVWNVTGNRLLYRVEGVRLKTRPSQCTDYVSIKKRVEMLAKMKVTHYQFA

LDWTSILPTGNLSKVNRQVLRYYRCVVSEGLKLGVFPMVTLYHPTHSHLG

LPLPLLSSGGWLNMNTAKAFQDYAELCFRELGDLVKLWITINEPNRLSDM

YNRTSNDTYRAAHNLSVIIAHAQVWHLYDRQYRPVQHGAVSLSLHCDWAEPA

NPFVDSHWKAAERFLQFEIAWFADPLFKTGDYPSVMKEYIASKNQRGLSS

SVLPRFTAKESRLVKGTVDFYALNHFTTRFV1HKQLNTNRSVADRDVQFL

QDITRLSSPSRLAVTPWGVRKLLAWIRRNYRDRDIYITANGIDDLALEDD

QIRKYYLEKYVQEALKAYLIDKVKIKGYYAFKLTEEKSKPRFGFFTSDFR

AKSSVQFYSKLISSSGLPAENRSPACGQPAEDTDC'TICSFLVEKJECPLIFF

GCCFISTLAVLLSITVFHHQKRRKFQKARNLQN1PLKKGHSRVFS (SEQ ID NO:456) (iii) huBeta_Kiotho( 194-1044)( muBetaKLOTHO 1-L193) MKTGCAAGSPGNEWIFFSSDERNTRSRKTMSNRALQRSAVLSAFVLLRAV TGFSGDGK.AIWDKKQYVSPVNPSQLFLYDTFPKNFSWGVGTGAFQVEGSW KTDGRGPSIWDRYVYSHLRGVNGTDRSTDSYIFLEKDLLALDFLGVSFYQ FSISWPRLFPNGTVAAVNAQGLRYYRALLDSLVLRNIEPIVTLYHWDLPL ALQEKYGGWKNDTIIDIFNDYATYCFQMFGDRVKYWIT1HNPYLVAWHGY GTGMHAPGEKGNLAAVYTVGHNLiKAHSKVWHNYNTHFRPHQKGWLSITL GSHWiEPNRSENTMDIFKCQQSMVSVLGWFANPIHGDGDYPEGMRKKLFS VLPIFSEAEKHEMRGTADFFAFSFGPNNFKPLNTIViAICMGQNVSLNLREAL NWIKLEYNNPRILIAENGWFTDSRVKTEDTTAIYMMKNFLSQVLQAIRLD EIRVFGYTAWSLLDGFEWQDAYT1RRGLFYVDFNSKQKERKPKSSAHYYK QIIRENGFSLKESTPDVQGQFPCDFSWGVTESVLICPESVASSPQFSDPHL YVWNATGNRLLHRVEGVRLKTRPAQCTDFVNIKKQLEMLARMKVTHYRFA LDWASVLPTGNLSAVNRQALRYYRCVVSEGLKLGISAMVTLYYPTHAHLG LPEPLLFIADGWLNPSTAEAFQAYAGLCFQELGDLVKLWITIMEPNRLSDI YNRSGNDTYGAAHNLLVAHALAWRLYDQQFRPSQRGAVSLSLHADWAEPA NPYADSHWRAAERFLQFEIAWFAEPLFKTGDYPAAMREYIASKHRRGLSS SALPRLTEAERRLLKGTVDFCALNHFTTRFVMHEQLAGSRYDSDRDIQFL QDITRLSSPTRLAVIPWGVRKLLRWVRRNYGDMDIYITASGIDDQALEDD RLRKYYLGKYLQEVLKAYLIDKVRIKGYYAFKLAEEKSKPRFGFFTSDFK AKSSIQFYNKVfSSRGFPFENSSSRCSQTQENTECTVCLFLVQKKPLIFL GCCFFSTLVLLLS1A1FQRQKRRKFWKAKNLQHIPLKKGKRVVS (SEQ ID NO:457)

(iv) huBcta_Kiotho( 1 -81,303-1044)(muBctaRLOTHO 82P-302S) MKPGCAAGSPGNEWIFFSTDEITTRYRNTMSNGGLQRSVILSALILLRAV TGFSGDGRA1WSKNPNFTPVNESQLFLYDTFPKNFSWGVGTGAFQVEGSW KTDGRGPSIWDRYVYSBLRGVNGTDRSTDSYIFLEKDLLALDFLGVSFYQ FSISWPRLFPNGTVAAVNAQGLRYYRALLDSLVLRNIEPIVTLYHWDLPL TLQEEYGGWKI9ATMIDLFNDYATYCFQTFGDRVKYW1TIHNPYLVAWFIGF GTGMHAPGEKGNLTAVYTVGHNLIKAHSKVWHF1YDKNFRPHQKGWLS1TL GSHWIEPNRSENTMDIFKCQQSMVSVLGWFANPiHGDGDYPEGMRKKLFS VLPIFSEAEKHEMRGTADFFAFSFGPNNFKPLNTMAKMGQNVSLNLREAL NWIKLEYNNPRIUAENGWFTDSRVKTEDTTAIYMMKNFLSQVLQAIRLD

E1RVFGYTAWSLLDGFEWQDAYTIRRGLFYVDFNSKQKERKPKSSAHYYK

QIIRENGFSLKESTPDVQGQFPCDFSWGVTESVLKPESVASSPQFSDPHL

YVWNATGNRLLHRVEGVRLKTRPAQCTDFVNiKKQLEMLARMKVTHYRFA

LDWASVLPTGNLSAVNRQALRYYRCVVSEGLKLGISAMVTLYYPTHAHLG

LPEPLLHADGWLNPSTAEAFQAYAGLCFQELGDLVKLWITINEPNRLSDI

YNRSGNDTYGAAHNLLVAHALAWRLYDQQFRPSQRGAVSLSLHADWAEPA

NPYADSHWRAAERFLQFEIAWFAEPLFKTGDYPAAMREYIASKHRRGLSS

SALPRLTEAERRLLICGTVDFCALNHFTTRFVMHEQLAGSRYDSDRDIQFL

QDITRLSSPTRLAVIPWGVRKLLRWVRRjNYGDMDIYITASGIDDQALEDD

RLRKYYLGKYLQEVLKAYLIDKVRIKGYYAFKLAEEKSKPRFGFFTSDFK

AKSSIQFYNKVISSRGFPFENSSSRCSQTQENTECTVCLFLVQKKPLIFL GCCFFSTLVLLLSIAIFQRQKRRKFWKAKNLQHIPLKKGKRVVS (SEQ ID NO:458) (v) huBeia_Klotho( 1 -193,419-1044)(muBetaKLOTH0 Y194-416G) MK.PGCAAGSPGNEWIFFSTDEITTRYRNTMSNGGLQRSVILSALILLRAV TGFSGDGRAIWSKNPNFTPVNESQLFLYDTFPKNFFWGIGTGALQVEGSW KKDGKGPSTWDHFIHTHLKNVSSTNGSSDSYIFLEKDLSALDFIGVSFYQ FSISWPRLFPDGIVTVANAKGLQYYSTLLDALVLRNIEPIVTLYHWDLPL TLQEEYGGWKNATMIDLFNDYATYCFQTFGDRVKYWITIHNPYLVAWHGF GTGMHAPGEKGNLTAVYTVGHNLIKAHSKVWHNYDKNFRPHQKGWLS1TL GSHWIEPNRTDNMEDVINCQHSMSSVLGWFANP1HGDGDYPEFMKTGAMI PEFSEAEK.EEVRGTADFFAFSFGPNNFRPSNTVVKMGQNVSLNLRQVLNW IKLEYDDPQILISENGWFTDSRVKTEDTTAIYMMKNFLSQVLQAIRLDEI RVFGYTAWSLLDGFEWQDAYTIRRGLFYVDFNSKQKERKPKSSAHYYKQI IRENGFSLKESTPDVQGQFPCDFSWGVTESVLKPESVASSPQFSDPHLYV WNATGNRLLHRVEGVRLKTRPAQCTDFVNIKKQLEMLARMKVTHYRFALD WASVLPTGNLSAVNRQALRYYRCVVSEGLKLGISAMVTLYYPTHAHLGLP EPLLHADGWLNPSTAEAFQAYAGLCFQELGDLVKLWITINEPNRLSDIYN RSGNDTYGAAHNLLVAHALAWRLYDQQFRPSQRGAVSLSLHADWAEPANP YADSHWRAAERFLQFEIAWFAEPLFKTGDYPAAMREYIASKHRRGLSSSA LPRLTEAERRLLKGTVDFCALNHFTTRFVMHEQLAGSRYDSDRDIQFLQD ITRLSSPTRLAVIPWGVRKLLRWVRRNYGDMDIYITASGIDDQALEDDRL RKYYLGKYLQEVLKAYLIDKVRIKGYYAFKXAEEKSKPRFGFFTSDFKAK SSIQFYNK.VISSRGFPFENSSSRCSQTQENTECTVCLFLVQKK.PLIFLGC CFFSTLVLLLSIAIFQRQKRRKFWKAKNLQHIPLKKGKRVVS (SEQ ID NO:459)

(vi) huBcta_Klotho( 1-301, 509-1044)(muBctaKLOTHO S302-F506) MKPGCAAGSPGNEWIFFSTDEITTRYRNTMSNGGLQRSVILSALILLRAV TGFSGDGRAIWSKNPNFTPVNESQLFLYDTFPKNFFWGIGTGALQVEGSW KKDGKGPSIWDHFIHTHLiCNVSSTNGSSDSYIFLEKDLSALDFlGVSFYQ FSISWPRLFPDGIVTVANAKGLQYYSTLLDALVLRNIEPIVTLYHWDLPL ALQEKYGGWKNDT1IDIFNDYATYCFQMFGDRVKYWITIHNPYLVAWHGY GTGMHAPGEKGNLAAVYTVGHNLIKAHSKVWHNYNTHFRPHQKGWLSITL GSHWIEPNRTDNMEDVINCQHSMSSVLGWFANPIHGDGDYPEFMKTGAMI PEFSEAEKEEVRGTADFFAFSFGPNNFRPSNTVVKMGQNVSLNLRQVLNW IKLEYDDPQILISENGWFTDSYIKTEDTTAIYMMKNFLNQVLQAIKFDEI RVFGYTAWTLLDGFEWQDAYTTRRGLFYVDFNSEQKERKPKSSAHYYKQ1

IQDNGFSLKESTPDVQGQFPCDFSWGVTESVLKPESVASSPQFSDPHLYV

WNATGNRLLHRVEGVRLKTRPAQCTDFVNIKKQLEMLARMKVTHYRFALD

WASVLPTGNLSAVNRQALRYYRCVVSEGLK.LGISAMVTLYYPTHAHLGLP

EPLLHADGWLNPSTAEAFQAYAGLCFQELGDLVKLWITINEPNRLSD1YN

RSGNDTYGAAHNLLVAHALAWRLYDQQFRPSQRGAVSLSLHADWAEPANP

YADSHWRAAERFLQFEIAWFAEPLFKTGDYPAAMREYIASKHRRGLSSSA

LPRLTEAERRLLKGTVDFCALNHFTTRFVMHEQLAGSRYDSDRDIQFLQD

ITRLSSPTRLAV1PWGVRKLLRWVRRNYGDMDIYITASGIDDQALEDDRL

RKYYLGKYLQEVLKAYL1DKVRIKGYYAFKLAEEKSKPRFGFFTSDFKAK

SS1QFYNKVISSRGFPFENSSSRCSQTQENTECTVCLFLVQKKPL1FLGC CFFSTLVLLLSIAIFQRQKRRKFWKAKNLQHIPLKXGFCRVVS (SEQ ID NO:460) (vii) huBeta_Klotho( 1-417, 522-1044)(muBetaKLOTHO G416-F519) MKPGCAAGSPGNEWIFFSTDE1TTRYRNTMSNGGLQRSV1LSALILLRAV TGFSGDGRA1WSKNPNFTPVNESQLFLYDTFPKMFFWGIGTGALQVEGSW KKDGKGPSIWDHF1HTHLKNVSSTNGSSDSY1FLEKDLSALDFIGVSFYQ FSISWPRLFPDGIVTVANAKGLQYYSTLLDALVLRNIEPIVTLYHWDLPL ALQEKYGGWKNDTUDIFNDYATYCFQMFGDRVKYWITIHNPYLVAWHGY GTGMHAPGEKGNLAAVYTVGHNLIKAHSKVWHNYNTHFRPHQKGWLS1TL GSHW1EPNRSENTMDIFKCQQSMVSVLGWFANP1HGDGDYPEGMRKKLFS VLPIFSEAEKHEMRGTADFFAFSFGPNNFKPLNTMAKMGQNVSLNLREAL NWIKLEYNNPRILIAENGWFTDSYIKTEDTTAIYMMKNFLNQVLQAIKFD EIRVFGYTAWTLLDGFEWQDAYTTRRGLFYVDFNSEQKERKPKSSAHYYK QIIQDNGFPLKESTPDMKGRFPCDFSWGVTESVLKPESVASSPQFSDPHL YVWNATGNRLLHRVEGVRLKTRPAQCTDFVNIKKQLEMLARMKVTHYRFA LDWASVLPTGNLSAVNRQALRYYRCVVSEGLKLG1SAMVTLYYPTHAHLG LPEPLLHADGWLNPSTAEAFQAYAGLCFQELGDLVKLWITINEPNRLSDI YNRSGNDTYGAAHNLLVAHALAWRLYDQQFRPSQRGAVSLSLHADWAEPA NPYADSHWRAAERFLQFEIAWFAEPLFKTGDYPAAMREYIASKHRRGLSS SALPRLTEAERRLLKGTVDFCALNHFTTRFVMHEQLAGSRYDSDRD1QFL QD1TRLSSPTRLAVIPWGVRKLLRWVRRNYGDMDIYITASGIDDQALEDD RLRKYYLGKYLQEVLKAYLIDKVR1KGYYAFKLAEEKSKPRFGFFTSDFK AKSS1QFYNKV1SSRGFPFENSSSRCSQTQENTECTVCLFLVQKKPL1FL GCCFFSTLVLLLSIAIFQRQKRRKFWKAKNLQHIPLKKGKRWS (SEQ ID NO:461)

(viii) huBeta_Klotho( 1-507, 635-1044)(muBcta KLOTHO F06-G632) MKPGCAAGSPGNEWIFFSTDE1TTRYRNTMSNGGLQRSV1LSALILLRAV TGFSGDGRAIWSKNPNFTPVNESQLFLYDTFPKNFFWGIGTGALQVEGSW KKDGKGPSIWDHFIHTHLKNVSSTNGSSDSYIFLEKDLSALDFIGVSFYQ FSISWPRLFPDGIVTVANAKGLQYYSTLLDALVLRN1EPIVTLYHWDLPL ALQEKYGGWKNDTI1DIFNDYATYCFQMFGDRVKYWITIHNPYLVAWHGY GTGMHAPGEKGNLAAVYTVGHNLIKAHSKVWHNYNTHFRPHQKGWLSITL GSHW1EPNRSENTMD1FKCQQSMVSVLGWFANPIHGDGDYPEGMRKKLFS VLP1FSEAEKHEMRGTADFFAFSFGPNNFKPLNTMAKMGQNVSLNLREAL NWIKLEYNNPRILIAENGWFTDSRVKTEDTTAIYMMKNFLSQVLQAIRLD

EIRVFGYTAWSLLDGFEWQDAYTIRRGLFYVDFNSKQKERKPKSSAHYYK

QIIRENGFPLKESTPDMKGRFPCDFSWGVTESVLKPEFTVSSPQFTDPHL

YVWNVTGNRLLYRVEGVRLKTRPSQCTDYVSIKKRVEMLAKMKVTHYQFA

LDWTS1LPTGNLSKVNRQVLRYYRCVVSEGLKLGISAMVTLYYPTHAHLG

LPEPLLHADGWLNPSTAEAFQAYAGLCFQELGDLVKLWITINEPNRLSDI

YNRSGNDTYGAAHNLLVAHALAWRLYDQQFRPSQRGAVSLSLHADWAEPA

NPYADSHWRAAERFLQFEIAWFAEPLFKTGDYPAAMREYIASKHRRGLSS

SALPRLTEAERRLLKGTVDFCALNHFTTRFVMHEQLAGSRYDSDRDIQFL

QDITRLSSPTRLAVIPWGVRKLLRWVRRNYGDMDIYITASGIDDQALEDD

RLRKYYLGKYLQEVLKAYL1DKVRIKGYYAFKLAEEKSKPRFGFFTSDFK

AKSSIQFYNKVISSRGFPFENSSSRCSQTQENTECTVCLFLVQKKPLIFL GCCFFSTLVLLLSIAIFQRQKRRKFWKAKNLQHIPLKKGKRVVS (SEQ ID NO:462) (ix) huBeta_Klotho(l -521,738-1044)(muBcta KLOTHO 520P-735A) MKPGCAAGSPGNEWIFFSTDEITTRYRNTMSNGGLQRSV1LSALILLRAV TGFSGDGRAIWSKNPNFTPVNESQLFLYDTFPKNFFWGIGTGALQVEGSW KKDGKGPSIWDHFIHTHLKNVSSTNGSSDSYIFLEKDLSALDFIGVSFYQ FSISWPRLFPDGIVTVANAKGLQYYSTLLDALVLRNIEPIVTLYHWDLPL ALQEKYGGWKNDTIIDIFNDYATYCFQMFGDRVKYWITIHNPYLVAWHGY GTGMHAPGEKGNLAAVYTVGHNLIKAHSKVWHNYNTHFRPHQKGWLSITL GSHWIEPNRSENTMDIFKCQQSMVSVLGWFANPIHGDGDYPEGMRKKLFS VLPIFSEAEKHEMRGTADFFAFSFGPNNFKPLNTMAKMGQNVSLNLREAL NWIKLEYNNPRILIAENGWFTDSRVKTEDTTAIYMMKNFLSQVLQAIRLD EIRVFGYTAWSLLDGFEWQDAYTIRRGLFYVDFNSKQKERKPKSSAHYYK QIIRENGFSLKESTPDVQGQFPCDFSWGVTESVLKPEFTVSSPQFTDPHL YVWNVTGNRLLYRVEGVRLKTRPSQCTDYVS1K.KRVEMLAKMKVTHYQFA LDWTSILPTGNLSKVNRQVLRYYRCVVSEGLKLGVFPMVTLYHPTHSHLG LPLPLLSSGGWLNMNTAKAFQDYAELCFRELGDLVKLWJTINEPNRLSDM YNRTSNDTYRAAHNLMIAHAQVWHLYDRQYRPVQHGAVSLSLHADWAEPA NPYADSHWRAAERFLQFEIAWFAEPLFKTGDYPAAMREY1ASKHRRGLSS SALPRLTEAERRLLKGTVDFCALNHFTTRFVMHEQLAGSRYDSDRDIQFL QDITRLSSPTRLAVIPWGVRKLLRWVRRNYGDMDIYITASGIDDQALEDD RLRKYYLGKYLQEVLKAYLIDKVRIKGYYAFKLAEEKSKPRFGFFTSDFK AKSSIQFYNKVISSRGFPFENSSSRCSQTQENTECTVCLFLVQKKPLIFL GCCFFSTLVLLLSIA1FQRQKRRKFWKAKNLQH1PLKKGKRWS (SEQ ID NO:463)

(x) huBeta_K!otho(I -633, 852-1044)(muBeta KLOTHO 632G-849Q) MKPGCAAGSPGNEWIFFSTDEITTRYRNTMSNGGLQRSVILSALILLRAV TGFSGDGRAIWSKNPNFTPVNESQLFLYDTFPKNFFWGIGTGALQVEGSW KKDGKGPSIWDHFIHTHLKNVSSTNGSSDSYIFLEKDLSALDFIGVSFYQ FSISWPRLFPDGIVTVANAKGLQYYSTLLDALVLRNIEPIVTLYHWDLPL ALQEKYGGWKNDTIID1FNDYATYCFQMFGDRVKYWITIHNPYLVAWHGY GTGMHAPGEKGNLAAVYTVGHNLIKAHSKVWHNYNTHFRPHQKGWLSITL GSHWIEPNRSENTMDIFKCQQSMVSVLGWFANPIHGDGDYPEGMRKKLFS VLPIFSEAEKHEMRGTADFFAFSFGPNNFKPLNTMAKMGQNVSLNLREAL NWIKLEYNNPRILIAENGWFTDSRVKTEDTTAIYMMKNFLSQVLQAIRLD

EIRVFGYTAWSLLDGFEWQDAYTIRRGLFYVDFNSKQKERKPKSSAHYYK

QIIRENGFSLKESTPDVQGQFPCDFSWGVTESVLKPESVASSPQFSDPHL

YVWNATGNRLLHRVEGVRLKTRPAQCTDFVNIKKQLEMLARMKVTHYRFA

LDWASVLPTGNLSAVNRQALRYYRCVVSEGLKLGVFPMVTLYHPTHSHLG

LPLPLLSSGGWLNMNTAKAFQDYAELCFRELGDLVKLWITINEPNRLSDM

YNRTSNDTYRAAHNLMIAHAQVWHLYDRQYRPVQHGAVSLSLHCDWAEPA

NPFVDSHWKAAERFLQFEJAWFADPLFKTGDYPSVMKEYIASKNQRGLSS

SVLPRFTAKESRLVKGTVDFYALNHFTTRFViHKQLNTNRSVADRDVQFL

QDITRLSSPTRLAVIPWGVRKLLRWVRRNYGDMDIYITASGIDDQALEDD

RLRKYYLGKYLQEVLKAYL1DKVRIKGYYAFKLAEEKSKPRFGFFTSDFK

AKSSIQFYNKVISSRGFPFENSSSRCSQTQENTECTVCLFLVQKKPL1FL GCCFFSTLVLLLSIA1FQRQKRRKFWKAKNLQHIPLKKGKRVVS (SEQ ID NO:464) (xi) huBetaJClotho( 1-736, 967-1044)(muBeta KLOTHO 735A-963S) MKPGCAAGSPGNEWIFFSTDE1TTRYRNTMSNGGLQRSVILSALILLRAV TGFSGDGRAIWSKNPNFTPVNESQLFLYDTFPKNFFWGIGTGALQVEGSW KKDGKGPSIWDHF1HTHLKNVSSTNGSSDSYIFLEKDLSALDFIGVSFYQ FSISWPRLFPDGIVTVANAKGLQYYSTLLDALVLRNIEPIVTLYHWDLPL ALQEKYGGWKNDTIJDIFNDYATYCFQMFGDRVKYWITIHNPYLVAWHGY GTGMHAPGEKGNLAAVYTVGHNLIKAHSKVWHNYNTHFRPHQKGWLSITL GSHWIEPNRSENTMDIFKCQQSMVSVLGWFANPIHGDGDYPEGMRKKLFS VLPIFSEAEKHEMRGTADFFAFSFGPNNFKPLNTMAKMGQNVSLNLREAL NWIKLEYNNPRILIAENGWFTDSRVKTEDTTAIYMMKNFLSQVLQAIRLD EIRVFGYTAWSLLDGFEWQDAYTIRRGLFYVDFNSKQKERKPKSSAHYYK QIIRENGFSLKESTPDVQGQFPCDFSWGVTESVLKPESVASSPQFSDPHL YVWNATGNRLLHRVEGVRLKTRPAQCTDFVNIKKQLEMLARMKVTHYRFA LDWASVLPTGNLSAVNRQALRYYRCVVSEGLKLGISAMVTLYYPTHAHLG LPEPLLHADGWLNPSTAEAFQAYAGLCFQELGDLVKLWITINEPNRLSD1 YNRSGNDTYGAAHNLLVAHALAWRLYDQQFRPSQRGAVSLSLHCDWAEPA NPFVDSHWKAAERFLQFEIAWFADPLFKTGDYPSVMKEY1ASKNQRGLSS SVLPRFTAKESRLVKGTVDFYALNHFTTRFVIHKQLNTNRSVADRDVQFL QDITRLSSPSRLAVTPWGVRKLLAWIRRNYRDRDIY1TANGIDDLALEDD QIRKYYLEKYVQEALKAYLIDKVK1KGYYAFKLTEEKSKPRFGFFTSDFR AKSSVQFYSKLISSSGFPFENSSSRCSQTQENTECTVCLFLVQKKPLIFL GCCFFSTLVLLLSIAIFQRQKRRKFWKAKNLQHIPLKKGKRWS (SEQ ID NO:465)

(xii) huBcta_Klotho(82-1044)(muBeta KLOTHO 1-81F) MKTGCAAGSPGNEWIFFSSDERNTRSRKTMSNRALQRSAVLSAFVLLRAV TGFSGDGKAIWDKKQYVSPVNPSQLFLYDTFPKNFFWGIGTGALQVEGSW KKDGKGPSIWDHFIHTHLKNVSSTNGSSDSYIFLEKDLSALDFIGVSFYQ FSISWPRLFPDG1VTVANAKGLQYYSTLLDALVLRNIEPIVTLYHWDLPL ALQEKYGGWKNDTIIDIFNDYATYCFQMFGDRVKYWITIHNPYLVAWHGY GTGMHAPGEBCGNLAAVYTVGHNLIKAHSKVWHNYNTHFRPHQKGWLSITL GSHWIEPNRSENTMDIFKCQQSMVSVLGWFANPIHGDGDYPEGMRKKLFS VLPIFSEAEKHEMRGTADFFAFSFGPNNFKPLNTMAKMGQNVSLNLREAL NWIKLEYNNPRILIAENGWFTDSRVKTEDTTAIYMMKNFLSQVLQAIRLD

EIRVFGYTAWSLLDGFEWQDAYTIRRGLFYVDFNSKQKERKPKSSAHYYK

QIIRENGFSLKESTPDVQGQFPCDFSWGVTESVLKPESVASSPQFSDPHL

YVWNATGNRLLHRVEGVRLKTRPAQCTDFVNIKKQLEMLARMKVTHYRFA

LDWASVLPTGNLSAVNRQALRYYRCVVSEGLKLGISAMVTLYYPTHAHLG

LPEPLLHADGWLNPSTAEAFQAYAGLCFQELGDLVKLWITINEPNRLSDI

YNRSGNDTYGAAHNLLVAHALAWRLYDQQFRPSQRGAVSLSLHADWAEPA

NPYADSHWRAAERFLQFE1AWFAEPLFKTGDYPAAMREYIASKHRRGLSS

SALPRLTEAERRLLKGTVDFCALNHFTTRFVMHEQLAGSRYDSDRDiQFL

QDITRLSSPTRLAVIPWGVRKLLRWVRRNYGDMDIYITASGIDDQALEDD

RLRKYYLGKYLQEVLKAYLJDKVRIKGYYAFKLAEEKSKPRFGFFTSDFK

AKSSIQFYNKVISSRGFPFENSSSRCSQTQENTECTVCLFLVQKKPLIFL GCCFFSTLVLLLS1AIFQRQKRRKFWKAKNLQHIPLKJCGKRVVS (SEQ ID NO:466) (xiii) huBcta_KIotho( 1-81, I94-1044)(muBeta KLOTHO 82P-193L)

MKPGCAAGSPGNEWIFFSTDE1TTRYRNTMSNGGLQRSVILSALILLRAV

TGFSGDGRAIWSKNPNFTPVNESQLFLYDTFPKNFSWGVGTGAFQVEGSW

KTDGRGPSIWDRYVYSHLRGVNGTDRSTDSY1FLEKDLLALDFLGVSFYQ

FSISWPRLFPNGTVAAVNAQGLRYYRALLDSLVLRNIEP1VTLYHWDLPL

ALQEKYGGWKNDTnDIFNDYATYCFQMFGDRVKYWITIHNPYLVAWHGY

GTGMHAPGEKGNLAAVYTVGHNL1KAHSKVWHNYNTHFRPHQKGWLSITL

GSHWIEPNRSENTMDIFKCQQSMVSVLGWFANPiHGDGDYPEGMRKKLFS

VLPIFSEAEKHEMRGTADFFAFSFGPNNFKPLNTMAKMGQNVSLNLREAL

NWIKLEYNNPRILIAENGWFTDSRVKTEDTTAIYMMKNFLSQVLQAIRLD

EIRVFGYTAWSLLDGFEWQDAYTIRRGLFYVDFNSKQKERKPKSSAHYYK

QI1RENGFSLKESTPDVQGQFPCDFSWGVTESVLKPESVASSPQFSDPHL

YVWNATGNRLLHRVEGVRLKTRPAQCTDFVNIKKQLEMLARMKVTHYRFA

LDWASVLPTGNLSAVNRQALRYYRCVVSEGLfCLGISAMVTLYYPTHAHLG

LPEPLLHADGWLNPSTAEAFQAYAGLCFQELGDLVKLW1TINEPNRLSDI

YNRSGNDTYGAAHNLLVAHALAWRLYDQQFRPSQRGAVSLSLHADWAEPA

NPYADSHWRAAERFLQFEIAWFAEPLFKTGDYPAAMREYIASKHRRGLSS

SALPRLTEAERRLLKGTVDFCALNHFTTRFVMHEQLAGSRYDSDRDIQFL

QDITRLSSPTRLAVIPWGVRKLLRWVRRNYGDMDIYITASGIDDQALEDD

RLRKYYLGKYLQEVLKAYL1DKVRIKGYYAFKLAEEKSKPRFGFFTSDFK

AKSSIQFYNKVISSRGFPFENSSSRCSQTQENTECTVCLFLVQKKPL1FL GCCFFSTLVLLLSIAIFQRQKRRKFWKAKNLQHIPLKKGKRWS (SEQ ID 140:467)

Various antigen binding proteins provided herein, as well as human FGF21, were tested for the ability to activate the chimeras in L6 cells. Figure 30 correlates the observed results with each tested molecule.

These data indicate that while human FGF2I was able to activate FGFRlc combined with ail of the human/mouse β-Klotho chimeras (“+” sign indicate activity on the receptor), the substitutions of mouse sequences into human β-Klotho affected the activities of 16F17, 37D3, and 39F7. See Figure 30. These results suggest that β-Klotho sequences 1-81, 302-522, and 849-1044 are important for the activities of agonistic antigen binding proteins and may represent an important epitope for their function. EXAMPLE 12

PROTEASE PROTECTION ANALYSIS

Regions of the human FGF21 receptor bound by the antigen binding proteins that bind human FGF2! receptor, e.g., FGFRlc, β-Kiotho or FGFRlc and β-Klotho complex can be identified by fragmenting human FGF21 receptor into peptides with specific proteases, e.g., AspN, Lys-C, chymotrypsin or trypsin. The sequence of the resulting human FGF21 receptor peptides (i.e., both disulfide- and non-disulfide-containing peptide fragments from FGFRlc and β-Klotho portions) can then be determined. In one example, soluble forms of a human FGF21 receptor, e.g., a complex comprising the FGFRlc ECD-Fc and β-KIotho ECD-Fc heterodimer described herein can be digested with AspN (which cleaves after aspartic acid and some glutamic acid residues at the amino end) by incubating about 100 ,ug of soluble FGF21 receptor at 1.0 mg/ml in 0.1 M sodium phosphate (pH 6.5) for 20 hrs at 37°C with 2 (ug of AspN. A peptide profile of the AspN digests can then be generated on HPLC chromatography while a control digestion with a similar amount of antibody is expected to be essentially resistant to AspN endoprotease. A protease protection assay can then be performed to determine the proteolytic digestion of human FGF21 receptor in the presence of the antigen binding proteins. The general principle of this assay is that binding of an antigen binding protein to the FGF21 receptor can result in protection of certain specific protease cleavage sites and this information can be used to determine the region or portion of FGF21 receptor where the antigen binding protein binds.

Briefly, the peptide digests can be subjected to HPLC peptide mapping; the individual peaks are collected, and the peptides are identified and mapped by on-line electrospray ionization LC-MS (ESI-LC-MS) analyses and/or by N-tcrminal sequencing. HPLC analyses for these studies can be performed using a narrow bore reverse-phase Cl8 column (Agilent Technologies) for off-line analysis and using a capillary reverse phase Cl8 column (The Separation Group) for LC-MS. HPLC peptide mapping can be performed with a linear gradient from 0.05% trifluoroacctic acid (mobile phase A) to 90% acetonitrile in 0.05% trifluoroacetic acid. Columns can be developed at desirable flow rate for narrow bore HPLC for off-line or on-line LC-MS analyses, and for capillary HPLC for on-line LC-MS analyses.

Sequence analyses can be conducted by on-line LC-MS/MS and by Edman sequencing on the peptide peaks recovered from HPLC. On-line ESI LC-MS analyses of the peptide digest can be performed to determine the precise mass and sequence of the peptides that are separated by HPLC. The identities of selected peptides present in the peptide peaks from the protease digestion can thus be determined, EXAMPLE 13

CYNOMOLGOUS MONKEY STUDY A construct encoding the antigen binding protein designated herein as 16H7 was generated using the methodology disclosed in Examples 1-3. 16H7 was expressed, purified and characterized as described in Examples 1-5 and was studied in vivo in obese cynomolgus monkeys. 16H7 is a fully human lgGl antibody and is described by the sequences provided in Tables 1 -4, supra.

Example 13.1

Study Design

The study was conducted in obese cynomolgus monkeys. The monkeys were 8-19 years old. Their body weights ranged from 7-14 kg and BMI ranged from 36-74 kg/m2. Monkeys were acclimated for 6 weeks prior to the initiation of compound administration. During the acclimation period, the monkeys were familiarized with study-related procedures, including chair-restraint, subcutaneous injection (PBS, 0.1 ml/kg), gavage (water, 10 ml/kg), and blood drawn for non-OGTT and OGTT samples. After 4 weeks of training, baseline OGTT and plasma metabolic parameters were measured. 20 monkeys were selected and randomized into two treatment groups to achieve similar baseline levels of body weight, glucose OGTT profiles, and plasma glucose and triglyceride levels.

The study was conducted in a blinded fashion. Vehicle (n=10), 16H7 (n-10). Compound was given every other week (5 mg/kg). On the week when animals were not injected with 16H7, they received vehicle injection instead. After 2 injections of 16H7, animals were monitored during an additional 6 weeks for compound washout and recovery from treatments.

Food intake, body weight, clinical chemistry and OGTT were monitored throughout the study. Food intake was measured every meal. Body weight was measured weekly. Blood samples were collected on different days in fasted or fed state to measure glucose, insulin and triglyceride levels. OGTTs were conducted every two weeks after the initiation of the study. The day starting the treatment is designated as 0 and the detailed study plan is shown in Figure 14.

The results presented in this Example represent data collected throughout the 68 days of the study.

Example 13,2

Effect of 16H7 on Food Intake

Animals were fed twice a day, with each animal receiving 120 g of formulated food established during the acclimation period. The remaining food was removed and weighed after each meal to calculate food intake. The feeding times were from 8:00 AM to 8:30 AM (±30 minutes) and then from 4:30PM to 5:00PM (±30 minutes). Fruit (150 g) was supplied to each animal at 11:30 to 12:30 PM (±30 minutes) every day.

Compared with vehicle, 16H7 reduced food intake in the monkeys. The effect diminished and the food intake returned to close to baseline or control levels after about 21 days of treatment. 16H7 did not have a significant effect on AM food intake (Figure 15) and only modestly reduced food intake on PM meal during the treatment (Figure 16). An increase in AM food intake was seen after day 49 (Figure 15). Throughout the study (and even during the acclimation period), fruit intake seemed lower in the 16H7 group compared to the vehicle group. Overall, 16H7 showed a significant effect on inhibiting food intake.

Example 13.3

Effect of 16H7 on Body Weight

Body weight was monitored weekly throughout the study. Over the course of the 4 week treatments, the body weight of animals treated with vehicle remained constant while body weight of animals treated with 16H7 progressively decreased. Body weight did not return to baseline by the end of the 6 weeks wash out period (Figure 17).

Example 13.4

Effect of 16H7 on Body Mass Index (BMP.

Abdominal Circumference (AO and Skin Fold Thickness (SFT) BMI, AC and SFT were monitored weekly throughout the study, both pre- and postadministration of test compound when the body weight was taken. BMI is defined as the individual's body weight divided by the square of his or her height. SFT is the thickness of a double layer of skin and the fat beneath it as measured with a caliper. BMI, SFT and AC are relatively accurate, simple, and inexpensive measurements of body composition, particularly indicative of subcutaneous fat. Animals treated with vehicle showed relatively stable BMI, SFT and AC throughout the study. Animals treated with 16H7 showed decreased levels of BMI, AC and SFT over the course of the 4 week study, suggesting that 16H7 compound resulted in reduction of fat mass. Results are shown in Figures 18-20, respectively. These measured parameters did not come back to baseline values at the end of the 6 weeks wash out period.

Example 13.5

Effect of 16117 on Ora! Glucose Tolerance Test iOGTTt OGTTs were conducted before and after initiation of treatments. Before 16H7 injections baseline values for glucose and insulin levels were measured throughout the OGTT (Figures 21 and 22, respectively) and were not statistically significantly different between the vehicle and 16H7 groups. Post-dose OGTTs were performed every two weeks during the treatment period and after 3 weeks of wash out period. 16H7 slightly improved glucose tolerance after 4 weeks of treatment and 3 weeks of wash out period. The animal model used is not glucose intolerant explaining the modest effects observed (Figure 21). insulin levels were statistically significantly decreased in animals treated with 16H7 (significance observed at time 0 during the OGTT performed after 2 weeks of treatment, at time 0 and 15 minutes during the OGTT performed after 4 weeks of treatment and at time 0 and 60 minutes during the OGTT performed after 2 weeks of treatment) (Figure 22).

Example 13.6

Effect of 16H7 on Fasting anti Fed Blood Glucose and Insulin Levels

Blood was collected from overnight fasted animals or in fed conditions after the AM feeding. In the fasted conditions, blood drawn was conducted weekly 5 days post each injection. In the fed conditions, blood drawn was conducted on days 2, I I, 16, 25 and 46 post first injection. 16H7 did not reduce fasting or fed blood glucose levels (Figures 23 and 25). No hypoglycemia was observed in any of the monkeys treated with 16H7. 16H7 did, however, result in a statistically significant decrease in fasting and fed plasma insulin levels (Figures 24 and 26).

Example 13.7

Effect of 16H7 on Triglyceride Levels

Measurements were made from the same samples collected for glucose and insulin measurements. Triglyceride levels were significantly reduced in animals treated with 16H7 when measured in fasted or fed conditions (Figures 27 and 28).

Example 13.8

Conclusions

In a study conducted in male obese cynoniolgus monkeys, animals treated with 16H7 showed improved metabolic parameters. Body weight was reduced and body composition was improved. Short-term reduction of food intake was observed and the effect diminished and the food intake recovered to baseline or control levels at 21 days into the study. Fasting insulin and triglyceride levels were also reduced by 16H7. Insulin levels measured during OGTT were also improved.

EXAMPLE 14 VARIANT FORMS OF ANTIGEN BINDING PROTEINS 16H7 AND 22H5

Antigen binding proteins 16H7 and 22H5, which are described herein in Tables 1-4, were mutated to impart different properties to the molecule, such as changes in solubility, pi, overall charge, immunogenicity in humans and in animal models, stability, etc. The mutations comprised additions, deletions or substitutions in either the light chain (designated “LC”, SEQ ID NO: 14) or heavy chain (designated “HC”, SEQ ID NO:32) of the molecule. The disclosed single point mutations were made individually or two or more mutations were combined.

Examples of mutations and combinations of mutations that were introduced into the 16H7 heavy and light chain sequences include the following: I83K (in 16H7 heavy chain) (SEQ ID NO:396) EI6Q (in 16H7 heavy chain) + V24F (in I6H7 heavy chain) * I83T (in 16H7 heavy chain)* S1001 (in 16H7 heavy chain)* T119L (in 16H7 heavy chain) (SEQ ID NO:395) D109S (in 16H7 heavy chain) (SEQ IDNO:401)

Deletion of Y107 (in 16H7 heavy chain) (SEQ ID NO:400)

Insertion of a Y residue on the N-terminal side of Y107 (in 16H7 heavy chain) (SEQ ID NO;405) D88R+ P89A+ V90E (in 16H7 heavy chain) (SEQ ID NO:398) D49Y (in 16H7 light chain) (SEQ ID NO:386) D49A (in 16H7 light chain) (SEQ ID NO:387) D91A (in 16H7 light chain) (SEQ ID NO:388) D49A (in 16H7 light chain)* D91A (in 16H7 light chain) (SEQ ID NO:389) Q16K (in 16H7 light chain) (SEQ ID NO:3S5)

Examples of mutations and combinations of mutations that were introduced into the 22H5 heavy and light chain sequences include the following: N92Q (in 22H5 light chain) (SEQ ID NO:402) S94A (in 22H5 light chain) (SEQ ID NO:403) C109S (in 22H5 heavy chain) (SEQ ID NO:404)

Summarily, the generated antigen binding proteins comprised the following pairs of 16H7 heavy and light chains: (i) 16H7 light chain (SEQ ID NO: 14) paired with a 16H7 heavy chain comprising I83K (SEQ ID NO:396); (ϋ) 16H7 light chain (SEQ ID NO: 14) paired with a 16H7 heavy chain comprising E16Q, V24F, I83T, SI00I, T119L (SEQ ID NO:395); (iii) 16H7 light chain (SEQ ID N0:!4) paired with a 16H7 heavy chain comprising DI09S (SEQ ID NO:401); (iv) I6H7 light chain (SEQ ID NO: 14) paired with a 16H7 heavy chain comprising the deletion of Y107 (SEQ ID NO:400); (v) 16H7 light chain (SEQ ID NO: 14) paired with a 16H7 heavy chain comprising the insertion of a Y residue on the N-tcrminal side of Y307 (SEQ ID NO:405); (vi) 16H7 light chain (SEQ ID NO: 14) paired with a 16H7 heavy chain comprising D88R, P89A, V90E, (SEQ ID NO:398); (vii) 16H7 heavy chain (SEQ ID NO:32) paired with a 16H7 light chain comprising D49Y (SEQ ID NO:386); (viii) I6H7 heavy chain (SEQ ID NO:32) paired with a 16H7 light chain comprising D49A (LC) (SEQ ID NO:387); (xi) 16H7 heavy chain (SEQ ID NO:32) paired with a 16H7 light chain comprising D91A (SEQ ID NO:388); (ix) 16H7 heavy chain (SEQ ID NO:32) paired with a I6H7 light chain comprising D49A, D9IA (SEQ ID NO:389); (x) I6H7 heavy chain (SEQ ID NO:32) paired with a I6H7 light chain comprising Q16K (LC) (SEQ ID NO:385); and the following pairs of 22H5 heavy and light chain sequences: (xi) 22H5 heavy chain (SEQ ID NO:31) paired with a 22H5 light chain comprising N92Q (LC) (SEQ ID NO:402); (xii) 22H5 heavy chain (SEQ ID NO:3I ) paired with a 22H5 light chain comprising S94A (LC) (SEQ ID NO:403); (xiii) 22H5 light chain (SEQ ID NO: 13) paired with a 22H5 heavy chain comprising CI09S (HC) (SEQ ID NO:404); (xiv) 22H5 light chain (SEQ ID NO: 13) paried with a 22H5 heavy chain comprising an insertion of of a tyrosine residue at position 107 (SEQ ID NO:405).

The amino acid sequences for the generated light chain variants are shown in Table 6:

Table 6A

Amino Acid Sequences of 16H7 and 22H5 Variants

Table 6B

Nucleic Acid Sequences of I6H7 and 22H5 Variants

Table 6C CDR Amino Acid Sequences of Variants

Table 6D CPR Nucleic Acid Sequences of Variants

Additionally, a “hcmibody” was generated and studied. This structure comprised the 16H7 light chain (L3; SEQ ID NO:50), which was paired with an engineered form of the 16H7 heavy chain; the engineered heavy chain comprised the 16H7 heavy chain (SEQ ID NO:32) joined via a (G^S)* linker (SEQ ID NO:44Q) to an IgG2 Fc sequence (SEQ ID NO:441), which paired with the Fc sequence of the 16H7 heavy chain. The component parts of the hemibody have the following sequences: 3 6H7 Heavy Chain

MDMRVPAQLLGLLLLWLRGARCQVTLKJESGPVLVICPTETLTLTCTVSGFSLNNARMGV

SWIRQPPGKALEWLAH1FSNDEKSYSTSLKSRLTISKDTSKSQVVL1MTNMDPVDTATYY

CARSWTGGYYYDGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVK

DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKP

SNTKVDKTVERKSSVECPPCPAPPVAGPSVFLFPPKPKDTLM1SRTPEVTCVVVDVSHED

PEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKG

LPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP

ENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP (SEQ ID NO:32)

Linker GGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ IDNO:440)

JgG2 Fc

ERKSSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYV

DGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTIS

KTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP

MLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:441)

The full hemibody heavy chain had the amino acid sequence shown below: MDMRVPAQLLGLLLLWLRGARCQVTLKESGPVLVKPTETLTLTCTVSGFSLNNARMGV SWIRQPPGKALEWLAH1FSNDEKSYSTSLKSRLT1SKDTSKSQVVLIMTNMDPVDTATYY CARSVVTGGYYYDGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKP SNTKVDKTVERKSSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKG LPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDJAVEWESNGQP ENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSERKSSVECPPCPAPPV AGPSVFLFPPKPKDTLM1SRTPEVTCVWDVSHEDPEVQFNWYVDGVEVHNAKTKPREE QFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPP SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:453) which is encoded by the follow sequence:

ATGGACATGAGGGTGCCCGCTCAGCTCCTGGGGCTCCTGCTGCTGTGGCTGAGAGGT

GCGCGCTGTCAGGTCACCTTGAAGGAGTCTGGTCCTGTGCTGGTGAAACCCACAGAG

ACCCTCACGCTGACCTGCACCGTGTCTGGGTTCTCACTCAACAATGCTAGAATGGGT

GTGAGCTGGATCCGTCAGCCCCCAGGGAAGGCCCTGGAGTGGCTTGCACACATTTTT

TCGAATGACGAAAAATCCTACAGCACATCTCTGAAGAGCAGGCTCACCATCTCCAA

GGACACCTCCAAAAGCCAGGTGGTCCTAATTATGACCAACATGGACCCTGTGGACA

CAGCCACATATTACTGTGCACGGTCAGTAGTAACTGGCGGCTACTACTACGACGGTA

TGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCTAGTGCCAGCACCAAGGGC

CCCTCCGTGTTCCCTCTGGCCCCCTGCAGCAGAAGCACCAGCGAGAGCACAGCC'GCC

CTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTCTTGGAACAGC

GGAGCCCTGACCAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCT

GTACAGCCTGAGCAGCGTGGTCACCGTGCCCAGCAGCAACTTCGGCACCCAGACCT

ACACCTGTAACGTGGACCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTGGA

GCGGAAGTCCAGCGTGGAGTGCCCTCCTTGTCCTGCCCCTCCTGTGGCCGGACCTAG

CGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCXGATGATCAGCCGGACCCCCGA

AGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCCGAGGTGCAGTTCAATT

GGTACGTGGACGGGGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAGGAACA

GTTCAACAGCACCTTCCGGGTGGTGTCCGTCCTCACCGTGGTGCACCAGGACTGGCT

GAACGGCAAAGAGTACAAGTGCAAGGTCTCCAACAAGGGCCTGCCTGCCCCCATCG

AGAAAACCATCAGCAAGACCAAGGGCCAGCCTCGGGAGCCTCAGGTGTACACCCTG

CCCCCCAGCCGGGAGGAAATGACCAAGAACCAGGTGTCCCTGACCTGCCTCGTGAA

GGGCTTCTACCCCAGCGATATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGA

ACAACTACAAGACCACCCCCCCCATGCTGGACAGCGACGGCAGCTTCTTCCTGTACT

CCAAACTGACCGTGGACAAGAGCCGGTGGCAGCAGGGCAACGTGTTCAGCTGTAGC

GTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGTCTCCT GGCGGAGGCGGAGGATCTGGCGGCGGAGGAAGTGGAGGGGGCGGATCTGGTGGTG GAGGCAGCGGCGGAGGTGGAAGTGGCGGTGGAGGATCCGGTGGAGGCGGCTCAGG TGGCGGCGGAAGCGAGAGAAAGTCCTCCGTGGAGTGTCCACCATGCCCTGCTCCAC CAGTGGCTGGCCCTTCCGTCTTTCTCTTTCCACCTAAACCTAAGGATACACTCATGAT CTCCAGAACTCCAGAGGTCACATGTGTGGTCGTCGATGTCAGTCATGAGGATCCTGA AGTCCAGTTTAACTGGTATGTGGATGGCGTCGAAGTCCATAATGCTAAGACAAAACC TCGCGAAGAACAGTTTAACTCCACCTTTAGAGTCGTGAGCGTGCTGACAGTCGTCCA TCAGGATTGGCTCAATGGGAAAGAATACAAATGTAAAGTCTCTAACAAAGGACTGC CCGCTCCTATCGAAAAGACCATCTCCAAAACAAAGGGGCAGCCCAGAGAGCCCCAG GTCTACACACTCCCACCCTCCAGAGAAGAGATGACAAAAAATCAGGTGTCACTCAC CTGTCTGGTCAAGGGGTTTTACCCCTCCGACATTGCCGTGGAATGGGAATCCAATGG GCAGCCTGAAAACAATTATAAGACTACACCTCCTATGCTCGACTCTGATGGGAGTTT CTTOTCTACTCTAAACTCACAGTGGATAAGTCTAGATGGCAGCAGGGGAATGTCTT TTCCTGCTCCGTCATGCATGAAGCTCTCCACAATCATTATACACAGAAGTCTTTGTCC CTGTCCCCCGGCAAG (SEQ ID NO:454)

Example 14.1 β-KIotho Binding ELISA for Engineered Antibodies

The engineered forms of 16H7 and 22H5 were tested for β-Klotho binding using an ELISA assay. Conditions for the ELISA were as follows.

Strcptavidin coated Maxisorp plates were incubated with 2pg/ml β-Klotho overnight at 4 degrees. Antibodies were added in 3-fold serial dilutions starting at ΙμΜ for 1 hour at room temp. HRP conjugated anti-human Fc was used as the detector antibody. Signal was developed with Lumiglo and read on Envision.

Results of the ELISA assay are shown in Figure 32A-32C and indicate that most variants of 16H7 bound to human β-Klotho except for a mutant carrying insertion of tyrosine at position 107.

Example 14.2

Engineered Variants of 16H7 and 22H5 Bind to Native Receptor Structure,

as Shown bv FACS A FACS binding assay was performed with several of the engineered forms of 16H7 and 22H5, The experiments were performed as follows, CHO cells stably expressing FGF21 receptor were treated with parent antibody 16H7 and 22H5 and also with engineered variants of them (Ipg per 1x10° cells in ΙΟΟμΙ PBS/0.5% BSA). Cells were incubated with the antibodies at 4°C followed by two washes with PBS/BSA. Cells were then treated with FITC-labeled secondary antibodies at 4”C followed by two washes. The cells were resuspended in 1ml PBS/BSA and antibody binding was analyzed using a FACS Calibur instrument.

Consistent with ELISA results, most of engineered variants of FGF2I receptor agonistic antibodies tested bind well to cell surface FGF2I receptor in FACS. This observation further confirmed that the guided engineering of FGF21 receptor agonistic antibodies maintain binding to the native structure, In one mutant, in which C'DR3 was engineered to include a tyrosine at position Y107, a complete loss of binding to cell surface receptor was observed, which is similar to the ELISA results. This observation points to the role of CDR3 loop in binding to native conformation.

Example 14.3

Activity' of 16H7 and 22H5 Variants in Primary Human Adipocytes FGF21 stimulates glucose uptake and lipolysis in cultured adipocytes and, therefore, adipocytes are often considered to be a physiologically relevant assay. A panel of the engineered variants of I6H7 and 22H5 was shown to exhibit Erk-phosphorylation activity similar to FGF2I in the human adipocyte assay with an estimated EC50 less than 10 nM, as shown in Table 7.

Table 7

Activity of Variants in Human Adinocvte Assay

Example 14,4

Biacore Binding Experiments and Off-rate Measurement

Binding of 16H7 and 22H5 variants to human β-Klotho was tested using Biacore assays. Briefly, mouse anti-His antibody (Qiagen, Valencia, CA) was immobilized on a C'M5 chip using amine coupling reagents (General Electronics, Piscataway, NJ). His-tagged human recombinant β-Klotho was captured on the second flow cell to -100RU. The first flow cell was used as a background control. lOOnM mAbs were diluted in PBS plus O.lmg/ml BSA, 0.005% P20 and injected over the β-Klotho captured on anti-His antibody surface. For kinetic measurement, 0.78~ΙΟΟηΜ mAbs diluted in PBS plus O.lmg/ml BSA, 0.005% P20 were injected over the β-Klotho surface.

The variants tested are summarized in Table 8:

Table 8

Variants Studied in Binding and Off-rate Experiments

Among the engineered mAbs tested, the majority of them showed tight binding to human β-Klotho, except #15 which showed no binding. Table 9 below shows lOOnM mAbs binding to β-Klotho captured on anti-His, Figure 33 shows the comparison to off-rate.

Table 9

Binding to β-Klotho

EXAMPLE 15

Combinations of Antigen Binding Proteins Show an Additive Effect Antigen binding proteins representing different binding bins (Fig 11a and b) were selected and tested in reporter assays in pairs to determine if the pair of molecules would behave in an additive fashion. Assays were run as follows.

On day one, AM-l/D FGFRlc+p-Klotho Luc clone was seeded in a 96-well plate at 20K cells/wcll in DMEM + 10% FBS medium. The plate was incubated overnight. The following day, the medium was replaced with assay medium (DMEM + 0.2% FBS) and incubated overnight. From an antibody working stock (1 mg/mL in PBS), each antibody under study was prepared at a dilution of 2 μg/ml in assay medium. 100 pL of each antibody to be tested was combined in a U-bottom plate. The assay medium was removed from the cells, and 50 pL of the antibody mixtures was transferred to the cells. The antibody mixtures were incubated on the cells for 5 hrs. Lastly, each sample was read-out with SteadyGlo Luciferase reagent (50 pl/well),pcr the manufacturer’s specifications.

Table 10 below is a summary of the activity (% of FGF21 activity from the reporter assay) observed from the study; Table 11 expresses the observed activities with respect to bins.

Table 10

Antibody Combination Activity (%)

Table Π

Antibody Combinations Expressed in Terms of Bins

Surprisingly, several pairs of molecules showed an additive effect. As shown in Figures 34 and 35, respectively, 39F11 and FGF21 showed an additive effect when measured in the reporter assay of Example 5, as did 16H7 and 39H11.

Summarizing the data from this set of experiments, it was observed that antigen binding proteins from the same binding bin, e.g., 16H7 when paired with 20D4 (both from Group A), the summed activity was not additive. This was also observed when 12E4 was paired with 26H11 (both from Group B). Additionally, paired antigen binding proteins from non-overlapping bins showed additive activities, e,g„ 16H7 (Group A) paired with 26H11 or 12E4 (Group B), or paired with 39F7 (Group C). Further, antigen binding proteins 26H11 and 12E4 (Group B) showed additive effect when combined with Abs from Group A but not Group C, suggesting there may be some overlap between the binding sites of Group B and Group C and/or that the activation conformations induced by the antigen binding proteins from Group B and Group C arc not mutually compatible. Finally, as expected, when a functional antigen binding protein is paired with a non-functional antigen binding protein (e.g., 2GI0) which binds to a distinct and non-overlapping binding site from Group A, B or C, there is no effect upon the activity from the functional antigen binding protein from Group A, B or C.

Collectively, this data suggests that the disclosed antigen binding proteins can be coadministered to enhance the effect that a given antigen binding protein may provide on its own.

Each reference cited herein is incorporated by reference in its entirety for all that it teaches and for all purposes.

The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended as illustrations of individual aspects of the disclosure, and functionally equivalent methods and components form aspects of the disclosure. Indeed, various modifications of the disclosure, in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications arc intended to fall within the scope of the appended claims.

Claims (15)

1. An isolated antibody or antibody fragment thereof that mimics FGF21-mediated signaling comprising: a heavy chain variable region CDR1 comprising the sequence set forth in SEQ ID NO: 122; a heavy chain variable region CDR2 comprising the sequence set forth in SEQ ID NO: 133; a heavy chain variable region CDR3 comprising the sequence set forth in SEQ ID NO: 148; a light chain variable region CDR1 comprising the sequence set forth in SEQ ID NO: 166; a light chain variable region CDR2 comprising the sequence set forth in SEQ ID NO: 176; and a light chain variable region CDR3 comprising the sequence set forth in SEQ ID NO: 188.

2. The isolated antibody or antibody fragment thereof according to claim 1 comprising a heavy chain variable region comprising the sequence set forth in SEQ ID NO: 68 and a light chain variable region comprising the sequence set forth in SEQ ID NO: 50.

3. The isolated antibody or antibody fragment thereof according to claim 2 having one of the following mutations: in the light chain variable region, Q16K, D49Y, D49A or in the heavy chain variable region, V24F or I83T; or combinations thereof.

4. An isolated antibody or antibody fragment thereof that mimics FGF21-mediated signalling, wherein said antibody or fragment thereof competes for binding to: b-Klotho; FGFRlc, FGFR2c, FGFR3c or FGFR4; or a complex comprising b-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4, with an antibody of any one of claims 1 to 3.

5. A pharmaceutical composition comprising one or more antibodies or antibody fragments thereof of any one of claims 1 to 4 in admixture with a pharmaceutically acceptable carrier.

6. A pharmaceutical composition comprising an antibody or antibody fragment thereof of any one of claims 1 to 4 in admixture with a pharmaceutically acceptable carrier.

7. An isolated nucleic acid comprising a polynucleotide sequence encoding the antibody or antibody fragment thereof of any one of claims 1 to 4.

8. An expression vector comprising the nucleic acid of claim 7.

9. An isolated cell comprising the nucleic acid of claim 7.

10. An isolated cell comprising the expression vector of claim 8.

11. A method of producing an antibody or antibody fragment thereof that specifically binds to (i) β-Klotho; (ii) FGFRlc, FGFR2c, FGFR3c or FGFR4; or (iii) a complex comprising β-Klotho and one of FGFRlc, FGFR2c, FGFR3c, and FGFR4, comprising incubating the cell of claim 9 or claim 10 under conditions that allow it to express the antibody.

12. An antibody or antibody fragment thereof when produced by the method of claim 11.

13. A method of preventing or treating a condition in a subject in need of such treatment comprising administering the antibody or antibody fragment thereof of any one of claims 1 to 4 or 12, or a therapeutically effective amount of the composition of claim 5 or claim 6 to the subject, wherein the condition is treatable by lowering blood glucose, insulin or serum lipid levels.

14. The method of claim 13, wherein the condition is type 2 diabetes, obesity, dyslipidemia, NASH, cardiovascular disease or metabolic syndrome.

15. Use of the antibody or antibody fragment thereof of any one of claims 1 to 4 or 12 in the manufacture of a medicament for preventing or treating a condition, wherein the condition is treatable by lowering blood glucose, insulin or serum lipid levels.