EP2015776A1 - Glp-1 compound/glucagon antibody compositions - Google Patents

Abstract

Compositions comprising antibodies that bind glucagon linked to GLP-I compounds are provided, as well as methods of using the compositions for treating, for example, metabolic disorders, enhancing insulin expression, and promoting insulin secretion in a subject.

Description

GLP-I COMPOUND/GLUCAGON ANTIBODY COMPOSITIONS

CROSS-REFERENCE TO RELATED APPLICATIONS

This applications claims the benefit of U.S. Provisional Application No. 60/793,690, filed April 20, 2006, which is incorporated herein by reference in its entirety for all purposes.

BACKGROUND

Glucagon-like peptide 1 (GLP-I) and the related peptide glucagon are produced via differential processing of proglucagon and have opposing biological activities. Proglucagon itself is produced in α-cells of the pancreas and in the enteroendocrine L- cells, which are located primarily in the distal small intestine and colon. In the pancreas, glucagon is selectively cleaved from proglucagon. In the intestine, in contrast, proglucagon is processed to form GLP-I and glucagon-like peptide 2 (GLP-2), which correspond to amino acid residues 78-107 and 126-158 of proglucagon, respectively (see, e.g., Irwin and Wong, 1995, MoI. Endocrinol. 9:267-277 and Bell et al, 1983, Nature 304:368-371). By convention, the numbering of the amino acids of GLP-I is based on the GLP-I (1-37) formed from cleavage of proglucagon. The biologically active forms are generated from further processing of this peptide, which, in one numbering convention, yields GLP-I (7-37)-OH and GLP-I (7-36)-NH₂. Both GLP-I (7-37)-OH (or simply GLP-I (7-37)) and GLP-I (7-36)-NH₂ have the same activities. For convenience, the term "GLP-I", is used to refer to both of these forms. The first amino acid of these processed peptides is His7 in this numbering convention. Another numbering convention recognized in the art, however, assumes that the numbering of the processed peptide begins with His as position 1 rather than position 7. Thus, in this numbering scheme, GLP-I (1-31) is the same as GLP-I (7-37), and GLP-l(l-30) is the same as GLP-I (7-36).

Glucagon is secreted from the α-cells of the pancreas in response to low blood sugar, with the main target organ for glucagon being the liver. Glucagon stimulates glycogen breakdown and inhibits glycogen biosynthesis. It also inhibits fatty acid synthesis, but enhances gluconeogenesis. The net result of these actions is to significantly increase the release of glucose to the liver. GLP-I, in contrast, lowers glucagon secretion, while stimulating insulin secretion, glucose uptake and cyclic-AMP (cAMP) formation in response to absorption of nutrients by the gut. Various clinical data provide evidence of these activities. The administration of GLP, for example, to poorly controlled type 2 diabetics normalized their fasting blood glucose levels (see, e.g., Gutniak, et al, 1992, New Eng. J. Med. 326: 1316-1322). GLP-I has a number of other important activities. For instance, GLP-I also inhibits gastric motility and gastric secretion (see, e.g., Tolessa, 1998, J. Clin. Invest. 102:764-774). This effect, sometimes referred to as the ileal brake effect, results in a lag phase in the availability of nutrients, thus significantly reducing the need for rapid insulin response. Studies also indicate that GLP-I can promote cell differentiation and replication, which in turn aids in the preservation of pancreatic islet cells and an increase in β-cell mass (See, e.g., Andreasen et al, 1994, Digestion 55:221-228; Wang, et al, 1997, J. Clin. Invest. 99:2883-2889; Mojsov, 1992, Int. J. Pep. Prot, Res. 40:333-343; and Xu et al, 1999, Diabetes 48:2270-2276). Evidence also indicates that GLP-I can increase satiety and decrease food intake (see, e.g., Toft-Nielsen et al, 1999, Diabetes Care 22: 1 137-1 143; Flint et al, 1998, J. Clin. Invest. 101:515-520; Gutswiller et al, 1999 Gut 44:81-86).

Other research indicates that GLP-I induces β-cell-specific genes, including GLUT-I transporter, insulin receptor and hexokinase-1 (see, e.g., Perfetti and Merkel, 2000, Eur. J. Endocrinol 143:717-725). Such induction could reverse glucose intolerance often associated with aging.

Because it plays a key role in regulating metabolic homeostasis, GLP-I is an attractive target for treating a variety of metabolic disorders, including diabetes, obesity and metabolic syndrome. Current treatments for diabetes include insulin injection and administration of sulfonylureas. Both approaches, however, have significant shortcomings. Insulin injections, for instance, require complicated dosing considerations, and treatment with sulfonylureas often becomes ineffective over time. Potential advantages of GLP-I therapy include: 1) increased safety because insulin secretion is dependent on hyperglycemia, 2) suppression of glucagon secretion which in turn suppresses excessive glucose output, and 3) slowing of gastric emptying, which in turn slows nutrient absorption and prevents sudden glucose increases. A key hurdle for effective treatment with GLP-I, however, has been the very short half-life of the peptide, which typically is only a few minutes (see, e.g., Hoist, 1994, Gastroenterology 107: 1848-1855). Various analogs have been developed with the goal of extending the half-life of the molecule. Some of these, however, have significant gastrointestinal side effects, including vomiting and nausea (see, e.g., Agerso et at, 2002, Diabetologia 45 : 195-202).

Accordingly, there thus remains a need for improved molecules that have GLP-I type activity, for use in the treatment of various metabolic diseases such as diabetes, obesity and metabolic syndrome.

SUMMARY

Compositions comprising an anti-glucagon antibody linked to a GLP-I compound are provided. In some compositions, the antibody specifically binds human glucagon. Methods for treating a variety of diseases by administering an effective amount of the compositions are also provided. Such methods can be used to treat, for example, diabetes, impaired glucose tolerance, insulin resistance, various lipid disorders, obesity, cardiovascular diseases and bone disorders.

Some compositions, for example, comprise an antibody that binds glucagon and a GLP-I compound linked to the antibody that binds glucagon, wherein the GLP-I compound has a GLP-I activity. In certain compositions, the antibody comprises (i) a heavy chain variable region and (ii) a light chain variable region; and the GLP-I compound is linked to either the heavy chain variable region or the light chain variable region. In some compositions, the carboxy terminus of the GLP-I compound is linked to the amino terminus of the light chain variable region, and/or the carboxy terminus of the GLP-I compound is linked to the amino terminus of the heavy chain variable region.

A variety of GLP-I compounds can be attached to the anti-glucagon antibody. In one aspect, a GLP-I compound in a composition as provided herein comprises a GLP-I peptide that has at least 90% sequence identity to SEQ ID NO: 1 and has a GLP-I activity. In certain compositions, the GLP-I compound comprises the amino acid sequence of formula I (SEQ ID NO: 92): Xaai-Xaa₂-Xaa₃-Xaa₄-Xaa₅-Xaa₆-Xaa₇-Xaa₈-Xaa₉-Xaaio-Xaai i-Xaai_2-Xaai₃-

Xaai₄-Xaai₅-Xaai6-Xaai₇-Xaai8-Xaai9-Xaa2₀-Xaa₂i-Xaa22-Xaa23-Xaa₂4.Xaa2₅-

Xaa₂₆-Xaa₂₇-Xaa₂₈-Xaa₂₉-Xaa₃₀ -Xaa₃i- Xaa₃₂-Xaa₃₃-Xaa₃₄-Xaa₃₅-Xaa₃₆-Xaa₃7-

C(O)-Ri (Formula I, SEQ ID NO: 92) wherein,

Ri is OR₂ or NR₂R₃;

R₂ and R₃ are independently hydrogen or (Ci-C₈)alkyl;

Xaa at position 1 is: L-histidine, D-histidine, desamino-histidine, 2-amino- histidine, 3-hydroxy-histidine, homohistidine, α-fluoromethyl-histidine or α- methyl-histidine;

Xaa at position 2 is GIy, bAla (2-aminopropionic acid), Asp, Ala, 1-amino- cylcopentanecarboxylic acid, 2-aminoisobutryic acid or alpha-alpha-disubstituted amino acids;

Xaa at position 3 is GIu, Asp, or Lys; Xaa at position 4 is GIy, Thr or His;

Xaa at position 5 is Thr, Ala, GIy, Ser, Leu, He, VaI, GIu, Asp, or Lys;

Xaa at position 6 is: His, Trp, Phe, or Tyr;

Xaa at position 7 is Thr or GIy;

Xaa at position 8 is Ser, Ala, GIy, Thr, Leu, He, VaI, GIu, Asp, or Lys; Xaa at position 9 is Asp, Asn or GIu;

Xaa at position 10 is VaI, Ala, GIy, Ser, Thr, Leu, He, Tyr, GIu, Asp, Trp, or Lys;

Xaa at position 1 1 is Ser, Ala, GIy, Thr, Leu, He, VaI, GIu, Asp, or Lys;

Xaa at position 12 is Ser, Ala, GIy, Thr, Leu, He, VaI, GIu, Asp, Trp, Tyr, Asn,

Lys, Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta- Homoglutamic acid, or homoglutamic acid;

Xaa at position 13 is Tyr, Phe, Trp, GIu, Asp, GIn, Lys, Homolysine, Ornithine,

4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, or homoglutamic acid;

Xaa at position 14 is Leu, Ala, GIy, Ser, Thr, He, VaI, GIu, Asp, Met, Trp, Tyr, Asn, GIn, Lys, Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, or homoglutamic acid; Xaa at position 15 is GIu, Asp, Lys, Homolysine, Ornithine, 4-carboxy- phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, or homoglutamic acid;

Xaa at position 16 is GIy, Ala, Ser, Thr, Leu, He, VaI, GIu, Asp, Asn, Lys, Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-

Homoglutamic acid, or homoglutamic acid;

Xaa at position 17 is GIn, Asn, Arg, GIu, Asp, Lys, Ornithine, 4-carboxy- phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, or homoglutamic acid; Xaa at position 18 is Ala, GIy, Ser, Thr, Leu, He, VaI, Arg, GIu, Asp, Asn, Lys,

Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta- Homoglutamic acid, or homoglutamic acid;

Xaa at position 19 is Ala, GIy, Ser, Thr, Leu, He, VaI, GIu, Asp, Asn, Lys, Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta- Homoglutamic acid, or homoglutamic acid;

Xaa at position 20 is Lys, Homolysine, Arg, GIn, GIu, Asp, Thr, His, Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, or homoglutamic acid; Xaa at position 21 is Leu, GIu, Asp, Thr, Lys, Homolysine, Ornithine, 4-carboxy- phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, or homoglutamic acid;

Xaa at position 22 is Phe, Trp, Asp, GIu, Lys, Homolysine, Ornithine, 4-carboxy- phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, or homoglutamic acid; Xaa at position 23 is He, Leu, VaI, Ala, Phe, Asp, GIu, Lys, Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, or homoglutamic acid;

Xaa at position 24 is Ala, GIy, Ser, Thr, Leu, He, VaI, GIu, Asp, Lys, Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, or homoglutamic acid; Xaa at position 25 is Trp, Phe, Tyr, GIu, Asp, Asn, or Lys;

Xaa at position 26 is Leu, GIy, Ala, Ser, Thr, He, VaI, GIu, Asp, or Lys; Xaa at position 27 is VaI, GIy, Ala, Ser, Thr, Leu, He, GIu, Asp, Asn, or Lys;

Xaa at position 28 is Asn, Lys, Arg, GIu, Asp, or His;

Xaa at position 29 is GIy, Ala, Ser, Thr, Leu, He, VaI, GIu, Asp, or Lys;

Xaa at position 30 is GIy, Arg, Lys, GIu, Asp, Thr, Asn, or His; Xaa at position 31 is Pro, GIy, Ala, Ser, Thr, Leu, He, VaI, GIu, Asp, or Lys;

Xaa at position 32 is Thr, GIy, Asn, Ser, Lys, or is omitted;

Xaa at position 33 is GIy, Asn, Ala, Ser, Thr, He, VaI, Leu, Phe, Pro, or is omitted;

Xaa at position 34 is GIy, Thr, or is omitted; Xaa at position 35 is Thr, Asn, GIy or is omitted;

Xaa at position 36 is GIy or is omitted;

Xaa at position 37 is GIy or is omitted; provided that when the amino acid at position 32, 33, 34, 35, 36 or 37 is omitted, then each amino acid downstream of that amino acid is also omitted, and wherein the compound has a GLP-I activity.

The GLP-I compound in some compositions thus comprises the amino acid sequence of any of SEQ ID NO: 1-35, SEQ ID NO: 126, or SEQ ID NO: 127. In some compositions, the antibody that is part of the composition as provided herein comprises: (a) one or more light chain (LC) CDRs selected from the group consisting of: (i) a

LC CDRl with at least 60% sequence identity to SEQ ID NO:76; (ii) a LC CDR2 with at least 60% sequence identity to SEQ ID NO:77; and (iii) a LC CDR3 with at least 60% sequence identity to SEQ ID NO: 78;

(b) one or more heavy chain (HC) CDRs selected from the group consisting of (i) a HC CDRl with at least 60% sequence identity to SEQ ID NO:84; (ii) a HC CDR2 with at least 60% sequence identity to SEQ ID NO:85; and (iii) a HC CDR3 with at least 60% sequence identity to SEQ ID NO: 86; or

(c) one or more LC CDRs of (a) and one or more HC CDRs of (b). The antibody can comprise two, three, four, five, or all six CDRs from the CDRs listed above in (a) and (b). In certain compositions, the antibody that is part of the composition includes an antibody that comprises:

(a) one or more LC CDRs selected from the group consisting of: (i) a LC CDRl with the amino acid sequence as set forth in SEQ ID NO: 76; (ii) a LC CDR2 with the amino acid sequence as set forth in SEQ ID NO: 77; and (iii) a LC CDR3 with the amino acid sequence as set forth in SEQ ID NO: 78;

(b) one or more HC CDRs selected from the group consisting of (i) a HC CDRl with the amino acid sequence as set forth in SEQ ID NO: 84; (ii) a HC CDR2 with the amino acid sequence as set forth in SEQ ID NO: 85; and (iii) a HC CDR3 with the amino acid sequence as set forth in SEQ ID NO: 86; or

(c) one or more LC CDRs of (a) and one or more HC CDRs of (b). The antibody can comprise two, three, four, five, or all six CDRs from the CDRs listed above in (a) and

(b).

In some instances, the antibody of a composition comprises the LC CDR3 with the amino acid sequence of SEQ ID NO: 78 and/or the HC CDR3 with the amino acid sequence of SEQ ID NO: 86. In other compositions, the antibody can comprise at least two or three CDRs from the CDRs listed above in (a) and (b).

Certain compositions include an antibody that comprises (a) a light chain variable region (VL) having at least 90% sequence identity with SEQ ID NO: 79; or (b) heavy chain variable region (VH) having at least 90% sequence identity with SEQ ID NO: 83; or (c) a VL of (a) and a VH of (b). The antibody in such compositions can consist of two identical VH and two identical VL.

In some compositions, the antibody comprises: (a) a light chain comprising the amino acid sequence of any of SEQ ID NOs: 40-81 ; (b) a heavy chain comprising the amino acid sequence of any of SEQ ID NOs: 39 or 82-91 ; or (c) a light chain comprising the amino acid sequence of any of SEQ ID NOs: 41-81 and a heavy chain comprising the amino acid sequence of any of SEQ ID NOs: 39 or 82-91.

Polypeptides are also provided herein that comprise a glucagon binding antibody light chain variable region linked to a GLP-I analog. Certain such polypeptides have the amino acid sequence of any of SEQ ID NO: 41-74. The invention further provides antibodies comprising a polypeptide having the amino acid sequence of any of SEQ ID

NO: 41-74.

The invention also provides polypeptides comprising a glucagon binding antibody heavy chain variable region linked to a GLP-I peptide. In certain aspects, the polypeptide is a fusion protein that comprises the amino acid sequence of SEQ ID NO:

83. In certain such fusion proteins, the GLP-I compound comprises SEQ ID NO: 1-35,

SEQ ID NO: 126, or SEQ ID NO: 127.

Pharmaceutical compositions are also provided that comprise a pharmaceutically acceptable carrier and an effective amount of a composition as provided herein. Methods for treating a subject with various diseases, including for example various metabolic disorders, are also disclosed. Such methods generally involve administering to the subject an effective amount of the pharmaceutical composition as provided herein.

Specific examples of metabolic diseases that can be treated include, but are not limited to, diabetes, obesity and metabolic syndrome. Also provided are methods for enhancing insulin expression and for promoting insulin secretion in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition as provided herein.

Furthermore, the invention provides methods for treating a subject by administering to the subject an effective amount of a composition comprising an antibody that binds glucagon and a GLP-I compound linked thereto, wherein the GLP-I compound has GLP-I activity. Diseases that can be treated with such compositions include those just listed above. Also described herein are methods for enhancing insulin expression and for promoting insulin secretion in a subject, comprising administering to the subject an effective amount of the composition comprising an antibody that binds glucagon; and a GLP-I compound linked thereto, wherein the GLP-I compound has

GLP-I activity.

Specific embodiments will become evident from the following more detailed description of certain preferred embodiments and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 depicts a graph showing an assay for GLP(A2G)-AG159LC:AG159 IgG2 (GLP(A2G)-AG159) to determine if the construct would maintain GLP-I receptor binding properties in the presence of glucagon. The ligand binding assay was performed as described in the Examples, with the addition of 0, 1, 10 or 100 nM glucagon. Figure 2 depicts a graph showing an assay to determine the GLP-I receptor agonist activity of GLP(A2G)-AG159LC:AG159 IgG2 (GLP(A2G)-AG159) in the presence of glucagon. Also depicted on the graph is the dose response curve of the activation of the GLP-I receptor by glucagon alone (without GLP(A2G)-AG159) ( ).

Figure 3 depicts a graph showing that the presence of GLP(A2G)- AG159LC:AG159 IgG2 (GLP(A2G)-AG159) dose-dependently decreases the activity induced by glucagon.

Figure 4 depicts a graph showing results for GLP(A2G)-AG159LC:AG159 IgG2 (a construct in which GLP(A2G) was fused to the light chain of the AGl 59 antibody) and various other antibody fusions. The antibody fusions included the following GLP-I peptides fused to the light chain (LC) of AGl 59: A2G/K28N/R30T (SEQ ID NO: 28), A2G/Q17N/A19T (SEQ ID NO: 23), A2G/V10Q/V27Q (SEQ ID NO: 9), and A2G/W25Q/V27Q (SEQ ID NO: 12). These LC fusions were paired with AGl 59 IgG2 heavy chains to give the following antibodies, which were tested: GLP(A2G/K28N/R30T)-AG159LC:AG159 IgG2, GLP(A2G/Q17N/A19T)- AG159LC:AG159 IgG2, GLP(A2G/V10Q/V27Q)-AG159LC:AG159 IgG2, and GLP(A2G/W25Q/V27Q)-AG159LC:AG159 IgG2. Dosage was 12 ug/mouse.

Figure 5 depicts a graph showing that, for each of the compositions tested, blood glucose was decreased for the first 6 hours after a single injection and returned to baseline levels 24 hours after a single injection. The antibody fusions tested included the following GLP-I peptides fused to the light chain (LC) of AGl 59: GLP(A2G) (SEQ IDNO: 126), GLP(A2G/G31N/+G32/+T33) (SEQ ID NO: 31), GLP(A2G/G29N/G31/T) (SEQ ID NO: 29) and GLP(A2G/K28N/R30T) (SEQ ID NO: 28). These LC fusions were paired with AGl 59 IgG2 heavy chains to give the following antibodies, which were tested: GLP(A2G)-AG159LC:AG159 IgG2, GLP(A2G/G31N/+G32/+T33)- AG159LC:AG159 IgG2, GLP(A2G/G29N/G31/T)-AGl 59LC:AG159 IgG2, and GLP(A2G/K28N/R3OT)-AG159LC:AG159 IgG2. Figure 6 depicts a graph showing that GLP(A2G)-AG159LC:AG159 IgG2 decreased blood glucose levels in a dose dependent fashion.

Figure 7 depicts a graph showing a dose response study conducted in normal mice challenged by a glucose tolerance test with a composition in which GLP(A2G/R30G) was fused to the light chain of AGl 59 and paired with the heavy chain of AGl 59 to give the antibody fusion GLP(A2G/R30G)-AG159LC:AG159 IgG2.

Figure 8 depicts a graph showing the differences in activity between the attachment of GLP(A2G) to either the LC or HC of AGl 59, such that the resulting antibody was respectively GLP(A2G)-AG159LC:AG159 IgG2 (referred to as GLP(A2G)-AG159 LC in FIG. 8) and AG159LC:GLP(A2G)-AG159 IgG2 (referred to as GLP(A2G)-AG159 HC in FIG. 8). Both constructs were equally effective in lowering blood glucose levels.

Figure 9 depicts a graph showing blood glucose levels in mice treated with GLP(A2G/R30G)-AG159LC:AG159 IgG2. Blood glucose was measured during a glucose tolerance test every 24 hours until blood glucose levels returned to the original values.

Figure 10 depicts a graph depicting results of a tachyphylaxis experiment in mice treated with GLP(A2G/R30G)-AG159LC:AG159 IgG2.

Figure 11 depicts a graph showing AGl 59 neutralization of glucagon stimulated reporter activity.

Figure 12 depicts a graph showing that AGl 59 disrupts ¹²⁵I-glucagon binding to the human glucagon receptor.

Figure 13 depicts a graph showing that AGl 59 reduces blood glucose in ob/ob mice.

DETAILED DESCRIPTION

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All references cited in this application are expressly incorporated by reference herein for any purpose.

I. Definitions As used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural references unless the content clearly dictates otherwise.

Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY (2d ed. 1994); THE CAMBRIDGE DICTIONARY OF SCIENCE AND TECFINOLOGY (Walker ed., 1988); THE GLOSSARY OF GENETICS, 5TH ED., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY (1991).

As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

"Insulinotropic activity" refers to the ability to increase insulin synthesis, release or secretion in a glucose-dependent manner. The insulinotropic effect can result from any of a number of different mechanisms, including, but not limited to, an increase in the number of insulin positive cells and/or due to an increase in the amount of insulin synthesized or released from existing insulin positive cells in a given time period. Insulinotropic activity can be assayed using methods known in the art, such as in vivo and in vitro experiments that measure GLP-I receptor binding activity or receptor activation (for example, assays using pancreatic islet cells or insulinoma cells as described in EP 619,322 and US Patent No. 5,120,712 and assays as described herein). In humans, insulinotropic activity can be measured by examining insulin levels or C-peptide levels.

The term "isolated protein" referred to herein means that a subject protein (1) is free of at least some other proteins with which it would typically be found in nature, (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 not associated (by covalent or noncovalent interaction) with portions of a protein with which the "isolated protein" is associated in nature, (6) is operably associated (by covalent or noncovalent interaction) with a polypeptide with which it is not associated in nature, or (7) does not occur in nature. Such an isolated protein can be encoded by genomic DNA, cDNA, mRNA or other RNA, of synthetic origin, or any combination thereof. 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 use (therapeutic, diagnostic, prophylactic, research or otherwise).

"Polypeptide" and "protein" are used interchangeably herein and include a molecular chain of amino acids linked through peptide bonds. The terms do not refer to a specific length of the product. Thus, "peptides," and "oligopeptides," are included within the definition of polypeptide. The terms include post-translational modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like. In addition, protein fragments, analogs, mutated or variant proteins, fusion proteins and the like are included within the meaning of polypeptide.

The term "polypeptide fragment" refers to a polypeptide, which can be monomeric or multimeric, having an amino-terminal deletion, a carboxyl-terminal deletion, and/or an internal deletion or substitution of a naturally-occurring or recombinantly-produced polypeptide. In certain embodiments, a polypeptide fragment can comprise an amino acid chain at least 5 to about 500 amino acids long. It will be appreciated that in certain embodiments, fragments are at least 5, 6, 8, 10, 14, 20, 50, 70, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids long. Particularly useful polypeptide fragments include functional domains, including binding domains. In the case of an antibody as provided herein, useful fragments include, but are not limited to: a CDR region, especially a CDR3 region of the heavy or light chain; 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 "antibody" or "antibody peptide" as used herein refer to a monomeric or multimeric protein comprising one or more polypeptide chains that can bind specifically to an antigen and may be able to inhibit or modulate the biological activity of the antigen. The terms as used herein thus include an intact immunoglobulin of any isotype, or a fragment thereof that can compete with the intact antibody for specific binding to the target antigen, and includes, for example, chimeric, humanized, fully human, and bispecific antibodies. An intact antibody generally will comprise at least two full-length heavy chains and two full-length light chains, but in some instances may include fewer chains such as antibodies naturally occurring in camelids that may comprise only heavy chains. Antibodies may be derived solely from a single source, or may be "chimeric," that is, different portions of the antibody may be derived from two different antibodies. For example, the CDR regions may be derived from a rat or murine source, while the framework region of the V region are derived from a different animal source, such as a human. Antibodies or binding fragments as described herein may be produced in hybridomas, by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Unless otherwise indicated, the term "antibody" includes, in addition to antibodies comprising two full-length heavy chains and two full-length light chains, derivatives, variants, fragments, and muteins thereof, examples of which are described below. Thus, the term includes a polypeptide that comprises all or part of a light and/or heavy chain variable region that can bind specifically to an antigen (e.g., glucagon). The term antibody thus includes immunologically functional fragments and include, for instance, F(ab), F(ab'), F(ab')_2, Fv, and single chain Fv fragments.

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_L, and a constant region domain, C_L. The variable region domain of the light chain is at the amino-terminus of the polypeptide. Light chains include kappa chains and lambda 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, V_H, and three constant region domains, C_HI , C_H2, and C_H3. The V_H domain is at the amino-terminus of the polypeptide, and the C_H domains are at the carboxyl -terminus, with the Cn3 being closest to the -COOH end. Heavy chains according to the invention may be of any isotype, including IgG (including IgGl, IgG2, IgG3 and IgG4 subtypes), IgA (including IgAi and IgA₂ subtypes), IgM and IgE.

The term "immunologically functional fragment" (or simply "fragment") of an immunoglobulin chain, as used herein, refers to a portion of an antibody light chain or heavy chain that lacks at least some of the amino acids present in a full-length chain but which is capable of binding specifically to an antigen. Such fragments are biologically active in that they bind specifically to the target antigen and can compete with intact antibodies for specific binding to a given epitope. In one aspect of the invention, 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 may be produced by recombinant DNA techniques, or may be produced by enzymatic or chemical cleavage of intact antibodies. Immunologically functional immunoglobulin fragments of the invention include, but are not limited to, Fab, Fab', F(ab')₂, Fv, domain antibodies and single-chain antibodies, and may 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 inventive antibodies, 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 serum half-life.

A "Fab fragment" is comprised of one light chain and the C_HI and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

A "Fab' fragment" contains one light chain and a portion of one heavy chain that contains the VH domain and the CHI domain, such that an interchain disulphide bond can be formed between the light chain and heavy chain.

A "F(ab')₂ fragment" contains two light chains and two heavy chains containing a portion of the constant region between the C_HI and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains. A F(ab')₂ 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.

"Single-chain antibodies" are Fv molecules in which the heavy and light chain variable regions have been connected by a flexible linker to form a single polypeptide chain, which forms an antigen-binding region. Single chain antibodies are discussed in detail in International Patent Application Publication No. WO 88/01649 and U.S. Patent Nos. 4,946,778 and 5,260,203, the disclosures of which are incorporated by reference.

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 V_H regions are covalently joined with a peptide linker to create a bivalent domain antibody. The two V_H regions of a bivalent domain antibody may target the same or different antigens.

A "bivalent antibody" comprises two antigen binding sites. In some instances, the two binding sites have the same antigen specificities. However, bivalent antibodies may be bispecific (see below).

A "multispecific antibody" is one that targets more than one antigen or epitope.

A "bispecific," "dual-specific" or "bifunctional" antibody is a hybrid antibody having two different antigen binding sites. Bispecific antibodies are a species of multispecific antibody and may be produced by a variety of methods including, but not limited to, fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai & Lachmann (1990), Clin. Exp. Immunol. 79:315-321; Kostelny et al. (1992), J. Immunol. 148:1547-1553. The two binding sites of a bispecific antibody will bind to two different epitopes, which may reside on the same or different protein targets. 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 antibody, and additionally capable of being used in an animal to produce antibodies capable of binding to an epitope of that antigen. An antigen may have one or more epitopes.

The term "epitope" includes any determinant, preferably a polypeptide determinant, capable of specific binding to an immunoglobulin or T-cell receptor. An epitope is a region of an antigen that is bound by an antibody. In certain embodiments, epitope determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments, may have specific three-dimensional structural characteristics, and/or specific charge characteristics. In certain embodiments, an antibody is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules. An antibody is said to specifically bind an antigen when the equilibrium dissociation constant is < 10^" or 10^" M. In some embodiments, the equilibrium dissociation constant may be < 10^"9 M or < 10^"10 M. The term "naturally-occurring" as used herein and applied to an object refers to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and that has not been intentionally modified by man is naturally-occurring. The term "polynucleotide" as referred to herein means single-stranded or double- stranded nucleic acid polymers of at least 10 bases in length. In certain embodiments, the nucleotides comprising the polynucleotide can be ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. Said modifications include base modifications such as bromuridine, ribose modifications such as arabinoside and 2',3'-dideoxyribose and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate and phosphoroamidate. The term "polynucleotide" specifically includes single and double stranded forms of DNA.

The term "isolated polynucleotide" as used herein shall mean a polynucleotide of genomic, cDNA, or synthetic origin or some combination thereof, which by virtue of its origin the isolated polynucleotide (1) is not associated with all or a portion of a polynucleotide in which the isolated polynucleotide is found in nature, (2) is linked to a polynucleotide to which it is not linked in nature, or (3) does not occur in nature as part of a larger sequence. The term "oligonucleotide" referred to herein includes naturally occurring, and modified nucleotides linked together by naturally occurring, and/or non-naturally occurring oligonucleotide linkages. Oligonucleotides are a polynucleotide subset comprising members that are generally single-stranded and have a length of 200 bases or fewer. In certain embodiments, oligonucleotides are 10 to 60 bases in length. In certain embodiments, oligonucleotides are 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 bases in length. Oligonucleotides may be single stranded or double stranded, e.g. for use in the construction of a gene mutant. Oligonucleotides as provided herein may be sense or antisense oligonucleotides with reference to a protein-coding sequence.

Unless specified otherwise, the left-hand end of single-stranded polynucleotide sequences is the 5' end; the 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 and which 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 and which are 3' to the 3' end of the RNA transcript are referred to as "downstream sequences".

The term "vector" is used to refer to any molecule (e.g., nucleic acid, plasmid, or virus) used to transfer coding information to a host cell.

The term "expression vector" refers to a vector that is suitable for transformation of a host cell and contains nucleic acid sequences that direct and/or control expression of inserted heterologous nucleic acid sequences. Expression includes, but is not limited to, processes such as transcription, translation, and RNA splicing, if introns are present.

The term "host cell" is used to refer to a cell into which has been introduced, or is capable of being introduced with a nucleic acid sequence and further expresses or is capable of expressing a selected 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, so long as the selected gene is present.

The term "identity," as known in the art, refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by comparing the sequences thereof. In the art, "identity" also means the degree of sequence relatedness between nucleic acid molecules or polypeptides, as the case may be, as determined by the match between strings of two or more nucleotide or two or more amino acid sequences. "Identity" measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (i.e., "algorithms"). The term "similarity" is used in the art with regard to a related concept, but in contrast to "identity," "similarity" refers to a measure of relatedness, which includes both identical matches and conservative substitution matches. If two polypeptide sequences have, for example, 10/20 identical amino acids, and the remainder are all non- conservative substitutions, then the percent identity and similarity would both be 50%. If in the same example, there are five more positions where there are conservative substitutions, then the percent identity remains 50%, but the percent similarity would be 75% (15/20). Therefore, in cases where there are conservative substitutions, the percent similarity between two polypeptides will be higher than the percent identity between those two polypeptides.

Identity and similarity of related nucleic acids and polypeptides can be readily calculated by known methods. Such methods include, but are not limited to, those described in COMPUTATIONAL MOLECULAR BIOLOGY, (Lesk, A.M., ed.), 1988, Oxford University Press, New York; BIOCOMPUTING: INFORMATICS AND GENOME PROJECTS, (Smith, D. W., ed.), 1993, Academic Press, New York; COMPUTER ANALYSIS OF SEQUENCE DATA, Part 1, (Griffin, A.M., and Griffin, H.G., eds.), 1994, Humana Press, New Jersey; von Heinje, G., SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, 1987, Academic Press; SEQUENCE ANALYSIS PRIMER, (Gribskov, M. and Devereux, J., eds.), 1991, M. Stockton Press, New York; Carillo et al, 1988, SIAM J. Applied Math., 48:1073; and Durbin et al, 1998, BIOLOGICAL SEQUENCE ANALYSIS, Cambridge University Press. Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity are described in publicly available computer programs. Preferred computer program methods to determine identity between two sequences include, but are not limited to, the GCG program package, including GAP (Devereux et al, 1984, Nucl. Acid. Res., L2:387; Genetics Computer Group, University of Wisconsin, Madison, WI), BLASTP, BLASTN, and FASTA (Altschul et al, 1990, J. MoI Biol, 215:403-410). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, MD 20894; Altschul et al, 1990, supra). The well-known Smith Waterman algorithm may also be used to determine identity. Certain alignment schemes for aligning two amino acid or polynucleotide sequences may result in matching of only a short region of the two sequences, and this small aligned region may have very high sequence identity even though there is no significant relationship between the two full-length sequences. Accordingly, in certain embodiments, the selected alignment method (GAP program) will result in an alignment that spans at least 50 contiguous amino acids of the target polypeptide. In some embodiments, the alignment can comprise at least 60, 70, 80, 90, 100, 110, or 120 amino acids of the target polypeptide. If polynucleotides are aligned using GAP, the alignment can span at least about 100, 150, or 200 nucleotides, which can be contiguous. For example, using the computer algorithm GAP (Genetics Computer Group,

University of Wisconsin, Madison, WI), two polypeptides for which the percent sequence identity is to be determined are aligned for optimal matching of their respective amino acids (the "matched span", as determined by the algorithm). In certain embodiments, a gap opening penalty (which is calculated as three-times the average diagonal; where 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 one-tenth of the gap opening penalty), as well as a comparison matrix such as PAM250 or BLOSUM 62 are used in conjunction with the algorithm. In certain embodiments, a standard comparison matrix {see Dayhoff et al, 1978, Atlas of Protein Sequence and Structure, 5:345-352 for the PAM 250 comparison matrix; Henikoff et al, 1992, Proc. Natl. Acad. Sci USA, 89: 10915-10919 for the BLOSUM 62 comparison matrix) is also used by the algorithm.

In certain embodiments, the parameters for a polypeptide sequence comparison include the following:

Algorithm: Needleman et al. , 1970, J. MoI. Biol., 48:443-453; Comparison matrix: BLOSUM 62 from Henikoff et al., 1992, supra; Gap Penalty: 12 Gap Length Penalty: 4 Threshold of Similarity: 0 The GAP program may be useful with the above parameters. For micleotide sequences, parameters can include a gap penalty of 50 and a gap length penalty of 3, that is a penalty of 3 for each symbol in each gap. In certain embodiments, the aforementioned parameters are the default parameters for polypeptide comparisons (along with no penalty for end gaps) using the GAP algorithm.

As used herein, the twenty conventional amino acids and their single and three letter abbreviations follow conventional usage. See IMMUNOLOGY-A SYNTHESIS, 2nd Edition, (E. S. Golub and D. R. Gren, Eds.), Sinauer Associates: Sunderland, MA, 1991, incorporated herein by reference for any purpose. Stereoisomers (e.g., D-amino acids) of the twenty conventional amino acids; unnatural amino acids such as α-, α- disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids may also be suitable components for polypeptides as provided herein. Examples of unconventional amino acids include: 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N- formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-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. The term "downstream" when used in reference to a GLP-I compound means positions that are located toward the carboxyl end of the polypeptide relative to the position being referenced, i.e., to the right of the position being referenced. The term "upstream" when used in reference to a GLP-I compound means positions that are located toward the amino terminal end of the polypeptide relative to the position being referenced, i.e., to the left of the position being referenced. The recommended IUPAC- IUB Nomenclature and Symbolism for Amino Acids and Peptides have been published in Biochem. J., 1984, 219, 345-373; Eur. J. Biochem., 1984, 138, 9-37; 1985, 152, 1 ; 1993, 213, 2; Internat. J. Pept. Prot. Res., 1984, 24, following p 84; J. Biol. Chem., 1985, 260,14-42; Pure Appl. Chem., 1984, 56, 595-624; Amino Acids and Peptides, 1985, 16, 387-410; Biochemical Nomenclature and Related Documents, 2nd edition, Portland Press, 1992, pages 39-69. In assessing antibody binding and neutralization according to the invention, an antibody binds specifically and/or substantially inhibits binding of glucagon to its receptor and/or prevents glucagon receptor activation, when an excess of antibody reduces the quantity of receptor bound to or activated by glucagon by at least about 20%, 40%, 60%, 80%, 85%, or more (as measured in an in vitro competitive binding assay or in vitro functional assay respectively). A specifically-binding antibody can be expected to have an equilibrium dissociation constant for binding to glucagon of less than or equal to than 10^"8 molar, optimally less than or equal to 10^~9 or 10^"10 molar.

The term "agent" is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials.

As used herein, the terms "label" or "labeled" refers to incorporation of a detectable marker, e.g., by incorporation of a radiolabeled amino acid, or attachment to a polypeptide or nucleic acid of a fluorescent marker, a chemiluminescent marker or an enzyme having a detectable activity, or attachment to a polypeptide of biotin moieties that can be detected by labeled avidin (e.g., streptavidin preferably comprising a detectable marker such as a fluorescent marker, a chemiluminescent marker or an enzymatic activity that can be detected, inter alia, by optical or colorimetric methods). In certain embodiments, the label can also be therapeutic. Various methods of labeling polypeptides and glycoproteins are known in the art and may be used advantageously in the methods disclosed herein. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides {e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ^99mTc, ^{11 1}In, ¹²⁵I, ¹³¹I), fluorescent labels {e.g., fluorescein isothiocyanate or FITC, rhodamine, or lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, β- galactosidase, luciferase, alkaline phosphatase), chemiluminescent labels, hapten labels such as biotinyl groups, and predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, or epitope tags). In certain embodiments, labels are attached by spacer arms (such as (CH₂),,, where n < about 20) of various lengths to reduce potential steric hindrance. The term "biological sample", as used herein, includes, but is not limited to, any quantity of a substance from a living thing or formerly living thing. Such living things include, but are not limited to, humans, mice, monkeys, rats, rabbits, and other animals.

Such substances include, but are not limited to, blood, serum, urine, cells, organs, tissues, bone, bone marrow, lymph nodes, and skin.

The term "pharmaceutical agent or drug" as used herein refers to a chemical compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient.

II. Overview

Compositions comprising an anti-glucagon antibody (i.e., an antibody that binds glucagon) that is linked to a GLP-I compound are provided herein. Typically the antibody not only binds glucagon but also neutralizes glucagon such that glucagon cannot activate the glucagon receptor. The antibody of the composition typically binds human glucagon and includes at least a portion of the heavy chain or light chain variable region. Results with certain compositions unexpectedly show that the antibody of the composition is able to bind glucagon while GLP-I is still able to bind to the GLP-I receptor, despite combining these two entities. Such compositions thus have dual activities. Also provided are polypeptides that include at least portion of the heavy chain or light chain variable region of a glucagon antibody fused to a GLP-I compound which optionally can be combined with one or more other light or heavy chains or fragments thereof to form an antibody.

The antibody of the composition may be a chimeric, a humanized or a fully human antibody, including immunologically functional fragments. Also disclosed herein are polypeptides that are capable of exhibiting immunological binding properties of antibody antigen-binding sites. The GLP-I compound that is linked to the anti-glucagon antibody can be native GLP-I, any of the GLP-I analogs that are known in the art, or one of the GLP-I analogs disclosed herein that have an activity of native GLP-I. In some compositions, the anti-glucagon antibody and the GLP-I compound are part of a fusion protein in which the two molecules are joined directly or via a peptide linker. In other compositions, the antibody and GLP-I compound are not part of a fusion protein and instead are joined via a non-peptide linker. Nucleic acids encoding the antibodies and polypeptides are also disclosed, as well as methods for expressing the antibodies using these nucleic acids.

As described in greater detail below, the GLP-I compounds that are provided can be administered therapeutically or prophylactically to treat a variety of diseases. Examples of diseases that can be treated with the compounds include, but are not limited to, diabetes, impaired glucose tolerance, insulin resistance, hyperglycemia, metabolic syndrome, various lipid disorders, obesity, coronary diseases, bone disorders, and irritable bowel syndrome.

III. GLP-I Compound/Antibody Compositions and Polypeptides

The compositions that are provided generally comprise an antibody that binds glucagon and one or more GLP-I compounds that are linked to the antibody. When used to describe the relationship between the anti-glucagon antibody and the GLP-I compound, the term "linked" means that the two molecules are joined together, with each molecule still retaining at least one of its native activities (e.g., the antibody maintains the ability to bind glucagon, and the GLP-I compound maintains a GLP-I activity). The antibody in some compositions is a fully human antibody. Typically the antibody is a neutralizing antibody that can bind glucagon and inhibit its ability to activate the glucagon receptor (e.g., in an in vitro or in vivo assay such as described herein). More than one GLP-I compound can be linked to the antibody, and these may be the same or different. The antibody and the compound(s) may or may not be joined via a linker. Thus, for example, the GLP-I compound(s) and the antibody may be chemically conjugated to one another via reactive groups naturally present in or introduced into the molecules without the use of a linker. In other instances, the compound and the antibody are fused to one another as part of a fusion protein, either directly or via a peptide linker. In other compositions, a peptide or synthetic linker is used to link the GLP-I compound and the antibody. Further details regarding options for linking the antibody and GLP-I compound(s) are listed below. The compound(s) can be linked to the N- or C-terminus of the antibody or both. In certain embodiments, the GLP-I compound is linked to the variable region of the heavy and/or light chain of the antibody that binds glucagon. Thus, in certain compositions, both the light and heavy chains of the antibody are linked to a GLP-I compound. The compound attached to the different chains may be the same or different. The antibodies of some compositions that are provided have a binding affinity

(K_a) for glucagon of at least 10⁴ or 10⁵/M x seconds. Other antibodies have a k_a of at least 10⁶, 10⁷, 10⁸ or 10⁹/M x seconds. Certain antibodies that are provided have a low disassociation rate. Some antibodies, for instance, have a K_Ofr of 1 x 10^"4S^"1, 1 x 10^" V or lower. Antibodies in some compositions have a half-life of at least one day in vitro or in vivo (e.g., when administered to a human subject). The antibody in certain compositions has a half-life of at least two or three days. In another embodiment, the antibody has a half-life of four days or longer. Still other antibodies have a half-life of seven or eight 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 under ivatized or unmodified antibody.

Also provided are polypeptides that contain glucagon binding sites optionally fused with one or more GLP-I compounds.

In certain embodiments, a GLP-I /antibody composition comprises at least one anti-glucagon antibody, at least one linker and at least one GLP-I compound. Exemplary linkers include, but are not limited to, a peptide linker, an alkyl linker, a PEG linker, and a linker that that results from a chemical or enzymatic process used to connect two polypeptides. In some compositions, at least one anti-glucagon antibody comprises two full-length heavy chains and two full-length light chains. In other compositions, at least one anti-glucagon antibody comprises at least one truncated heavy chain and/or at least one truncated light chain. Thus, for example, in certain compositions, the antibody is a fragment that retains the ability to bind glucagon. Certain exemplary antibody fragments include, but are not limited to, a Fab, a Fab', a F(ab')₂, an Fv, and a single-chain Fv (scFv). The C-terminus of the GLP-I compound in some compositions is linked to the N- terminus of the light and/or heavy chain of the antibody, whereas in other compositions the N-terminus of the GLP-I compound is linked to the C-terminus of the light and/or heavy chain. In some compositions, the GLP-I compound is linked via its N- or C- terminus to another molecule via its N- or C- terminus.

Some GLP-I /antibody compositions comprise one anti-glucagon antibody and one GLP-I compound. Other compositions comprise one antibody and two compounds.

Still other compositions comprise one antibody and more than two GLP-I compounds.

Certain compositions comprise more than one antibody and one GLP-I compound.

Other compositions comprise more than one antibody and more than one compound.

In certain compositions, a first GLP-I compound is linked to the heavy chain of a anti-glucagon antibody and a second GLP-I compound having the same or different amino acid sequence as the first compound is linked to the light chain of the antibody. The antibody and compound in some compositions of this type are linked via a linker.

In other compositions, at least one GLP-I compound is fused to the heavy chain of an anti-glucagon antibody. In still other compositions, the GLP-I compound is fused to the light chain of a anti-glucagon antibody. In some compositions, a first GLP-I compound is fused to the heavy chain of an anti-glucagon antibody and a second GLP-I compound having the same or different amino acid sequence as the first compound is fused to the light chain of the antibody. In certain embodiments, the heavy chain of an anti-glucagon antibody is fused to at least two GLP-I compounds having the same or different sequence. In certain embodiments, the light chain of the antibody is fused to at least two GLP-I compounds having the same or different sequence. In certain embodiments, the heavy chain of the antibody is fused to at least two first GLP-I compounds having the same or different sequence and the light chain of the antibody is fused to at least two second GLP-I compounds having the same or different sequence. Certain compositions have a ratio of two GLP-I compounds per one anti- glucagon antibody. Thus, in some embodiments, the composition comprises a first GLP- 1 compound linked to a first heavy chain of anti-glucagon antibody and a second GLP-I compound linked to a second heavy chain of the antibody. In certain embodiments, such a composition comprises a first GLP-I compound linked to a first light chain of a glucagon antibody and a second GLP-I compound linked to a second light chain of the antibody. Other compositions comprising a ratio of two GLP-I compounds per one glucagon antibody will be apparent to those of ordinary skill in the art.

Some compositions comprise four GLP-I compounds per anti-glucagon antibody. Thus, in some embodiments, a composition comprises a first GLP-I compound linked to a first heavy chain of a glucagon antibody, a second compound linked to a second heavy chain of the antibody, a third compound linked to a first light chain of the antibody, and a fourth compound linked to a second light chain of the antibody. In certain compositions, such a composition comprises a first GLP-I compound linked to the N-terminus of a first heavy chain of a glucagon antibody, a second compound linked to the C-terminus of the first heavy chain of the antibody, a third compound linked to the N-terminus of a second heavy chain of the antibody, and a fourth compound linked to the C-terminus of the second heavy chain of the antibody. In other compositions, a first GLP-I compound and a second GLP-I compound are linked to the N-terminus of a first heavy chain of an anti- glucagon antibody, and a third GLP-I compound and a fourth GLP-I compound are linked to the N-terminus of a second heavy chain of the antibody. Other various compositions are also included herein as one skilled in the art can design additional compositions that comprise a ratio of four GLP-I compounds per one anti-glucagon antibody.

Still other compositions comprise eight GLP-I compounds per one anti-glucagon antibody. For example, some compositions comprise a first GLP-I compound linked to the N-terminus of a first heavy chain of an anti-glucagon antibody, a second compound linked to the C-terminus of the first heavy chain of the antibody, a third compound linked to the N-terminus of a second heavy chain of the antibody, a fourth compound linked to the C-terminus of the second heavy chain of the antibody, a fifth compound linked to the N-terminus of a first light chain of the antibody, a sixth compound linked to the C- terminus of the first light chain of the antibody, a seventh compound linked to the N- terminus of a second light chain of the antibody, and an eighth compound linked to the C- terminus of the second light chain of the antibody. Other compositions comprise a first and second GLP-I compound linked to the N-terminus of a first heavy chain of an anti- glucagon antibody, a third and a fourth compound linked to the N-terminus of a second heavy chain of the antibody, a fifth and a sixth compound linked to the N-terminus of a first light chain of the antibody, and a seventh and an eighth compound linked to the N- terminus of a second light chain of the antibody. Other additional combinations that include similar ratios are included as one skilled in the art can design additional GLP- 1 /antibody compositions that comprise a ratio of eight GLP-I compounds per one anti- glucagon antibody.

A. Antibodies

The antibody of the composition may be a chimeric, a humanized or a fully human antibody, as well as an immunologically functional fragment of such antibodies (e.g., a F(ab), F(ab'), F(ab')_2i Fv, single chain Fv fragment, a domain antibody or an immunoadhesion). The composition may also include a polypeptide that has the capacity to bind glucagon (e.g., a polypeptide that includes antibody antigen-binding sites).

One exemplary antibody that binds glucagon that is useful in some of the compositions that are provided is referred to as AGl 59. This antibody is a fully human antibody. The full length light and heavy chain sequences, the light and heavy chain variable region sequences and the light and heavy chain CDRs are set forth in Tables 1 and 2 below. One of skill in the art can generate and identify additional antibodies that bind glucagon using the methods and techniques described herein. For example, exemplary glucagon antibodies and methods for making them are described in US Patent No. 5,770,445.

1. Exemplary Naturally Occurring Antibodies

Some compositions include an antibody that has 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 and one full-length "heavy" chain. 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-terminal 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 are classified as kappa and lambda light chains, and each of these contains one variable domain and one constant domain. Heavy chains are 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, IgGi, IgG₂, IgG₃, and IgG₄. IgM subtypes include IgMi and IgM₂. IgA subtypes include IgAi and IgA₂. 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 may 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 C_HI , C_H2 and C_H3. The antibodies that are provided can have any of these isotypes and subtypes.

In full-length light and heavy chains, the variable and constant regions are 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, e.g., Fundamental Immunology, 2nd ed., 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 light/heavy chain pair typically form the antigen binding site.

The variable regions typically exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs from the two chains of each pair are typically embedded within the framework regions, which may enable binding to a specific epitope. From N-terminal to C-terminal, both light and heavy chain variable regions typically comprise the domains FRl, CDRl, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is typically in accordance with the definitions of Kabat et al., as explained in more detail below. Kabat et al., Sequences of Proteins of Immunological Interest (1991, National Institutes of Health, Bethesda, Md.); see also Chothia & Lesk, 1987, J. MoI. Biol 196:901-917; Chothia et al, 1989, Nature 342:878-883, CDRs constitute the major surface contact points for antigen binding. See e.g. Chothia and Lesk, supra. Further, CDR3 of the light chain and, especially, CDR3 of the heavy chain may constitute the most important determinants in antigen binding within the light and heavy chain variable regions. See e.g. Chothia and Lesk, supra; Desiderio et al. (2001), J. MoI. Biol. 310: 603-15; Xu and Davis (2000), Immunity 13(1): 37-45; Desmyter et al. (2001), J. Biol. Chem. 276(28): 26285-90; and Muyldermans (2001), J. Biotechnol. 74(4): 277-302. In some antibodies, the heavy chain CDR3 appears to constitute the major area of contact between the antigen and the antibody. Desmyter et al, supra. In vitro selection schemes in which CDR3 alone is varied can be used to vary the binding properties of an antibody. Muyldermans, supra; Desiderio, supra.

CDRs can be located in a heavy chain variable region sequence in the following way. CDRl starts at approximately residue 31 of the mature antibody and is usually about 5-7 amino acids long, and it is almost always preceded by a Cys-Xxx-Xxx-Xxx- Xxx-Xxx-Xxx-Xxx-Xxx (SEQ ID NO: 93) (where "Xxx" is any amino acid). The residue following the heavy chain CDRl is almost always a tryptophan, often a Typ-Val, a Trp-Ile, or a Trp-Ala. Fourteen amino acids are almost always between the last residue in CDRl and the first in CDR2, and CDR2 typically contains 16 to 19 amino acids. CDR2 may be immediately preceded by Leu-Glu-Trp-Ile-Gly (SEQ ID NO: 94) and may be immediately followed by Lys/Arg-Leu/Ile/Val/Phe/Thr/Ala-Thr/Ser/Ile/Ala. Other amino acids may precede or follow CDR2. Thirty-two amino acids are almost always between the last residue in CDR2 and the first in CDR3, and CDR3 can be from about 3 to 25 residues long. A Cys-Xxx-Xxx almost always immediately precedes CDR3, and a Trp-Gly-Xxx-Gly (SEQ ID NO: 95) almost always follows CDR3. Light chain CDRs can be located in a light chain sequence in the following way.

CDRl starts at approximately residue 24 of the mature antibody and is usually about 10 to 17 residues long. It is almost always preceded by a Cys. There are almost always 15 amino acids between the last residue of CDRl and the first residue of CDR2, and CDR2 is almost always 7 residues long. CDR2 is typically preceded by Ue-Tyr, Val-Tyr, He- Lys, or Ile-Phe. There are almost always 32 residues between the light chain CDR2 and CDR3, and CDR3 is usually about 7 to 10 amino acids long. CDR3 is almost always preceded by Cys and usually followed by Phe-Gly-Xxx-Gly (SEQ ID NO: 96).

One of skill in the art will realize that the lengths of framework regions surrounding the CDRs can contain insertions or deletions that make their length differ from what is typical. As meant herein, the length of heavy chain framework regions fall within the following ranges: FRl, 0 to 41 amino acids; FR2, 5 to 24 amino acids; FR3, 13 to 42 amino acids; and FR4, 0 to 21 amino acids. Further, the invention contemplates that the lengths of light chain framework regions fall within the following ranges: FRl, 6 to 35 amino acids; FR2, 4 to 25 amino acids; FR3, 2 to 42 amino acids; and FR4, 0 to 23 amino acids.

Naturally occurring antibodies typically include a signal sequence, which directs the antibody into the cellular pathway for protein secretion and which is not present in the mature antibody. A polynucleotide encoding an antibody as provided herein may encode a naturally occurring signal sequence or a heterologous signal sequence as described below.

Antibodies can be matured in vitro to produce antibodies with altered properties, such as a higher affinity for an antigen or a lower dissociation constant. Variation of only residues within the CDRs, particularly the CDR3s, can result in altered antibodies that bind to the same antigen, but with greater affinity. See e.g. Schier et al, 1996, J. MoI. Biol. 263:551-67; Yang et al, 1995, J MoI Biol. 254:392-403. The invention encompasses antibodies created by a variety of in vitro selection schemes, such as affinity maturation and/or chain shuffling (Kang et al, 1991, Proc. Natl. Acad. Sci. 88:1 1120- 23), or DNA shuffling (Stemmer, 1994, Nature 370:389-391), by which antibodies may be selected to have advantageous properties. In many schemes, a known antibody is randomized at certain positions, often within the CDRs, in vitro and subjected to a selection process whereby antibodies with desired properties, such as increased affinity for a certain antigen, can be isolated. See e.g. van den Beucken et al, 2001, J MoI Biol. 310:591-601 ; Desiderio et al, 2001, J. MoI Biol. 310:603-15; Yang et al, 1995, J MoI Biol 254:392-403; Schier et al, 1996, J. MoI Biol 263:551-67. Typically, such mutated antibodies may comprise several altered residues in one or more CDRs, depending on the design of the mutagenesis and selection steps. See e.g. van den Beucken et al, supra. Specific examples of some of the full length light and heavy chains of the antibodies that are provided and their corresponding amino acid sequence include those listed in Table 1 , which provides the light and heavy chain sequences of AGl 59. Additional sequences related to the light and heavy chains are listed in Table 2. The C- terminus of some heavy chain sequences can end . . . SLSPGK or . . . SLSPG depending upon the host in which the protein is expressed. That the C-terminal lysine may or may not be present is indicated is indicated in Tables 1 and 2 by enclosing the symbol for lysine in parentheses, i.e., (K). In CHO cells, for instance, the C-terminal lysine is cleaved, resulting in the C-terminus sequence of . . . SLSPG rather than . . . SLSPGK.

Table 1 : Light and Heavy Chains

Table 2

The light chain listed in Table 1 can be combined with any of the heavy chains shown in Table 1 to form an antibody. Thus, antibodies included in certain compositions include those in which Ll is combined with either Hl or H2. In some instances, the antibodies include at least one heavy chain and one light chain from those listed in Table 1. In other instances, the antibodies contain two identical light chains and two identical heavy chains. As an example, an antibody may include two Ll light chains and two Hl heavy chains, or two Ll light chains and two H2 heavy chains.

Other compositions include antibodies that are variants of antibodies formed by combination of the heavy and light chains shown in Table 1 and comprise light and/or heavy chains that each have at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97% 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

2. Antibody Variable Domains

Certain GLP-I compound/antibody compositions include an antibody that comprises a light chain variable region having the amino acid sequence of SEQ ID NO:79 and/or a heavy chain variable region having the amino acid sequence of SEQ ID NO: 83, and immunologically functional fragments, derivatives, muteins and variants of these light chain and heavy chain variable regions. The variable domain sequences are shown in Table 3. Table 3

The antibody of some compositions comprises a light chain variable domain comprising a sequence of amino acids that differs from the sequence of SEQ ID NO:79 at only 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 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. The light chain variable region in some antibodies comprises a sequence of amino acids that has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or

99% sequence identity to the amino acid sequences of the light chain variable region of SEQ ID NO:79.

Certain compositions that are provided include an antibody that comprises a heavy chain variable domain that comprises a sequence of amino acids that differs from the sequence of SEQ ID NO:83 at only 1, 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. The heavy chain variable region in some antibodies comprises a sequence of amino acids that has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% sequence identity to the amino acid sequences of SEQ ID NO:83.

3. CDRs of Antibodies

Complementarity determining regions (CDRs) and framework regions (FR) of a given antibody may be identified using the system described by Kabat el 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. Certain antibodies of the composition that are disclosed herein comprise one or more amino acid sequences that are identical or have substantial sequence identity to the amino acid sequences of one or more of the CDRs as summarized in Table 4.

Table 4: CDRs

The antibodies of certain GLP-I compound/antibody compositions that are provided can include one, two, three, four, five or all six of the CDRs listed above. Some antibodies include both the light chain CDR3 and/or the heavy chain CDR3. Certain antibodies have variant forms of the CDRs listed in Table 4, with one or more (i.e., 2, 3, 4, 5 or 6) of the CDRs each having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%, sequence identity to a CDR sequence listed in Table 4. For example, the antibody or fragment can include both a light chain CDR3 and a heavy chain CDR3 that each have at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%, sequence identity to the light chain CDR3 sequence and the heavy chain CDR3, respectively, listed in Table 4. The CDR sequences of some of the antibodies that are provided may also differ from the CDR sequences listed in Table 4 such that the amino acid sequence for any given CDR differs from the sequence listed in Table 4 by no more than 1, 2, 3, 4 or 5 amino acid residues. Differences from the listed sequences usually are conservative substitutions (see below). Polypeptides comprising one or more of the light or heavy chain CDRs may be produced by using a suitable vector to express the polypeptides in a suitable host cell as described in greater detail below.

The heavy and light chain variable regions and the CDRs that are disclosed in Table 3 and 4 can be used to prepare any of the various types of immunologically functional fragments that are known in the art including, but not limited to, domain antibodies, Fab fragments, Fab' fragments, F(ab')₂ fragments, Fv fragments, single-chain antibodies and scFvs.

4. Monoclonal Antibodies

Certain GLP-I compound/antibody compositions that are provided include a monoclonal antibody that binds glucagon (e.g., human glucagon). Monoclonal antibodies may 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/l.Ag 4 1, Sp210-Agl4, FO, NSO/U, MPC-I l, MPC11-X45-GTG 1.7 and S194/5XX0 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 are U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6. In some instances, a hybridoma cell line is produced by immunizing an animal

(e.g., a transgenic animal having human immunoglobulin sequences) with a glucagon immunogen; 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 a glucagon. Monoclonal antibodies secreted by a hybridoma cell line can be purified using any technique known in the art. Hybridomas or mAbs may be further screened to identify mAbs with particular properties, such as the ability to block a glucagon induced activity. Examples of such screens are provided in the examples below.

5. Chimeric and Humanized Antibodies

Other GLP-I compound/antibody compositions include a chimeric or humanized antibody. Monoclonal antibodies for use as therapeutic agents may be modified in various ways prior to use. 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, U.S. Patent No. 4,816,567; and Morrison et al, Proc. Nail. Acad. ScL USA 81 :6851-6855 (1985), which are hereby incorporated by reference. CDR grafting is described, for example, in U.S. Patent Nos. 6,180,370, 5,693,762, 5,693,761, 5,585,089, and 5,530,101, which are all hereby incorporated by reference for all purposes.

Generally, the goal of making a chimeric antibody is to create a chimera in which the number of amino acids from the intended patient species is maximized. One example is the "CDR-grafted" 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/are 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 V region or selected CDRs from a rodent antibody often are grafted into a human antibody, replacing the naturally-occurring V 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, are 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., U.S. Patent Nos. 5,585,089, and 5,693,762; Jones et al, 1986, Nature 321 :522-25; Riechmann et al, 1988, Nature 332:323-27; Verhoeyen et al, 1988, Science 239:1534-36).

6. Fully Human Antibodies

Compositions in which the antibody is a fully human antibody are also provided. As noted above, the AGl 59 Ab disclosed herein is an example of a fully human anti- glucagon antibody. Methods are available for making other fully human antibodies specific for glucagon without exposing human beings to the antigen ("fully human antibodies"). One means 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-derivatized Mabs to humans as therapeutic agents.

Fully human antibodies can be produced by immunizing transgenic animals (usually 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, for example, Jakobovits et al, 1993, Proc. Natl. Acad. ScI USA

90:2551-2555; Jakobovits et al, 1993, Nature 362:255-258; and Bruggermann et al, 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 all of the desired immune system modifications. When administered an immunogen, these transgenic animals produce antibodies that are immunospecific for the immunogen but have human rather than murine amino acid sequences, including the variable regions. For further details of such methods, see, for example, WO96/33735 and WO94/02602, which are hereby incorporated by reference. Additional methods relating to transgenic mice for making human antibodies are described in U.S. Patent Nos. 5,545,807; 6,713,610; 6,673,986; 6,162,963; 5,545,807; 6,300,129; 6,255,458; 5,877,397; 5,874,299 and 5,545,806; in PCT publications WO91/10741, WO90/04036, and in EP 546073B1 and EP 546073A1, all of which are hereby incorporated by reference in their entirety for all purposes.

The transgenic mice described above, referred to herein as "HuMab" mice, contain a human immunoglobulin gene minilocus that encodes unrearranged human heavy (μ and γ) and K light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous μ and K chain loci (Lonberg et al , 1994, Nature 368: 856-859). Accordingly, the mice exhibit reduced expression of mouse IgM or K 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 monoclonal antibodies (Lonberg et al, supra.; Lonberg and Huszar, 1995, Intern. Rev. Immunol, 13: 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; Chen et al, 1993, International Immunology 5: 647-656; Tuaillon et al, 1994, J. Immunol. 152: 2912-2920; Lonberg et al, 1994, Nature 368: 856-859; Lonberg, 1994, Handbook of Exp. Pharmacology 1 13: 49-101; Taylor et al, 1994, International Immunology 6: 579-591 ; Lonberg and Huszar, 1995, Intern. Rev. Immunol. 13: 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 U.S. Patent Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; and 5,770,429; as well as U.S. 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 ah, 1997, Nature Genetics 15: 146- 156, which are hereby incorporated by reference. For example, the HCo7 and HCo 12 transgenic mice strains can be used to generate human anti-glucagon antibodies. 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 may be cloned and expressed using a suitable vector and host cell (see, for instance, the Examples below), or the antibodies can be harvested from cultured hybridoma cells. Fully human antibodies can also be derived from phage-display libraries (as disclosed in Hoogenboom et ah, 1991, J. MoI. Biol. 227:381 ; and Marks et ah, 1991, J. MoI. 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. WO99/10494 (hereby incorporated by reference), which describes the isolation of high affinity and functional agonistic antibodies for MPL- and msk- receptors using such an approach.

7. Bispecific or Bifunctional Antibodies Antibodies included in the compositions disclosed herein can also be bispecific and bifunctional antibodies that include one or more CDRs or one or more variable regions as described above. A bispecific or bifunctional antibody in some instances is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites. Bispecific antibodies may be produced by a variety of methods including, but not limited to, fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai & Lachmann, 1990, Clin. Exp. Immunol. 79: 315-321 ; Kostelny et ah, 1992, J. Immunol. 148: 1547-1553.

8. Exemplary Variant Antibodies The antibodies of certain compositions that are provided herein are variant forms of the antibodies disclosed above (e.g., those having the sequences listed in Tables 1 and 2). For instance, some of the antibodies are ones having 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 and 2. Naturally-occurring amino acids may be divided into classes based on common side chain properties:

1) hydrophobic: norleucine, Met, Ala, VaI, Leu, He;

2) neutral hydrophilic: Cys, Ser, Thr, Asn, GIn;

3) acidic: Asp, GIu; 4) basic: His, Lys, Arg;

5) residues that influence chain orientation: GIy, Pro; and

6) aromatic: Trp, Tyr, Phe.

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

Non-conservative substitutions may involve the exchange of a member of one of the above classes for a member from another class. Such substituted residues may 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 may 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: isoleucine (+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); proline (-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, for example, Kyte et ah, 1982, J. MoI, Biol, 157:105-131). It is known that certain amino acids may 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 are within ±2 is included. In some aspects, those which are within ±1 are included, and in other aspects, those within ±0.5 are 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 (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); 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 are within ±2 is included, in other embodiments, those which are within ±1 are included, and in still other embodiments, those within ±0.5 are included. In some instances, one may 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

Amino Acid Substitutions

A skilled artisan will be able to determine suitable variants of the polypeptide chains as set forth herein using well-known techniques. One skilled in the art may identify suitable areas of the molecule that may 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 may be important for biological activity or for structure may 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 are 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 may opt for chemically similar amino acid substitutions for such predicted important amino acid residues.

One skilled in the art can also analyze the three-dimensional structure and amino acid sequence in relation to that structure in similar polypeptides. In view of such information, one skilled in the art may predict the alignment of amino acid residues of an antibody with respect to its three dimensional structure. One skilled in the art may choose not to make radical changes to amino acid residues predicted to be on the surface of the protein, since such residues may be involved in important interactions with other molecules. Moreover, one skilled in the art may generate test variants containing a single amino acid substitution at each desired amino acid residue. These variants can then be screened using assays for glucagon activity such as described herein, (see examples below) 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, Curr. Op. in Biotech. 7:422-427; Chou et al,

1974, Biochemistry 13:222-245; Chou et al, 1974, Biochemistry 1 13:211-222; Chou et al, 1978, Adv. Enzymol Relat. Areas MoI Biol 47:45-148; Chou et al, 1979, Ann. Rev. Biochem. 47:251-276; and Chou et al, 1979, Biophys. J. 26:367-384. Moreover, computer programs are 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% often have similar structural topologies. The recent 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 al, 1999, Nucl. Acid. Res. 27:244-247. It has been suggested (Brenner et al, 1997, Curr. 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, Curr. Opin. Struct. Biol. 7:377-87; Sippl et al, 1996, Structure 4:15-19), "profile analysis" (Bowie et al., 1991, Science 253:164-170; Gribskov et al, 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 of the invention, amino acid substitutions are made that: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter ligand 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) may be made in the naturally- occurring sequence. Substitutions can be made in that portion of the antibody that lies outside the domain(s) forming intermolecular 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 antibody). 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.

Antibody variants used in some compositions can include antibodies comprising a modified Fc fragment or a modified heavy chain constant region. An Fc fragment, which stands for "fragment that crystallizes," or a heavy chain constant region can be modified by mutation to confer on an antibody altered characteristics. See, for example, Burton and Woof, 1992, Advances in Immunology 5V. 1-84; Ravetch and Holland, 2001, Annu. Rev. Immunol. 19: 275-90; Shields et al, 2001, Journal of Biol Chem. 276: 6591-6604; Telleman and Junghans, 2000, Immunology 100: 245-251 ; Medesan et al., 1998, Eur. J. Immunol. 28: 2092-2100; all of which are incorporated herein by reference). Such mutations can include substitutions, additions, deletions, or any combination thereof, and are typically produced by site-directed mutagenesis using one or more mutagenic oligonucleotide(s) according to methods described herein, as well as according to methods known in the art (see, for example, Maniatis et al., MOLECULAR CLONING: A LABORATORY MANUAL, 3rd Ed., 2001, Cold Spring Harbor, N.Y. and Berger and Kimmel, METHODS IN ENZYMOLOGY, Volume 152, Guide to Molecular Cloning Techniques, 1987, Academic Press, Inc., San Diego, CA., which are incorporated herein by reference).

The antibodies in some compositions disclosed herein encompass glycosylation variants of the antibodies disclosed herein 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-linked glycosylation site is characterized by the sequence: Asn-X-Ser or Asn-X-Thr, wherein the amino acid residue designated as X may 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-linked 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-linked glycosylation site in the Fc region.

Additional preferred antibody variants include cysteine variants wherein one or more cysteine residues in the parent or native amino acid sequence are 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 may 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 to glucagon (e.g., human glucagon). For example, one or more of the

CDRs listed in Table 2 can be incorporated into a molecule (e.g., a polypeptide) covalently or noncovalently to make an immunoadhesin. An immunoadhesin may incorporate the CDR(s) as part of a larger polypeptide chain, may covalently link the CDR(s) to another polypeptide chain, or may incorporate the CDR(s) noncovalently. The

CDR(s) enable the immunoadhesin to bind specifically to a particular antigen of interest

(e.g., glucagon or an epitope thereof).

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. Fauchere,

1986, Adv. Drug Res. 15: 29; Veber and Freidinger, 1985, TINS p.392; and Evans et al,

1987, J. Med. Chem. 30: 1229, which are incorporated herein by reference for any purpose. Peptide mimetics that are structurally similar to therapeutically useful peptides may be used to produce a similar therapeutic or prophylactic effect. Such compounds are often developed with the aid of computerized molecular modeling. Generally, peptidomimetics of the invention are proteins that are structurally similar to an antibody displaying a desired biological activity, 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) may be used in certain embodiments of the invention to generate more stable proteins. In addition, constrained peptides comprising a consensus sequence or a substantially identical consensus sequence variation may be generated by methods known in the art (Rizo and Gierasch, 1992, Ann. Rev. Biochem. 61 : 387), incorporated herein by reference), for example, by adding internal cysteine residues capable of forming intramolecular disulfide bridges which cyclize the peptide.

Oligomers that contain one or more anti-glucagon antibody polypeptides may be used in some compositions. Oligomers may be in the form of covalently-linked or non- covalently-linked dimers, trimers, or higher oligomers. Oligomers comprising two or more anti- glucagon antibody polypeptides are contemplated for use, with one example being a homodimer. Other oligomers include heterodimers, homotrimers, heterotrimers, homotetramers, heterotetramers, etc.

B. GLP-I Compounds

A variety of GLP-I compounds can be linked to the anti-glucagon antibody of the composition, including GLP-I itself and a wide variety of GLP-I analogs.

As used herein, the term "GLP-I" refers to glucagon-like peptide 1 as described in the Background above. The carboxyl terminus of GLP-I (1-3I)-OH can be cleaved to produce GLP-I (1-3O)-NH₂. As discussed above, both GLP-I (1-3I)-OH, also referred to as GLP-I (1-31), and GLP-I (l-30)-NH_2> have the same activities. For convenience, the terms "GLP-I" and "native GLP-I" are used to refer to both of these biologically active forms. As discussed above, there are two different numbering conventions used in the art. The numbering convention adopted herein is the one in which the N-terminal histidine of GLP-I is considered as residue number one. Thus, native GLP-I (i.e., GLP- 1(1-3 I)-OH) has the following amino acid sequence:

¹ His-²Ala-³Glu-⁴Gly-⁵Thr-⁶Phe-⁷Thr-⁸Ser-⁹Asp- ' ⁰VaI- ' ' Ser- ' ²Ser- ' ³Tyr- ' ⁴Leu- ¹⁵Glu-¹⁶Gly-¹V_Jln-¹⁸Ala-¹⁹Ala-²Vs-²¹Glu-²¥he-²³Ile-²⁴Ala-²⁵Tφ-²⁶Leu-²Val- ²⁸Lys-²⁹Gly-³⁰Arg-³¹Gly (SEQ ID NO: 1). The amino acids located between the N-terminus and C-terminus are numbered consecutively as shown. Thus, for example, the amino acid at position 2 is Ala and the amino acid at position 20 is Lys. Likewise, when reference is made herein to making a substitution at a specified position, the same numbering system applies. Hence, for example, a substitution of Ala at position 16 means that the GIy at position 16 has been substituted with Ala. If amino acids are added at the amino terminus of GLP-I (1-31), the positions are consecutively numbered in decreasing order, such that the amino acid immediately upstream of position 1 is amino acid -1, and the next upstream amino acid is at position -2 and so on. If amino acids are added at the carboxyl terminus of GLP-I, the positions are consecutively numbered in increasing order, such that the amino acid immediately downstream of position 31 is amino acid 32, and the next downstream amino acid is at position 33, and so on. Alterations to the native GLP-I sequence are indicated in parentheses and have the form: x PositionNo y, where x is the amino acid at the indicated position number in the native GLP-I sequence and y is the amino acid substituted at this position. Thus, for instance, A2G, means that the alanine at position 2 of the native GLP-I sequence has been substituted with glycine. Multiple substitutions are separated by a forward slash (/). Amino acids added to the C-terminus are indicated with a plus sign (+) followed by the location of the addition.

A "GLP-I compound" as used herein refers to a molecule that comprises a GLP-I peptide and may include one or more additional components (e.g., a component that extends the half-life of the compound in vivo).

The term "GLP-I peptide" as used herein refers to native GLP-I or a peptide with one or more alterations in the amino acid sequence of native GLP-I (1-3I)-OH or GLP-I (l-30)-NH2 but that retains at least one activity of native GLP-I . The term also includes members of the exendin family such as exendin-3 and exendin-4 (see, e.g., U.S. Patent No. 5,424,286) or peptides with one or more alterations in the amino acid sequence of the exendin, provided the peptide retains at least one GLP-I activity. Exendin-4, for example, has the following amino acid sequence:

His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala- Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro- Ser (SEQ ID NO: 127). Exendin-3 has the following amino acid sequence:

His Ser Asp GIy Thr Phe Thr Ser Asp Leu Ser Lys GIn Met GIu GIu GIu Ala VaI Arg Leu Phe He GIu Tip Leu Lys Asn GIy GIy Pro Ser Ser GIy Ala Pro Pro Pro Ser

The phrase "GLP-I activity" or grammatical equivalents thereof refers broadly to any activity associated with GLP-I and the exendins. Examples of such activities include, but are not limited to, insulinotropic activity, inhibition of gastric motility, inhibition of gastric secretion, promotion of β-cell proliferation and replication, increase in β-cell mass, increase in satiety and decrease in food intake when administered to a subject. The term "GLP-I peptide" also includes variants, fragments and derivatives of the

GLP-I peptides that are functional equivalents to one of the GLP-I peptides that is disclosed herein in that the variant, fragment or derivative has a similar amino acid sequence (e.g. comprising conservative substitutions) and retains, to some extent, at least one activity of the GLP-I peptide. "GLP-I variants" include peptides that are "substantially identical" (see definition supra) to the GLP-I peptides described herein. Such variants include proteins having amino acid alterations such as deletions, insertions and/or substitutions. Typically, such alterations are conservative in nature (see, e.g., Creighton, 1984, Proteins, W. H. Freeman and Company) such that the activity of the variant protein is substantially similar to one of the GLP-I peptides that are disclosed herein. In the case of substitutions, the amino acid replacing another amino acid usually has similar structural and/or chemical properties. A GLP-I variant can have at least 60%, 70%, or 75%, preferably at least 85%, more preferably at least 90%, 95%, 96%, 97%, 98%, or 99% amino acid identity with a GLP-I peptide as described herein, provided the variant still has a GLP-I activity. A "GLP-I derivative" as used herein refers to one of the GLP-I peptides in which one or more amino acids has been: 1) substituted with the corresponding D-amino acid, 2) altered to a non-naturally occurring amino acid residue, and/or 3) chemically modified. Examples of chemical modification include, but are not limited to alkylation, acylation, deamidation, esterification, phosphorylation, and glycosylation of the peptide backbone and/or amino acid side chains. A "GLP-I fragment" refers to truncated forms of the GLP-I peptides listed herein or variants or derivatives thereof. The fragments typically are truncated by 1, 2, 3, 4 or 5 amino acids relative to the GLP-I peptides set forth herein. Truncation can be at either the amino and/or carboxyl terminus. Numerous examples of GLP-I peptides that are suitable for use in certain compositions are described, for example, in U.S. Patent Nos. 6,329,336; 6,703,365;

5,705,483; 5,977,071; 6,133,235; 6,410,513; 6,388,053; 6,358,924; 5,512,549; 6,006,753;

5,545,618; 5,118,666; 5,120,712; 5,614,492; 5,958,909; 6,162,907; 6,849,708; 6,828,303;

6,284,727; 6,344,180; 6,506,724; 6,858,576; 6,884,579; 6,528,486; 5,846,937; 5,990,077; 6,770,620; 6,620,910; 5,545,618; 6,569,832; and 6,268,343, each of which is incorporated herein by reference in its entirety. Other GLP-I peptides that can be linked to the anti-glucagon antibody in certain compositions are disclosed, for example, in the following published U.S. patent applications: US 2004/0053370; US 2004/0127399;

US 2003/0221201 ; US 2003/0226155; US 2004/0023334; US 2004/0143104; US 2005/0107318; US 2004/0106547; US 2004/0176307; US 2004/0052862;

US 2004/0082507; US 2004/0146985; US 2004/0053370; US 2003/0199672; and

US 2001/001 1071, each of which is incorporated herein by reference in its entirety. Still other GLP-I peptides that can be used in certain compositions are described for example in the following published PCT applications: WO 00/34331 ; WO 0034332; WO 02/46227; WO 03/060071 ; WO 2005/003296; WO 03/018516; WO 01/98331 ;

WO 03/059934; WO 2004/078777; WO 99/30731 ; WO 98/43658; WO 00/16797;

WO 00/15224; WO 03/103572; WO 03/087139; WO 2004/110472; WO 03/018516;

WO 2005/000892; WO 03/028626; WO 2004/020404; WO 2004/020405;

WO 2004/019872; WO 03/020746; WO 2004/094461 ; WO 91/1 1457; WO 87/06941 ; WO 90/11296; WO 00/34332; WO 2004/093823; WO 03/040309; WO 2004/022004;

WO 99/64061 ; WO 03/011892; WO 2004/029081 ; WO 2004/005342; WO 90/01540;

WO 02/22151 ; WO 99/43341 ; WO 96/29342; WO 98/08871 ; WO 99/43705;

WO 99/43706; WO 99/43707; WO 99/43708; WO 2004/105781 ; WO 2004/105790;

WO 2005/027978; WO 04/074315; WO 2005/028516; and WO 2005/046716. Additional GLP-I peptides that can be used in some compositions are described in European Patent Nos. 0 733 644; 1 364 967; 0 699,686; 0 619 322; 1 083 924; 0 512 042; and 1 061 946, each of which is incorporated herein by reference in its entirety.

Certain GLP-I peptides that are in some compositions comprise the amino acid sequence of formula I (SEQ ID NO: 92): Xaai -Xaa₂-Xaa₃-Xaa₄-Xaa₅-Xaa₆-Xaa₇-Xaa₈-Xaa₉-Xaai o-Xaai i -Xaai _2-Xaai ₃-

Xaai₄-Xaai₅-Xaai₆-Xaai₇-Xaai₈-Xaai₉-Xaa₂o-Xaa₂i-Xaa₂₂-Xaa₂₃-Xaa_24-Xaa₂₅-

Xaa26-Xaa2_7-Xaa28-Xaa29-Xaa₃₀ -Xaa₃i- Xaa₃2-Xaa₃₃-Xaa₃4-Xaa35-Xaa36-Xaa3₇-

C(O)-R, (Formula I, SEQ ID NO:92) wherein, R, is OR₂ or NR₂R₃;

R₂ and R₃ are independently hydrogen or (Ci-Cg)alkyl;

Xaa at position 1 is: L-histidine, D-histidine, desamino-histidine, 2-amino- histidine, 3-hydroxy-histidine, homohistidine, α-fluoromethyl-histidine or α- methyl-histidine; Xaa at position 2 is GIy, bAla (2-aminopropionic acid), Asp, Ala, 1-amino- cylcopentanecarboxylic acid, 2-aminoisobutryic acid or alpha-alpha-disubstituted amino acids;

Xaa at position 3 is GIu, Asp, or Lys;

Xaa at position 4 is GIy, Thr or His; Xaa at position 5 is Thr, Ala, GIy, Ser, Leu, He, VaI, GIu, Asp, or Lys;

Xaa at position 6 is: His, Trp, Phe, or Tyr;

Xaa at position 7 is Thr or GIy;

Xaa at position 8 is Ser, Ala, GIy, Thr, Leu, He, VaI, GIu, Asp, or Lys;

Xaa at position 9 is Asp, Asn or GIu; Xaa at position 10 is VaI, Ala, GIy, Ser, Thr, Leu, He, Tyr, GIu, Asp, Trp, or Lys;

Xaa at position 11 is Ser, Ala, GIy, Thr, Leu, He, VaI, GIu, Asp, or Lys;

Xaa at position 12 is Ser, Ala, GIy, Thr, Leu, He, VaI, GIu, Asp, Trp, Tyr, Asn,

Lys, Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-

Homoglutamic acid, or homoglutamic acid; Xaa at position 13 is Tyr, Phe, Tip, GIu, Asp, GIn, Lys, Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, or homoglutamic acid;

Xaa at position 14 is Leu, Ala, GIy, Ser, Thr, He, VaI, GIu, Asp, Met, Trp, Tyr, Asn, GIn, Lys, Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, or homoglutamic acid;

Xaa at position 15 is GIu, Asp, Lys, Homolysine, Ornithine, 4-carboxy- phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, or homoglutamic acid; Xaa at position 16 is GIy, Ala, Ser, Thr, Leu, He, VaI, GIu, Asp, Asn, Lys,

Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta- Homoglutamic acid, or homoglutamic acid;

Xaa at position 17 is GIn, Asn, Arg, GIu, Asp, Lys, Ornithine, 4-carboxy- phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, or homoglutamic acid;

Xaa at position 18 is Ala, GIy, Ser, Thr, Leu, He, VaI, Arg, GIu, Asp, Asn, Lys, Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta- Homoglutamic acid, or homoglutamic acid; Xaa at position 19 is Ala, GIy, Ser, Thr, Leu, He, VaI, GIu, Asp, Asn, Lys, Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-

Homoglutamic acid, or homoglutamic acid;

Xaa at position 20 is Lys, Homolysine, Arg, GIn, GIu, Asp, Thr, His, Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, or homoglutamic acid; Xaa at position 21 is Leu, GIu, Asp, Thr, Lys, Homolysine, Ornithine, 4-carboxy- phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, or homoglutamic acid;

Xaa at position 22 is Phe, Trp, Asp, GIu, Lys, Homolysine, Ornithine, 4-carboxy- phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, or homoglutamic acid; Xaa at position 23 is He, Leu, VaI, Ala, Phe, Asp, GIu, Lys, Homolysine,

Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, or homoglutamic acid;

Xaa at position 24 is Ala, GIy, Ser, Thr, Leu, He, VaI, GIu, Asp, Lys, Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, or homoglutamic acid;

Xaa at position 25 is Trp, Phe, Tyr, GIu, Asp, Asn, or Lys;

Xaa at position 26 is Leu, GIy, Ala, Ser, Thr, Ue, VaI, GIu, Asp, or Lys;

Xaa at position 27 is VaI, GIy, Ala, Ser, Thr, Leu, He, GIu, Asp, Asn, or Lys; Xaa at position 28 is Asn, Lys, Arg, GIu, Asp, or His;

Xaa at position 29 is GIy, Ala, Ser, Thr, Leu, He, VaI, GIu, Asp, or Lys;

Xaa at position 30 is GIy, Arg, Lys, GIu, Asp, Thr, Asn, or His;

Xaa at position 31 is Pro, GIy, Ala, Ser, Thr, Leu, He, VaI, GIu, Asp, or Lys;

Xaa at position 32 is Thr, GIy, Asn, Ser, Lys, or is omitted; Xaa at position 33 is GIy, Asn, Ala, Ser, Thr, He, VaI, Leu, Phe, Pro, or is omitted;

Xaa at position 34 is GIy, Thr, or is omitted;

Xaa at position 35 is Thr, Asn, GIy or is omitted;

Xaa at position 36 is GIy or is omitted; Xaa at position 37 is GIy or is omitted; provided that when the amino acid at position 32, 33, 34, 35, 36 or 37 is omitted, then each amino acid downstream of that amino acid is also omitted, and wherein the compound has a GLP-I activity. Thus, for example, if the amino acid at position 32 is omitted, then there are also no amino acids at positions 33-37. Similarly, if the amino acid at position 33 is omitted, there there are also no amino acids at positions 34-37. And if the amino acid at position 34 is omitted, then there is no amino acid at position 35-37, and so on.

In certain compositions, the GLP-I peptide comprises the amino acid sequence of any of SEQ ID NO: 1-35, or SEQ ID NO: 126 as shown in Table 6 below, or exendin-3 or exendin-4 (SEQ ID NO: 127). The GLP-I peptide in some other compositions comprises SEQ ID NO: 1-35, SEQ ID NO: 126, or SEQ ID NO: 127 with no more than 1, 2, 3, 4 or 5 conservative amino acid substitutions, provided that the variant has a GLP- 1 activity (e.g., insulinotropic activity). In still other compositions, the GLP-I peptide has at least 60%, 70%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO: 1- 35, SEQ ID NO: 126, or SEQ ID NO: 127.

Table 6

C. Linking Antibody and GLP-I Compound

As noted above, when the term "linked" is used in reference to the anti-glucagon antibody and the GLP-I compound, these two molecules may or may not be joined by a linker. In certain embodiments, if a linker is used to serve as a spacer between the antibody and compound, a variety of different chemical structures can be used. For instance, in certain embodiments, a linker comprises amino acid residues linked together by peptide bonds, i.e., a linker comprises a peptide. Thus, in certain embodiments, a linker is a peptide having between 1 and 20 amino acids residues, including all numbers between those endpoints. The amino acid residues used in linkers may be conventional or unconventional amino acid residues. In certain embodiments, amino acid residues in a linker may be glycosylated and/or derivatized in another manner. In certain embodiments, the amino acid residues in a linker are selected from glycine, alanine, proline, asparagine, glutamine, and lysine. In certain embodiments, a linker comprises a majority of amino acid residues that are sterically unhindered, such as glycine and/or alanine. Thus, in certain embodiments, a linker is selected from a polyglycine (e.g.,

(GIy)₄, (GIy)₅), a poly(Gly-Ala), and a polyalanine. Certain exemplary linkers include, but are not limited to: (GIy)₃LyS(GIy)₄ (SEQ ID NO:);

(GIy)₃ASnGIySeT(GIy)₂ (SEQ ID NO:); (GIy)₃CyS(GIy)₄ (SEQ ID NO:); GlyProAsnGlyGly (SEQ ID NO:); and GlyGlyGlyAlaPro (SEQ ID NO:).

To explain the above nomenclature, for example, (Gly)₃Lys(Gly)₄ means GIy- Gly-Gly-Lys-Gly-Gly-Gly-Gly. In certain embodiments, a linker comprises a combination of GIy and Ala residues. In certain embodiments, a linker comprises 10 or fewer amino acid residues. In certain embodiments, a linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues. In certain embodiments, a linker comprises 11-30 amino acid residues, including all numbers between those endpoints.

Additional examples of specific linkers that can be used in the compositions as provided herein include GSGSATGGSGSTASSGSGSATGGGGGG (SEQ ID NO: 36); GSGGGGSGGGGSGGGGSGGGGSGGGGG (SEQ ID NO: 37); and SGGGGSGGGGSGGGGSGGGGSGGGGG (SEQ ID NO: 38)

In certain embodiments, a peptide linker may result from the restriction enzyme sites used to clone two polypeptides into a single coding sequence. In certain embodiments, the restriction enzyme sites are added to the coding sequence of one or both of the polypeptides. In certain embodiments, the amino acid sequence of such linkers is dictated, at least in part, by the restriction enzyme sites selected for the cloning procedures.

In certain embodiments, non-peptide linkers are provided. Certain exemplary non-peptide linkers include, but are not limited to, alkyl linkers such as -NH-(CH₂V C(O)-, wherein s = 2-20. Such alkyl linkers may, in certain embodiments, further comprise substitutions including, but not limited to, non-sterically hindering group such as lower alkyl (e.g., Ci-C₆) lower acyl, halogen (e.g., Cl, Br), CN, NH₂, phenyl, etc. A non-limiting exemplary non-peptide linker is a PEG linker,

wherein n is a number such that the linker has a molecular weight of 100 to 5000 kD. In certain embodiments, n is a number such that the linker has a molecular weight of 100 to 500 kD, including all points between those endpoints.

In certain embodiments, a linker may result from a chemical and/or enzymatic process used to connect two polypeptides to one another. Certain exemplary chemical and/or enzymatic processes for connecting polypeptides are described, e.g., in the Pierce

Applications Handbook and Catalog (2003/2004) (Pierce Biotechnology, Inc., Rockford,

IL). D. Specific Examples of GLP-I Compound/Antibody Compositions

Some specific examples of compositions that are provided are ones in which the antibody comprises one or more of the light chain CDRs (SEQ ID NOs:76-78) and/or one or more of the heavy chain CDRs of AGl 59 (SEQ ID Nos:84-86), with the light chain variable region and/or the heavy chain variable region linked (e.g., fused) to a GLP-I peptide having the amino acid sequence of SEQ ID NO: 1-35, SEQ ID NO: 126, or SEQ ID NO: 127 or any of the other GLP-I peptides disclosed herein. In other compositions, the antibody comprises a light chain variable region and/or heavy chain variable region (SEQ ID NO:79 and 83, respectively) of AG159, with the light chain variable region and/or the heavy chain variable region fused to a GLP-I peptide having the amino acid sequence of SEQ ID NO: 1-35, SEQ ID NO: 126, or SEQ ID NO: 127, or any of the other GLP-I peptides disclosed herein. In still other compositions, the antibody comprises the mature heavy chain (SEQ ID NO: 82 or 89) and/or mature light chain (SEQ ID NO:40) of AG 159, with the light chain variable region and/or the heavy chain variable region fused to a GLP-I peptide having the amino acid sequence of SEQ ID NO:l-35, SEQ ID NO: 126, or SEQ ID NO: 127, or any of the other GLP-I peptides disclosed herein.

As already described at length above, these GLP-I peptides can be linked to the AG 159 antibody or fragments in a variety of different ways, including, for example, such that multiple GLP-I peptides (same or different from one another) are attached in varying numbers and locations to the antibody.

Certain exemplary compositions that are provided are listed in Table 6. It should be understood that these particular compositions are provided simply to illustrate specific examples of the general compositions described herein and that the compositions are not limited to these particular forms. The first column of the Table 6 indicates the general structure of the composition. In general, but not always, the shorthand form adopted here is LC:HC, with the light chain form listed before the colon and the heavy chain form listed after the colon. As indicated above, alterations to the native GLP-I sequence are indicated in parentheses and have the form: x PositionNo y, where x is the amino acid at the indicated position number in the native GLP-I sequence and y is the amino acid substituted at this position. Multiple substitutions are separated by a forward slash (/). Amino acids added to the C-terminus are indicated with a plus sign (+) followed by the location of the addition. If the GLP-I peptide is attached to the LC, this is indicated as GLP-AGl 59LC:AG15, with the GLP-I peptide being listed to the left of the colon. If the GLP-I peptide is attached to the HC, this is indicated as AG159LC:GLP-AG159, i.e., with the GLP-I peptide being listed to the right of the colon. The GLP-I peptide and the light or heavy chain of the AGl 59 antibody are fused together via a linker (e.g. SEQ ID Nos: 36-38).

In these specific compositions, the AGl 59 antibody is shown to be of either the IgGl or IgG2 isotype, but could be of any of the other immunoglobulin isotypes. In these fusions, the carboxy terminus of the GLP-I peptide is fused to the amino terminus of the AGl 59 antibody via a linker (e.g., SEQ ID Nos:36-38). However, as noted above, the GLP-I peptides can be attached at other locations and in other orientations.

Certain compositions comprise a light chain polypeptide fusion having the amino acid sequence of SEQ ID NO:41-74 or SEQ ID NO:129. In certain compositions the light chain polypeptide fusion is paired with a heavy chain of AGl 59 (SEQ ID NO: 82 or 89). Some compositions contain two identical pairs of the light chain fusion of SEQ ID NO:41-74 and two identical pairs of the heavy chain of AGl 59 to form an antibody with tetrameric structure.

Other compositions are similar except that the GLP-I peptide (e.g., SEQ ID NO: 1-35, SEQ ID NO: 126, or SEQ ID NO: 127) is fused to the heavy chain polypeptide of AGl 59 (SEQ ID NO: 82 or 89) instead of the light chain. Such heavy chain polypeptide fusions can be paired with the light chain of AGl 59 (SEQ ID NO:40 or 75), and some compositions contain two identical pairs of the heavy chain fusion and two identical pairs of the light chain of AGl 59 to form an antibody with tetrameric structure.

As described in detail above, the GLP-I compound/antibody compositions provided herein include those in which the antibody and/or the GLP-I peptide are variants of those listed in the tables herein. The alterations can be in the antibody and/or the GLP-I peptide. Thus, for example, certain compositions include a polypeptide chain that has at least 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% identity to the amino acid sequences of the chains listed in Tables 1-4 or 7. Other compositions include a polypeptide from Table 7, with no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, which typically are conservative substitutions as described above. In another embodiment, polypeptides are provided that comprise an amino acid sequence as set forth in any one of SEQ ID NOS: 41-74 or SEQ ID NO: 128 or 129.

Table 7

GLP(A2G/+G32/ HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRGGSGNGTSGGGGSGGGGSGGGGSGGGGS 74

+S33/+G34/+N3 GGGGGEIVLTQSPATLSLSPGDRATLSCRASQSVSSYLAWYQQKPGQAPRLLISDASN

5/+G36/+T36) / RATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWITFGQGTRLEIKRTVAA

-AG159 PSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD

LC: AGl59 IgG2 STYSLSSTLTLSKADYEKHKVΎACEVTHQGLSSPVTKSFNRGEC

E. Optional Components

In some compositions, the GLP-I compound and/or the anti-glucagon antibody are modified to include additional components. For instance, the GLP-I compound or the antibody may be linked to one or more water-soluble polymers. Suitable water- soluble polymers or mixtures thereof include, but are not limited to, N-linked or O-linked carbohydrates, sugars (e.g. various polysaccharides such as chitosan, xanthan gum, cellulose and its derivatives, acacia gum, karaya gum, guar gum, carrageenan, and agarose), phosphates, polyethylene glycol (PEG) (including the forms of PEG that have been used to derivatize proteins, including mono-(Ci-Cio), alkoxy-, or aryloxy- polyethylene glycol), monomethoxy-polyethylene glycol, dextran (such as low molecular weight dextran of, for example, about 6 kD), cellulose, or other carbohydrate based polymers, poly-(N-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyoxyethylene-polyoxypropylene, polyvinyl alcohol, and copolymers of the foregoing.

In certain compositions, the GLP-I compound is complexed with suitable divalent metal cations. Divalent metal complexes of GLP-I compounds can be administered subcutaneously as suspensions, and have a decreased rate of release in vivo, because such complexes of GLP-I compounds are generally insoluble in aqueous solutions of about physiological pH. Non-limiting examples of divalent metal cations suitable for complexing with a GLP-I compound include Zn⁺⁺, Mn⁺⁺, Fe⁺⁺, Ca⁺⁺, Co^, Cd⁺⁺, Ni⁺⁺, and the like. Divalent metal complexes of GLP-I compounds can be obtained, for example, using techniques as described in WO 01/98331, which is incorporated herein by reference. IV. Nucleic Acids

Nucleic acids that encode one or both chains of an antibody or the fusion of a GLP-I peptide and a chain of an anti-glucagon antibody as described herein are also provided, as well as nucleic acids encoding a fragment, derivative, mutein, or variant of such antibodies or fusions. Also provided are polynucleotides sufficient for use as hybridization probes, PCR primers or sequencing primers for identifying, analyzing, mutating or amplifying a polynucleotide encoding a polypeptide or antibody chain. The nucleic acids can be any length. They can be, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1,000, 1,500, 3,000, 5,000 or more nucleotides in length, and/or can comprise one or more additional sequences, for example, regulatory sequences, and/or be part of a larger nucleic acid, for example, a vector. The nucleic acids can be single-stranded or double-stranded and can comprise RNA and/or DNA nucleotides, and artificial variants thereof (e.g., peptide nucleic acids). DNA encoding antibody polypeptides (e.g., heavy or light chain, variable domain only, or full length) may be isolated from B-cells of mice that have been immunized with glucagon or an immunogenic fragment thereof. The DNA may be isolated by conventional procedures such as polymerase chain reaction (PCR). Phage display is another example of a known technique whereby derivatives of antibodies may be prepared. In one approach, polypeptides that are components of an antibody of interest are expressed in any suitable recombinant expression system, and the expressed polypeptides are allowed to assemble to form antibody molecules.

In another aspect, vectors comprising a nucleic acid encoding a polypeptide of the invention or a portion thereof (e.g., a fragment containing one or more CDRs or one or more variable region domains) are provided. Examples of vectors include, but are not limited to, plasmids, viral vectors, non-episomal mammalian vectors and expression vectors, for example, recombinant expression vectors. The recombinant expression vectors of the invention can comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell. The recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operably linked to the nucleic acid sequence to be expressed. Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells (e.g., SV40 early gene enhancer, Rous sarcoma virus promoter and cytomegalovirus promoter), those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences, see Voss el ah, 1986, Trends Biochem. Sci. 1 1 :287, Maniatis et ah, 1987, Science 236:1237, incorporated by reference herein in their entireties), and those that direct inducible expression of a nucleotide sequence in response to particular treatment or condition {e.g., the metallothionin promoter in mammalian cells and the tet-responsive and/or streptomycin responsive promoter in both prokaryotic and eukaryotic systems (see id.). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein. In another aspect, the present invention provides host cells into which a recombinant expression vector of the invention has been introduced. A host cell can be any prokaryotic cell (for example, E. colϊ) or eukaryotic cell (for example, yeast, insect, or mammalian cells {e.g., CHO cells)). Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., for resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Preferred selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die), among other methods. V. Preparation of Antibodies

The 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., cynomologous 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 of the invention, the antibodies may be produced by immunizing with human glucagon. The antibodies may be polyclonal, monoclonal, or may be synthesized in host cells by expressing recombinant DNA.

Fully human antibodies may 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.

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 Milstein, 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 cells from immunized mice are 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 may be used. Fusion procedures for making hybridomas also are well known.

The single chain antibodies that are provided may be formed by linking heavy and light chain variable domain (Fv region) fragments (see, e.g., SEQ ID NO:79 and 83) via an amino acid bridge (short peptide linker), resulting in a single polypeptide chain. Such single-chain Fvs (scFvs) may be prepared by fusing DNA encoding a peptide linker between DNAs encoding the two variable domain polypeptides (V_L and V_H). 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 al, 1997, Prot. Eng. 10:423; Kortt et al, 2001, Biomol. Eng. 18:95-108). By combining different V_L and V_H- comprising polypeptides, one can form multimeric scFvs that bind to different epitopes (Kriangkum et al, 2001, Biomol. Eng. 18:31-40). Techniques developed for the production of single chain antibodies include those described in U.S. Patent No. 4,946,778; Bird, 1988, Science 242:423; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879; Ward et al, 1989, Nature 334:544, de Graaf et al, 2002, Methods MoI Biol. 178:379-87.

Antibodies provided herein that are of one subclass can be changed to antibodies from a different subclass using subclass switching methods. Thus, IgG antibodies may 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 may be employed. Cloned DNA encoding particular antibody polypeptides may 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 MoI. Biol.178:303-16. Moreover, if an IgG4 is desired, it may also be desired to introduce a point mutation (CPSCP -> CPPCP) 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 IgG4 antibodies.

Techniques for deriving antibodies having different properties {i.e., varying affinities for the antigen to which they bind) are 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-phenyloxazol-5-one, as described by Marks et al, 1992, BioTechnology, 10:779. Substantial modifications in the functional and/or biochemical characteristics of the antibodies and fragments described herein may 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" may involve a substitution of a native amino acid residue with a normative residue that has little or no effect on the polarity or charge of the amino acid residue at that position. Furthermore, any native residue in the polypeptide may 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 human glucagon.

VI. Expression of Anti-Glucagon Antibodies

The anti-glucagon antibodies can be prepared by any of a number of conventional techniques. For example, anti-glucagon antibodies may be produced by recombinant expression systems, using any technique known in the art. See, for example, Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Kennet 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, NY (1988). Antibodies of the present invention can be expressed in hybridoma cell lines 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 U.S. Pat. Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455 (which patents are hereby incorporated herein by reference for any purpose). 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 are 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 microinjection of the DNA into nuclei. Recombinant expression constructs of the invention typically comprise a nucleic acid molecule encoding a polypeptide comprising one or more of the following: a heavy chain constant region; a heavy chain variable region; a light chain constant region; a light chain variable region; one or more CDRs of the light or heavy chain of the anti-glucagon antibody. The vector is typically selected to be functional in the particular host cell employed (i.e., the vector is compatible with the host cell machinery, permitting amplification and/or expression of the gene can occur). In some embodiments, vectors are used that employ protein-fragment complementation assays using protein reporters, such as dihydro folate reductase (see, for example, U.S. Patent 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 of the invention include those described in Bianchi and McGrew, Biotech Biotechnol Bioeng 84(4):439-44 (2003), which is hereby incorporated by reference. Additional suitable expression vectors are discussed, for example, in Methods Enzymol, vol. 185 (D. V. Goeddel, ed.), 1990, New York: Academic Press, which is hereby incorporated by reference.

Typically, expression vectors used in any of the host cells contain sequences for plasmid or virus maintenance and for cloning and expression of exogenous nucleotide sequences. Such sequences, collectively referred to as "flanking sequences" typically include one or more of the following operatively linked 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 element. Optionally, the vector may contain a "tag"-encoding sequence, that is, an oligonucleotide molecule located at the 5' or 3' end of the coding sequence, the oligonucleotide sequence encoding polyHis (such as hexaHis), or another "tag" for which commercially available antibodies exist, such as FLAG^®, HA (hemaglutinin from influenza virus), or myc. The tag is typically fused to the antibody protein upon expression, and can serve as a means for affinity purification of the antibody 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 antibody polypeptide by various means such as using certain peptidases for cleavage. Flanking sequences in the expression vector may be homologous (i.e., from the same species and/or strain as the host cell), heterologous (i.e., from a species other than the host cell species or strain), hybrid (i.e., a combination of flanking sequences from more than one source), synthetic or native. As such, the source of a flanking sequence may 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 of this invention may 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 may be known. Here, the flanking sequence may be synthesized using the methods described herein for nucleic acid synthesis or cloning.

Where all or only a portion of the flanking sequence is known, it may be obtained using PCR and/or by screening a genomic library with a suitable 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 may be isolated from a larger piece of DNA that may contain, for example, a coding sequence or even another gene or genes. Isolation may be accomplished by restriction endonuclease digestion to produce the proper DNA fragment followed by isolation using agarose gel purification, Qiagen^® 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 those skilled in the art.

An origin of replication is typically a part of prokaryotic expression vectors, particularly those purchased commercially, and the origin aids in the amplification of the vector in a host cell. If the vector of choice does not contain an origin of replication site, one may 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 origins (e.g., SV40, polyoma, adenovirus, vesicular stomatitis virus (VSV), or papillomaviruses such as HPV or BPV) are useful for cloning vectors in mammalian cells. Generally, a mammalian origin of replication is not needed for mammalian expression vectors (for example, the SV40 origin is often used only because it contains the early promoter).

The expression and cloning vectors of the present invention will typically contain a promoter that is recognized by the host organism and operably linked to nucleic acid encoding the anti-glucagon antibody. Promoters are untranscribed sequences located upstream (i.e., 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 the other hand, initiate continuous gene product production; that is, there is little or no experimental control over gene expression. A large number of promoters, recognized by a variety of potential host cells, are well known. A suitable promoter is operably linked to the DNA encoding anti-glucagon antibody by removing the promoter from the source DNA by restriction enzyme digestion or amplifying the promoter by polymerase chain reaction and inserting the desired promoter sequence into the vector.

Suitable promoters for use with yeast hosts are also well known in the art. Yeast enhancers are advantageously used with yeast promoters. Suitable promoters for use with mammalian host cells are well known and include, but are not limited to, those obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as

Adenovirus T), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis-B virus and most preferably Simian Vims 40 (SV40). Other suitable mammalian promoters include heterologous mammalian promoters, for example, heat-shock promoters and the actin promoter.

Particular promoters useful in the practice of the recombinant expression vectors of the invention include, but are not limited to: the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290: 304-10); the CMV promoter; the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, et al, 1980, Cell 22: 787- 97); the herpes thymidine kinase promoter (Wagner et al, 1981, Proc. Natl, Acad. ScL U.S.A. 78: 1444-45); the regulatory sequences of the metallothionine gene (Brinster et al, 1982, Nature 296: 39-42); prokaryotic expression vectors such as the beta-lactamase promoter (Villa-Kamaroff et al, 1978, Proc. Natl. Acad. ScL U.S.A., 75: 3727-31); or the tac promoter (DeBoer et al, 1983, Proc. Natl Acad. ScL U.S.A. 80: 21-25). Also available for use 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: 639-46; 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: 1 15-22); the mouse mammary tumor virus control region that is active in testicular, breast, lymphoid and mast cells (Leder et al, 1986, Cell 45: 485-95); the albumin gene control region that is active in liver (Pinkert et al, 1987, Genes and Devel Jh 268-76); the alpha-feto-protein gene control region that is active in liver (Krumlauf et al, 1985, MoI Cell. Biol. 5: 1639-48; Hammer et al, 1987, Science 235: 53-58); the alpha 1 -antitrypsin gene control region that is active in the liver (Kelsey et al, 1987, Genes and Devel 1 : 161-71); the beta-globin gene control region that is active in myeloid cells (Mogram et al, 1985, Nature 315: 338-40; Kollias et al, 1986, Cell 46: 89-94); the myelin basic protein gene control region that is active in oligodendrocyte cells in the brain (Readhead et al, 1987, Cell 48: 703-12); the myosin light chain-2 gene control region that is active in skeletal muscle (Sani, 1985, Nature 314: 283-86); the gonadotropic releasing hormone gene control region that is active in the hypothalamus (Mason et al, 1986, Science 234: 1372-78); and most particularly the immunoglobulin gene control region that is active in lymphoid cells (Grosschedl et al, 1984, Cell 38: 647-58; Adames et al, 1985, Nature 318: 533-38; Alexander et al, 1987, MoI Cell Biol. 7: 1436-44). An enhancer sequence may be inserted into the vector to increase the transcription in higher eukaryotes of a nucleic acid encoding an anti-glucagon antibody. Enhancers are cis-acting elements of DNA, usually about 10-300 bp in length, that act on promoters to increase transcription. Enhancers are relatively orientation and position independent. They have been found 5' and 3' to the transcription unit. Several enhancer sequences available from mammalian genes are known {e.g., globin, elastase, albumin, alpha-feto- protein and insulin). An enhancer sequence from a virus also can be used. The SV40 enhancer, the cytomegalovirus early promoter enhancer, the polyoma enhancer, and adenovirus enhancers are exemplary enhancing elements for the activation of eukaryotic promoters. While an enhancer may be spliced into the vector at a position 5' or 3' to a nucleic acid molecule, it is typically placed at a site 5' to the promoter.

In expression vectors, a transcription termination sequence is typically located 3' of the end of a polypeptide-coding region and serves to terminate transcription. A transcription termination sequence used for expression in prokaryotic cells typically 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 part of a vector, it can also be readily synthesized using methods for nucleic acid synthesis such as those described herein.

A selectable marker gene element encodes a protein necessary for the survival and growth of a host cell grown in a selective culture medium. Typical selection marker genes used in expression vectors 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 media. Examples of selectable markers include the kanamycin resistance gene, the ampicillin resistance gene and the tetracycline resistance gene. A bacterial neomycin resistance gene can also be used for selection in both prokaryotic and eukaryotic host cells.

Other selection genes can be used to amplify the gene that will be expressed. Amplification is a process whereby genes that cannot in single copy be expressed at high enough levels to permit survival and growth of cells under certain selection conditions are reiterated in tandem within the chromosomes of successive generations of recombinant cells. Examples of suitable amplifiable selectable markers for mammalian cells include dihydrofolate reductase (DHFR) and promoterless thymidine kinase. In the use of these markers mammalian cell transformants are placed under selection pressure wherein only the transformants are uniquely adapted to survive by virtue of the selection 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 permitting survival of only those cells in which the selection gene has been amplified. Under these circumstances, DNA adjacent to the selection gene, such as DNA encoding an antibody of the invention, is co-amplified with the selection gene. As a result, increased quantities of anti-glucagon polypeptide are synthesized from the amplified DNA.

A ribosome-binding site is usually necessary for translation initiation of mRNA and is characterized by a Shine-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, for example where glycosylation is desired in a eukaryotic host cell expression system, various presequences can be manipulated to improve glycosylation or yield. For example, the peptidase cleavage site of a particular signal peptide can be altered, or pro-sequences added, which also may affect glycosylation. The final protein product may 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 may 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 may result in a slightly truncated yet active form of the desired polypeptide, if the enzyme cuts at such area within the mature polypeptide.

Where a commercially available expression vector lacks some of the desired flanking sequences as described above, the vector can be modified by individually ligating these sequences into the vector. After the vector has been chosen and modified as desired, a nucleic acid molecule encoding an anti-glucagon antibody is inserted into the proper site of the vector.

The completed vector containing sequences encoding the inventive antibody or immunologically functional fragment thereof is inserted into a suitable host cell for amplification and/or polypeptide expression. The transformation of an expression vector for an anti-glucagon- 1 antibody into a selected host cell may be accomplished by well- known methods including methods such as transfection, infection, calcium chloride, electroporation, microinjection, lipofection, DEAE-dextran method, 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 are well known to the skilled artisan.

The transformed host cell, when cultured under appropriate conditions, synthesizes an anti-glucagon antibody 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, many immortalized cell lines available from the

American Type Culture Collection (ATCC), such as Chinese hamster ovary (CHO) cells,

HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and a number of other cell lines. In certain embodiments, the best cell line for expressing a particular DNA construct may be selected by testing various cell lines to determine which ones have the highest levels of expression levels and produce antibodies with glucagon binding properties.

VII. Exemplary Therapeutic Utilities

In view of the various activities associated with GLP-I (see Background), the compositions comprising GLP-I compounds that are described herein can be used generally to: 1) stimulate insulin release, 2) reduce blood glucose levels, 3) increase plasma insulin levels, 4) stimulate transcription of β-cell-specific genes (e.g., GLUT-I transporter, insulin receptor and hexokinase-1), 5) increase β-cell mass by inhibiting β- cell apoptosis and increasing β-cell proliferation and replication, 6) induce satiety thereby reducing food intake and promoting weight loss, 7) reduce gastric secretion, 8) delay gastric emptying, and 9) reduce gastric motility.

The compositions comprising GLP-I compounds can thus be used to treat a number of different forms of diabetes or diseases closely related thereto, including but not limited to, diabetes mellitus of Type I or Type II, impaired glucose tolerance, insulin resistance, latent autoimmune diabetes Adult (LADA), gestational diabetes, metabolic syndrome, and maturity-onset diabetes of the young (MODY). Thus, the compositions comprising GLP-I compounds can be used to treat individuals having decreased sensitivity to insulin due to infection, stress, stroke, or due to a decreased sensitivity induced during pregnancy. Other types of diabetes that can be treated are those in which diabetes is linked to another endocrine disease such as glucagonoma, primary aldosteronism, Cushing's syndrome and somatostatinoma, or diabetes that arises due to administration of certain drugs or hormones (e.g., estrogen-containing pharmaceuticals, psychoactive drugs, antihypertensive drugs, and thiazide diuretics).

The compositions comprising GLP-I compounds can also be used to treat various coronary diseases and diseases associated with lipid disorders, including, for instance, hypertension, coronary artery disease, hyperlipidemia, cardiovascular disease, atherosclerosis and hypercholesteremia and myocardial infarction. Bone disorders, osteoporosis and other related diseases can also be treated with the compositions comprising GLP-I compounds. Additional diseases that can be treated with the compositions comprising GLP-I compounds include: obesity, irritable bowel syndrome, stroke, catabolic changes after surgery, myocardial infarction,), and hyperglycemia. The GLP-I compounds can also be used as a sedative. The compositions comprising GLP-I compounds can also be used prophylactically, including treating individuals at risk for developing a disease such as listed above. As a specific example, the compounds can be administered prophylactially to an individual at risk for non-insulin dependent diabetes or becoming obese. Such individuals include, for instance, those that have impaired glucose tolerance, those that are overweight and those with a genetic predisposition to the foregoing diseases (e.g., individuals from families with a history of diabetes).

A variety of different subjects can be treated with the compositions comprising GLP-I compounds. The term "subject" or "patient" as used herein, typically refers to a mammal, and often, but not necessarily, is a human that has or is at risk for one of the foregoing diseases. The subject, however, can also be a non-human primate (e.g., ape, monkey, gorilla, chimpanzee). The subject can also be a mammal other than a primate such as a veterinarian animal (e.g., a horse, bovine, sheep or pig), a domestic animal (e.g., cat or dog) or a laboratory animal (e.g., mouse or rat).

VII. Pharmaceutical Compositions A. Composition

The GLP-I compound/antibody compositions that are provided herein can be used as the active ingredient in pharmaceutical compositions formulated for the treatment of the diseases listed in the section on therapeutic utilities. Thus, the GLP-I /antibody compositions that are disclosed can be used in the preparation of a medicament for use in various therapeutic applications, including those listed supra.

In addition to the GLP-I compound/antibody composition, pharmaceutical compositions can also include one or more other therapeutic agents that are useful in treating one or more of the various disorders for which the GLP-I compounds have utility. General classes of other therapeutic agents that can be combined with certain

GLP-I compound/antibody compositions include, but are not limited to, insulin releasing agents, inhibitors of glucagon secretion, protease inhibitors, glucagon antagonists, anti- obesity agents, compounds that reduce caloric intake, selective estrogen receptor modulators, steroid or non-steroid hormones, growth factors, and dietary nutrients.

Such additional therapeutic agents can include, for instance, agents for treating hyperglycemia, diabetes, hypertension, obesity and bone disorders. Examples of other therapeutic agents for treating diabetes that can be included in the compositions include those used in treating lipid disorders. Specific examples of such agents include, but are not limited to, bile acid sequestrants (e.g., cholestyramine, lipostabil, tetrahydrolipstatin), HMG-CoA reductase inhibitors (see, e.g., U.S. Patent Nos. 4,346,227; 5,354,772; 5,177,080; 5,385,929; and 5,753,675), nicotinic acid, MTP inhibitors (see, e.g., U.S. Patent Nos. 5,595,872; 5,760,246; 5,885,983; and 5,962,440), lipoxygenase inhibitors, fibric acid derivatives, cholesterol absorption inhibitors, squalene synthetase inhibitors (see, e.g., U.S. Patent Nos. 4,871,721 ; 5,712,396; and 4,924,024) and inhibitors of the ileal sodium/bile acid cotransporter. Other anti-diabetic agents that can be incorporated into the compositions include meglitinides, thiazolidinediones, biguanides, insulin secretagogues, insulin sensitizers, glycogen phosphorylase inhibitors, PPAR-alpha agonists, PPAR-gamma agonists.

An inhibitor of dipeptidylpeptidase IV activity can also be included to inhibit cleavage at the N-terminus of the GLP-I analog. In some embodiments, the pharmaceutical compositions comprise an effective amount of one or a plurality of the GLP-I comopund/antibody compositions together with a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative, and/or adjuvant. Preferably, acceptable formulation materials are nontoxic to recipients at the dosages and concentrations employed. In preferred embodiments, pharmaceutical compositions comprising a therapeutically effective amount of the GLP-I compound/antibody composition are provided.

In certain embodiments, acceptable formulation materials preferably are nontoxic to recipients at the dosages and concentrations employed.

In certain embodiments, the pharmaceutical composition may 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-sulfϊte); buffers (such as borate, bicarbonate, Tris- HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum 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 benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, 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 80, 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, 18^th Edition, (A.R. Gennaro, ed.), 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 may influence the physical state, stability, rate of in vivo release and rate of in vivo clearance of the antibodies.

In certain embodiments, the primary vehicle or carrier in a pharmaceutical composition may be either aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier may 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 are further exemplary vehicles. In preferred embodiments, pharmaceutical compositions comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0- 5.5, and may further include sorbitol or a suitable substitute therefor. In certain embodiments, anti- glucagon antibody compositions may 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, the GLP-I /antibody compositions may be formulated as a lyophilizate using appropriate excipients such as sucrose.

The pharmaceutical compositions can be selected for parenteral delivery. Alternatively, the compositions may 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 for use in this invention may be provided in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising the desired composition comprising GLP-I compound in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water in which the GLP-I /antibody composition 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-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads or liposomes, that may provide controlled or sustained release of the product which can be delivered via depot injection. In certain embodiments, hyaluronic acid may also be used, having the effect of promoting sustained duration in the circulation. In certain embodiments, implantable drug delivery devices may be used to introduce the desired GLP-I /antibody composition.

Pharmaceutical compositions can be formulated for inhalation. In these embodiments, pharmaceutical compositions comprising GLP-I compound/antibody compositions are advantageously formulated as a dry, inhalable powder. In preferred embodiments, pharmaceutical compositions may also be formulated with a propellant for aerosol delivery. In certain embodiments, solutions may be nebulized. Pulmonary administration and formulation methods therefore are further described in International Patent Application No. PC17US94/001875, which is incorporated by reference and describes pulmonary delivery of chemically modified proteins.

It is also contemplated that formulations can be administered orally. Pharmaceutical compositions comprising the GLP-I compound/antibody compositions that are 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 may 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 the compositions. Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders may also be employed.

A pharmaceutical composition is preferably provided to comprise an effective quantity of one or a plurality of GLP-I compound/antibody compositions 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 may 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 GLP-I compound/antibody compositions in sustained- or controlled-delivery formulations. Techniques for formulating a variety of other sustained- or controlled-delivery means, such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are 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 may include semipermeable polymer matrices in the form of shaped articles, e.g. films, or microcapsules. Sustained release matrices may 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-glutamic acid and gamma ethyl-L-glutamate (Sidman et al, 1983, Biopolymers 22:547-556), poly (2-hydroxyethyl-methacrylate) (Langer et al, 1981, J. Biomed. Mater. Res. 15: 167-277 and Langer, 1982, Chem. Tech. 12:98-105), ethylene vinyl acetate (Langer et al, supra) or poly-D(-)-3-hydroxybutyric acid (European Patent Application Publication No. EP 133,988). Sustained release compositions may 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. ScL USA 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 lyophilized, sterilization using this method may be conducted either prior to or following lyophilization and reconstitution. Pharmaceutical compositions for parenteral administration can be stored in lyophilized 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 pierceable by a hypodermic injection needle.

Once the pharmaceutical composition has been formulated, it may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, crystal, or as a dehydrated or lyophilized powder. Such formulations may 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. The kits may each contain both a first container having a dried protein and a second container having an aqueous formulation. In certain embodiments of this invention, kits containing single and multi-chambered pre-filled syringes (e.g., liquid syringes and lyosyringes) are provided.

B. Dosage

As noted above, the pharmaceutical compositions can be administered for prophylactic and/or therapeutic treatments. An "effective amount" refers generally to an amount that is a sufficient, but non-toxic, amount of the active ingredient (e.g., the GLP-I compound/antibody composition) to achieve the desired effect, which is a reduction or elimination in the severity and/or frequency of symptoms and/or improvement or remediation of damage. A "therapeutically effective amount" refers to an amount that is sufficient to remedy a disease state or symptoms, or otherwise prevent, hinder, retard or reverse the progression of a disease or any other undeirable symptom. A "prophylactically effective amount" refers to an amount that is effective to prevent, hinder, or retard the onset of a disease state or symptom.

In general, toxicity and therapeutic efficacy of the GLP-I compound/antibody composition can be determined according to standard pharmaceutical procedures in cell cultures and/or experimental animals, including, for example, determining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD_5O/ED₅o. Compositions that exhibit large therapeutic indices are desirable. The data obtained from cell culture and/or animal studies can be used in formulating a range of dosages for humans. The dosage of the active ingredient typically lines within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. The amount of active ingredient administered will depend upon various factors that can be assessed by the attending clinician, such as the severity of the disease, the age and size of the subject to be treated and the particular disease itself. In general, however, the total amount of the GLP-I compound/antibody composition itself that is administered typically ranges from 1 μg/kg body weight/day to 100 mg/kg/day. In some instances, the dosage ranges from 10 μg/kg /day to 10 mg/kg/day. In other treatment regimens, the GLP-I compound/antibody composition is administered at 50 ug/kg/day to 5 mg/kg/day or from 100 ug/kg/day to 1 mg/kg/day.

Dosing frequency will depend upon the pharmacokinetic parameters of the particular composition in the formulation used. Typically, a clinician administers the composition until a dosage is reached that achieves the desired effect. The composition may therefore be administered as a single dose, or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via an implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them. Appropriate dosages may be ascertained through use of appropriate dose-response data. In certain embodiments, the pharmaceutical compositions can be administered to patients throughout an extended time period. Chronic administration of an antibody in a composition minimizes the adverse immune or allergic response commonly associated with antibodies that are 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.

C. Administration

The pharmaceutical compositions described herein can be administered in a variety of different ways. Examples include administering a composition containing a pharmaceutically acceptable carrier via oral, intranasal, rectal, topical, intraperitoneal, intravenous, intramuscular, subcutaneous, subdermal, transdermal, intrathecal, and intracranial methods.

For oral administration, the active ingredient can be administered in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs, syrups, and suspensions. The active component(s) can be encapsulated in gelatin capsules together with inactive ingredients and powdered carriers, such as glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talcum, magnesium carbonate. Examples of additional inactive ingredients that may be added to provide desirable color, taste, stability, buffering capacity, dispersion or other known desirable features are red iron oxide, silica gel, sodium lauryl sulfate, titanium dioxide, and edible white ink. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric-coated for selective disintegration in the gastrointestinal tract. Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance.

The active ingredient, alone or in combination with other suitable components, can be made into aerosol formulations (i.e., they can be "nebulized") to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen.

Suitable formulations for rectal administration include, for example, suppositories, which consist of the packaged active ingredient with a suppository base. Suitable suppository bases include natural or synthetic triglycerides or paraffin hydrocarbons. In addition, it is also possible to use gelatin rectal capsules which consist of a combination of the packaged active ingredient with a base, including, for example, liquid triglycerides, polyethylene glycols, and paraffin hydrocarbons.

Formulations suitable for parenteral administration, such as, for example, by intraarticular (in the joints), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and nonaqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.

The components used to formulate the pharmaceutical compositions are preferably of high purity and are substantially free of potentially harmful contaminants (e.g., at least National Food (NF) grade, generally at least analytical grade, and more typically at least pharmaceutical grade). Moreover, compositions intended for in vivo use are usually sterile. To the extent that a given compound must be synthesized prior to use, the resulting product is typically substantially free of any potentially toxic agents, particularly any endotoxins, which may be present during the synthesis or purification process. Compositions for parental administration are also sterile, substantially isotonic and made under GMP conditions.

EXAMPLES

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

Example 1; Production of Human Monoclonal Antibodies Against Glucagon Fully human monoclonal antibodies to glucagon were prepared by Medarex using strains of transgenic mice, each of which expressed human antibody genes. Methods for preparing such monoclonal antibodies are described in Chen et al. (1993, EMBO J. 12:81 1-820), and in Example 1 of International Patent Application Publication No. WO 01/09187 (incorporated by reference). See also Fishwild et al. (1996, Nature Biotechnology 14:845-851), U.S. Patent Nos. 5,545,806, 5,625,825, and 5,545,807, and Example 2 of International Patent Application Publication No. WO 01/09187 (incorporated by reference).

To generate fully human monoclonal antibodies to glucagon, HuMab mice were immunized with purified recombinant human glucagon. Methods for immunization are described in International Patent Application Publication No. WO 04/035747; Lonberg et al. (1994, Nature 368:856-859; Fishwild et al, supra., and International Patent Application Publication No. WO 98/24884, the teachings of each of which are incorporated by reference).

Mice with sufficient titers of anti-glucagon human immunoglobulin were used to produce monoclonal antibodies in hybridoma cells. Methods for producing such hybridomas are discussed in International Patent Application Publication No. WO 04/035747. The antibody selected from a screen of the hybridomas was designated AGl 59. The antibody was selected in part because it could neutralize glucagon in vitro and in vivo.

Example 2: Cloning of the Anti-GIucagon Antibody Light and Heavy Chains

The hybridoma expressing glucagon binding monoclonal antibody AGl 59 was used as a source to isolate total RNA using TRIzol® reagent (Invitrogen). First strand cDNA was synthesized using a random primer with and extension adaptor (5'- GGC CGG ATA GGC CTC CAN NNN NNT -3'; SEQ ID NO: 101) and a 5' RACE (rapid amplification of cDNA ends) was performed using the GeneRacer™ Kit (Invitrogen).

For preparing complete light chain encoding cDNA, the forward primer was the GeneRacer™ nested primer (5' GGA CAC TGA CAT GGA CTG AAG GAG TA -3'; SEQ ID NO: 102) and a reverse primer designed to recognize a conserved region of the cDNA sequence found in the 3' untranslated region of human kappa chains (5'- GGG GTC AGG CTG GAA CTG AGG -3'; SEQ ID NO: 103).

For preparing variable region heavy chain encoding cDNA, the forward primer was the GeneRacer™ nested primer (5' GGA CAC TGA CAT GGA CTG AAG GAG TA -3'; SEQ ID NO: 104) and a reverse primer designed to recognize a conserved region in the coding sequence in the Fc region of human IgG chains (5'- TGA GGA CGC TGA CCA CAC G -3'; SEQ ID NO: 105).

The RACE products were cloned into pCR4-TOPO and the DNA sequences were determined. Consensus DNA sequences were determined and used to design primers for full-length kappa chain and variable region heavy chain PCR amplification.

A series of primers was used to extend the DNA sequence coding for the mature light chain to include a VK-I signal peptide sequence (MDMRVPAQLL GLLLLWLRGA RC; SEQ ID NO: 106). The first 5' primer encoded the last seven amino acids of the signal peptide and 14 amino acids of the mature light chain (5'- GTG GTT GAG AGG TGC CAG ATG TGA AAT TGT GCT GAC CCA GTC TCC AGC CAC CCT GTC TTT GTC TC-3'; SEQ ID NO: 107) and the 3' reverse primer encoded the carboxyl terminus and termination codon as well as a Sail restriction site (5'- CTT GTC GAC TCA ACA CTC TCC CCT GTT GAA GCT C-3'; SEQ ID NO: 108). The resulting product was further amplified using a 5' primer which encoded 15 amino acids of the signal peptide (5'-CCG CTC AGC TCC TGG GGC TCC TGC TGC TGT GGC TGA GAG GTG CCA GAT-3'; SEQ ID NO: 109) and the same reverse primer as used previously. The final reaction was performed a 5' primer which encoded the amino terminus of the signal sequence, an Xbal restriction endonuclease site and an optimized Kozak sequence (5'- CAG CAG AAG CTT CTA GAC CAC CAT GGA CAT GAG GGT GCC CGC TCA GCT CCT GGG-3'; SEQ ID NO: 1 10) and the same reverse primer. The resulting PCR product was purified, digested with Xbal and Sail, gel isolated and ligated into the mammalian expression vector pDSRal9 (see International Application, Publication No. WO 90/41363, which is herein incorporate by reference for any purpose).

A series of primers was used to extend the DNA sequence coding for the mature heavy chain to include a VK-I signal peptide sequence (MDMRVP AQLL GLLLLWLRGA RC; SEQ ID NO: 106). The first 5' primer encoded the last seven amino acids of the signal peptide and 6 amino acids of the mature heavy chain (5'- GTG GTT GAG AGG TGC CAG ATG TCA GGT GCA GCT GGT GGA G-3'; SEQ ID NO: 11 1) and the 3' reverse primer encoded the carboxyl end of the variable region, including a naturally occurring sense strand BsmBl site (5'- GTG GAG GCA CTA GAG ACG GTG ACC AGG GTT CC-3'; SEQ ID NO: 112). The resulting product was further amplified using a 5' primer which encoded 15 amino acids of the signal peptide (5'-CCG CTC AGC TCC TGG GGC TCC TGC TGC TGT GGC TGA GAG GTG CCA GAT-3'; SEQ ID NO: 1 13) and the same reverse primer as used previously. The final reaction was performed a 5' primer which encoded the amino terminus of the signal sequence, an Xbal restriction endonuclease site and an optimized Kozak sequence (5'- CAG CAG AAG CTT CTA GAC CAC CAT GGA CAT GAG GGT GCC CGC TCA GCT CCT GGG-3'; SEQ ID NO: 1 14) and the previous reverse primer. The resulting PCR product was purified, digested with Xbal and BsmBl, gel isolated and ligated into the mammalian expression vector pDSRal9 containing the human IgGl constant region. Construction of the GLP-I (A2G) AG159antibody chain fusion genes

A DNA sequence encoding the upstream Xbal site, optimized Kozak sequence, GLP-I (A2G) peptide and a linker sequence and part of the AGl 59 LC or HC cDNA containing a unique restriction site were synthesized by Picoscript (Houston, TX) (MDMRVPAQLLGLLLLWLRGARCHGEGTFTSDVSSYLEGQAAKEFIAWLVKGR GGSGSATGGSGSTASSGSGSATGGGGGG; SEQ ID NO: 1 15). Utilizing a naturally occurring unique Kpnl site for the kappa chain, the Xbal-Kpnl fragment from the synthesized gene was used to replace the synonymous fragment in the AGl 59 kappa chain pDSRal9 construct, resulting in the GLP-I (A2G)-AG159 LC fusion gene. Similarly, utilizing a naturally occurring unique Pvull site in the AGl 59 heavy chain DNA sequence, the synthesized GLP-1(A2G) DNA sequence was cut with Xbal and PvwII and used to replace the synonymous fragment in the AGl 59 heavy chain construct to create the GLP- 1(A2G)-AG 159 IgGl heavy chain fusion gene. The isotype of the heavy chain constant region of AGl 59 was switched from

IgGl to IgG2 by replacing the BsmBl-Sall fragment containing the IgGl constant region with the IgG2 constant region that had been amplified by PCR to produce similar restriction sites on the 5' and 3' ends of the gene.

Construction of AGl 59 Kappa Light Chain and GLP-I (A2G)-AG159 Kappa Light Chain Expression Plasmids

The full length AGl 59 LC was amplified by PCR with primers to introduce new restriction sites on either end of the gene for cloning purposes. The 5' primer included a Sail restriction site, a optimized Kozak sequence and the amino terminus of the VK-I signal sequence (5'-AAC CTC GAG GTC GAC TAG ACC ACC ATG GAC ATG AGG GTG CCC GCT-3'; SEQ ID NO: 1 16) while the 3' primer encoded the carboxyl terminus and termination codon, as well as a Notl restriction site (5'-AAC CGT TTA AAC GCG GCC GCT CAA CAC TCT CCC CTG TTG AA-3'; SEQ ID NO: 1 17). The resulting fragment was purified, digested with Sail and Notl and cloned into the expression vectors pDC323 and pDSRot24. The same process was performed utilizing the GLP-1(A2G)- AGl 59 LC construct as a template. Construction of AGl 59 IgGl, AGl 59 IgG2, GLP-I (A2G)-AG 159 IgGl and GLP- 1(A2G)-AG159 IgG2 Heavy Chain Expression Plasmids The AGl 59 IgGl heavy chain variable region fragment, described above, amplified by PCR using a 5' primer encoding a Sail site, optimized Kozak sequence and the amino terminus of the signal sequence (SEQ ID 106 described above) and a 3' primer encoding the carboxyl terminus, stop codon and Notl restriction site (5'-AAC CGT TTA AAC GCG GCC GCT CAT TTA CCC GGA GAC AGG GA-3' ; SEQ ID NO: 1 18). The resulting PCR fragment was purified, digested with Sail and Notl, gel isolated and cloned in the expression vectors pDC324 and pDSRα24. The same process was performed utilizing the GLP- 1(A2G)-AG 159 IgGl, AG159 IgG2 and GLP-1(A2G)- AGl 59 IgG2 constructs described above as templates.

Construction of different GLP-I (A2G)-AG 159 kappa chain mutants

To protect the GLP-1(A2G) peptide against proteolytic cleavage from the fusion protein, changes were made by site directed mutageneis using PCR. Using the GLP- 1(A2G)-AG159 kappa chain DNA sequence as a template, PCR was done to introduce a novel BamHl restriction site in the DNA sequence coding for the linker peptide. A forward primer containing a BamHl site (5'-GCT TGG CTG GTT AAA GGT CGT GGC GGA TCC GGC AGC GCT-3'; SEQ ID NO: 1 19) and a reverse primer that annealled to the sequence region containing the previously mentioned Kpnl site (5'-AGG TTT CTG TTG GTA CCA GGC-3'; SEQ ID NO: 120) were used to amplify a region that coded for the linker and the first 35 amino acids of the AGl 59 LC. A 5' primer that annealed in the vector promoter region upstream of the Xbal restriction site (5'-TTT CAG GTC CCG GAT CCG GTG-3"; SEQ ID NO: 121) was paired with specific 3' primers containing the desired changes and a BamHl restriction site (5'-GCT GCC GGA TCC GCC ACC ACC ATT TTT CAG CCA AGC GAT GAA-3'; SEQ ID NO: 122), (5'-GCT GCC GGA TCC GCC ACC ACC TTT AAC CAG CCA-3'; SEQ ID NO: 123), (5'-GCT GCC GGA TCC CAG CCA AGC GAT GAA TTC TTT AGC-3'; SEQ ID NO: 124), (5'-GCT GCC GGA TCC GCT GGG AGG CGG AGC ACC ACT ACT CGG TCC GCC GTT CTT CAG CCA AGC GAT GAA TTC-3'; SEQ ID NO: 125). The separate PCR products were purified, digested with tha appropriate restriction enzymes (Xbal and BamHl, or with BamHl and Kpnl) and ligated into the expression vector pDSRα20 containing the Xbal and Kpnl digested AGl 59 LC DNA.

Example 3: In vitro assays

Glucagon Receptor Functional Reporter Assay

In order to identify the compound/antibody compositions with neutralizing activity, reporter cell lines expressing human or rat glucagon receptors were generated. Increased cAMP levels were measured through enhanced expression of a luciferase reporter gene. Briefly, CHOKl cells expressing the rat or human glucagon receptor, in addition to harboring a luciferase reporter gene construct regulated by cyclic AMP levels, were plated 2 days prior to the assay, then cultured at 37⁰C, 5% CO₂. The evening prior to assay, the cells were washed, the medium replaced with serum-free medium containing 0.5% protease-free bovine serum albumin (BSA), and then cultured overnight. Cells were exposed to a range of concentrations of test composition with 1 nM or 0.1 nM glucagon, for human or rat glucagon receptor expressing cells respectively, for a period of 6 hours at 37⁰C in medium containing 0.5% protease-free BSA and 100 μM IBMX. Cell lysates were assayed for luciferase activity using the Luciferase Assay System (Promega Corporation, Madison, WI). Luciferase activity was measured using a Luminoskan Ascent (Thermo Electron Corporation, Marietta, OH). Nonlinear regression analyses of resultant compound concentration curves were performed using GraphPad Prism (GraphPad Software, Inc., San Diego, CA). The "IC₅o" represents the concentration of the compound/antibody composition at which the glucagon stimulated activity is reduced by 50%.

GLP-IR Functional Reporter Assay

In order to compare the potency of the GLP-I compound/antibody compositions, reporter cell lines expressing human or mouse GLP-I receptors were generated.

Increased cAMP levels were measured through enhanced expression of a luciferase reporter gene. Briefly, CHOKl cells expressing the mouse or human GLP-I receptor, in addition to harboring a luciferase reporter gene construct regulated by cyclic AMP levels, were plated 2 days prior to the assay, then cultured at 37⁰C, 5% CO₂. The evening prior to assay, the cells were washed, the medium replaced with serum-free medium containing 0.5% protease-free bovine serum albumin (BSA), and then cultured overnight. Cells were exposed to a range of concentrations of test composition or GLP-I for a period of 6 hours at 37⁰C in medium containing 0.5% protease-free BSA and 100 μM IBMX. Cell lysates were assayed for luciferase activity using the Luciferase Assay System (Promega Corporation, Madison, WI). Luciferase activity was measured using a Luminoskan Ascent (Thermo Electron Corporation, Marietta, OH). Nonlinear regression analyses of resultant compound concentration curves were performed using GraphPad Prism (GraphPad Software, Inc., San Diego, CA). The "EC₅₀" represents the concentration of the GLP- 1 compound/antibody composition at which 50 percent of the maximal activity is achieved.

Membrane Preparation

CHOKl cells expressing either human or mouse GLP-I receptor (or rat or human glucagon receptor) were harvested from 150 mm culture dishes using PBS. Cells were sedimented at 1500 rpm for 10 minutes. The resulting pellets were homogenized in 15 mis of ice cold sucrose buffer (25 mM Tris-HCl, 0.32 M Sucrose, 0.25 g/L sodium azide, pH 7.4) with a motorized, glass fitted, Teflon homogenizer. The homogenate was centrifuged at 48,000 X g at 4° C for 10 minutes, resuspended in 25 ml assay buffer (50 mM Tris-HCl, 5 mM MgCl₂, 10 mg/ml protease-free BSA, 0.1 mg/ml STI, and 0.1 mg/ml Pefabloc, pH 7.4) with a Tissue-Tearor (Biospec Products), then centrifuged again at 48,000 X g for 10 minutes. The pellets were homogenized for a third time in 15 ml assay buffer using the Tissue-Tearor and again centrifuged at 48,000 X g for 10 minutes. The resulting pellet was resuspended in assay buffer at a wet weight concentration of 4 mg/ml.

Ligand Binding Assays Binding assays were performed in 96-well U-bottom plates. Membranes (200 μg tissue) were incubated at room temperature for 2 hours in assay buffer containing 0.2 nM ¹²⁵I-GLP-I (or 0.2 nM ¹²⁵I-Glucagon) (PerkinElmer Life Sciences, Boston, MA) and with a range of concentrations of test composition or GLP-I (or glucagon) in a total volume of 100 μl. In addition, non-specific binding was assessed in the presence of 1 μM unlabeled GLP-I . The reaction was terminated by rapid filtration through Unfilter-96 GF/C glass fiber filter plates (FilterMate 196 Packard Harvester, PerkinElmer, Shelton, CT) pre- soaked in 0.5% polyethylenimine, followed by three washes with 300 μl of cold 50 mM Tris-HCl, pH 7.4. Bound radioactivity was determined using a TopCount microplate scintillation and luminescence counter (Packard Instrument Company, PerkinElmer, Shelton, CT). Nonlinear regression analyses of resulting concentration curves were performed using GraphPad Prism (GraphPad Software, Inc., San Diego, CA). The "IC₅o" represents the concentration of compound which reduces the maximal specific ¹²⁵I-GLP-I (or ¹²⁵I-glucagon) binding by 50 percent.

Example 4: Glucagon Neutralizing Activity of AG159 Antibody

The antibody variable regions were cloned from cDNA and confirmed by mass spectrophotometric analysis of antibody purified from the hybridoma. The cloned AGl 59 was verified to have glucagon neutralizing activity in receptor binding and receptor activation assays, as well as reducing blood glucose in a diabetic mouse model (Figures 11-13). In Figure 1 1, AG159 neutralization of glucagon stimulated reporter activity is shown. Cells expressing recombinant glucagon receptor were incubated with 0.1 nM glucagon in addition to a range of concentrations of AGl 59 or control IgG. The IC₅₀ for AGl 59 in this assay is 4 nM (Figure 1 1). In Figure 12, crude membrane fractions of cells recombinantly expressing the glucagon receptor were incubated with 200 pM ¹²⁵I-glucagon with a dose range of AGl 59 or control antibody. The IC₅₀ for AGl 59 in this ¹²⁵I-glucagon binding assay is 0.2 nM (Figure 12). Figure 13 shows glucose levels measured in ob/ob mice in response to AGl 59. Example 5: In vivo Assays

The db/db diabetic mouse model was used in this screen to further examine compositions in regard to fed blood glucose and monitored this measurement at 1 , 2, 4, 6 and 24h or every 24 hours until blood glucose levels were back to baseline levels. The db/db mice are commercially available from The Jackson Laboratory JAX® GEMM® Strain - Spontaneous Mutation Congenic Mice, and are homozygous for the diabetes spontaneous mutation (Lepr^c ). These mice become identifiably obese around 3 to 4 weeks of age. Our criterion of selection for each mouse to enter the study is blood glucose of at least 300 mg/dL. Db/db mice at 8.5 weeks of age (for a chronic l-2wk study) to about 10-1 1 weeks of age (for an acute 1-3 day study) were injected once with each tested molecule (acute experiment) or multiple times (chronic experiment). On the day of the experiment, the mice are bled at 9 am (baseline value) and then immediately handed over to the injector, who then injects the appropriate GLP-I construct or +/- control. The mice are then placed in a fresh cage without any chow, so as to limit any variability in blood glucose levels associated with eating behaviors. Blood glucose levels at lhr, 4hr, 6hr, and 24hr are normally measured. When at the 24hour time point, blood glucose values are below where they started, blood glucose levels are measured every 24hrs until blood glucose return to the baseline levels. Mice were fed normal chow after the 6hr time point. C57B16 (normal lean) mice were used at 10 to 12 weeks of age. These mice are commercially available through any vendor, such as Jackson Laboratories or Charles River, and are considered to be normal. The term "lean" is used to contrast these mice to obese db/db mice. C57B16 mice were randomized on body weight. 9 am bleed was performed to determine baseline blood glucose and compounds or PBS was administered prior to place the mice in a cage without food. After 4-5hrs, an intraperitoneal glucose tolerance test (glucose tolerance test measures the body's ability to metabolize glucose) was performed using 2g/kg of glucose dose. Blood glucose levels were measured 30 min and 90 minutes after the glucose load was administered and 24 hours or until blood glucose levels were back to the original values. From these studies, the enhanced effect of GLP-I action in utilizing glucose can be seen as opposed to (-) control PBS. Example 6: Binding Activity of AG159 Antibody

The antibody variable regions were cloned from cDNA and confirmed by mass spectrophotometric analysis of antibody purified from the hybridoma. The cloned AGl 59 was verified to have glucagon neutralizing activity in receptor binding and receptor activation assays. GLP(A2G) (SEQ ID NO: 126) was fused to the N-terminus of either the light or heavy chain of AGl 59 (see Example 2 above). Plasmids were co-transfected into CHO cells to produce full-length fusion proteins. The resulting antibody structure was AG159LC:GLP(A2G)-AG159 IgG2 and GLP(A2G)-AG159 LC:AG159 IgG2. By in vitro analysis (see Example 3), the two fusion proteins were shown to have equal binding (IC₅₀5-10 nM to human receptor) and activation (EC₅₀ = 0.1 nM for activation of human receptor and 5 nM for the murine receptor). Further, the antibody construct in which GLP(A2G) was fused to the light chain was shown to also activate the human receptor in the presence of glucagon. In addition, we established that the fused antibody is still active in neutralizing glucagon in the presence of GLP-I receptor. These in vitro data demonstrated that the fusion GLP-I analogue was bi-functional.

More specifically, GLP(A26)-AG159LC:AG159 IgG2 (GLP(A2G)-AG159) was assayed to determine if the construct would maintain GLP-I receptor binding properties in the presence of glucagon. The ligand binding assay was performed as described, with the addition of 0, 1, 10 or 100 nM glucagon. As shown in Figure 1, GLP(A2G)-AG159 competes for I-GLP-1 binding to the human GLP-I receptor in the presence of glucagon.

In another experiment, GLP(A2G)-AG159 was evaluated for GLP-I receptor agonist activity in the presence of glucagon. The GLP-IR reporter assay was performed as described above in Example 3, with the addition of a range of glucagon concentrations. As shown in Figure 2, GLP(A2G)-AG159 activates the human GLP-I receptor in the presence of glucagon. Also depicted on the graph is the dose response curve of the activation of the GLP-I receptor by glucagon alone (without GLP(A2G)-AG159) ( ).

The data presented in Figure 2 is shown in a different form in Figure 3. In figure 3, the activity attributable to a specific GLP (A2 G)- AG 159 concentration (without glucagon) was subtracted from the total activity for all doses of glucagon with the respective GLP(A2G)-AG159 concentration, such that the remaining activity was attributable to glucagon. As shown in figure 3, the presence of GLP(A2G)-AG159 dose- dependently decreases the activity induced by glucagon.

Example 7: Results with Antibody Fusions Including Different GLP-I Analogs

A variety of fusions between a GLP-I compound and AGl 59 were prepared and tested for their binding capacity as described in the examples above. Table 8 below shows results for a number of fusion proteins in which the C-terminus of native GLP-I had been modified (sequences for the fusions are listed in Tables 1 and 7 ).

Table 8

= not determined

To reduce proteolysis, a set of substitution mutant GLP-I analogs were designed as described above with the goal of investigating whether introduction of glycosylation concensus sites (NTX) would protect cleavage sites. In addition, glutamine residues were substituted at selected positions with the theory that glutamine' s propensity for helical structure would promote a more stable peptide with purification and protection from cleavage characteristics. These GLP-I analogs were fused to the light chain of AG 159 and tested (sequences for these fusions are listed in Tables 1 and 7). Results are shown in Table 9.

Table 9: Glutamine and Glycosylation GLP-I Compound/Ab fusions

Example 8: In vivo Results Experiments were conducted with a number of different fusion molecules that included AGl 59 and different GLP-I analogs to determine their ability to reduce blood glucose levels as a function of time. These experiments were conducted as described in

Example 3, with Db/db mice being injected once with different compositions (sequences of the different analogues are described in Tables 1 and 7).

Figure 4 shows results for a variety of GLP-I compound/AG159 fusions. The antibody fusions included either GLP(A2G) or one of the following GLP-I peptides fused to the light chain (LC) of AGl 59: A2G/K28N/R30T (SEQ ID NO: 28), A2G/Q17N/A19T (SEQ ID NO: 23), A2G/V10Q/V27Q (SEQ ID NO: 9), and A2G/W25Q/V27Q (SEQ ID NO: 12). These LC fusions were paired with AGl 59 IgG2 heavy chains to give the following antibodies, which were tested: GLP(A2G/K28N/R30T)-AG159LC:AG159 IgG2, GLP(A2G/Q17N/A19T)-

AG159LC:AG159 IgG2, GLP(A2G/V10Q/V27Q)-AG159LC:AG159 IgG2, and GLP(A2G/W25Q/V27Q)-AG159LC:AG159 IgG2. Dosage was 12 ug/mouse. The sequences for these fusions are listed in Tables 1 and 7 above.

As can be seen in figure 4, blood glucose was decreased at 1 hour with most of the analogs, with GLP(A2G)-AG159LC:AG159 IgG2 and GLP(A2G/K28N/R30T)- AG159LC:AG159 IgG2 showing the most blood glucose lowering effect. Blood glucose levels returned back to baseline levels 24 after the single injection. The maximal effect was observed between 4 and 6 hours after the single injection.

Another set of experiments were conducted with another set of GLP-I peptides fused to AGl 59LC. The antibody fusions tested included the following GLP-I peptides: GLP(A2G) (SEQ ID NO: 126), GLP(A2G/G31N/+G32/+T33) (SEQ ID NO: 31), GLP(A2G/G29N/G31/T) (SEQ ID NO: 29) and GLP(A2G/K28N/R30T) (SEQ ID NO: 28). These LC fusions were paired with AGl 59 IgG2 heavy chains to give the following antibodies, which were tested: GLP(A2G)-AG159LC:AG159 IgG2,

GLP(A2G/G31N/+G32/+T33)-AG159LC:AG159 IgG2, GLP(A2G/G29N/G31/T)- AG159LC:AG159 IgG2, and GLP(A2G/K28N/R30T)-AG159LC:AG159 IgG2. Dosage in this instance was 1 mg/kg. As can be seen in Figure 5, for each of the analogs tested, blood glucose was decreased for the first 6 hours after a single injection and returned to baseline levels 24 hours after a single injection. The maximal effect was observed between 4 and 6 hours after injection.

Example 9: Response Curve An in vivo dose response determination was conducted using db/db mice as described in Example 3. The composition used in one experiment was one in which GLP-I (A2G) was fused to the LC of AGl 59, to give the antibody GLP(A2G)- AG159LC:AG159 IgG2 (see Table 8 for sequence). Dosage was either 7 ug, 12 ug or 25 ug. As can be seen in Figure 6, blood glucose levels were found to decrease in a dose dependent fashion. The results also demonstrate that the composition operating on the mechanism of action.

Another dose response study was conducted in normal mice with a composition in which a GLP(A2G/R30G) analog (SEQ ID NO:4) was fused to AGl 59 LC to give the antibody GLP(A2G/R30G)AG159LC:AG159 HC IgGl. The dose response of this composition was more apparent at the 30 min time point post glucose injection during the glucose tolerance test (Figure 7) and after 24 and 48 hours after injection of the antibody composition. There is a somewhat minimal effect because this dose response was performed in normoglycemic mice. It is more challenging to decrease blood glucose in an animal model where blood glucose is normal than in a hyperglycemic animal model (db/db mice).

Example 10: In vivo Comparison of GLP-I fused to LC v HC

This experiment was designed to determine whether the attachment of a GLP-I compound to the LC or HC of AGl 59 resulted in differences in activity. The constructs tested were ones in which GLP(A2G) was attached to either the LC or HC of AGl 59 to give the following two antibody constructs GLP(A2G) AG159LC:AG159 IgG2

(GLP(A2G)-AG159 LC) and AG159LC:GLP-1-AG159 IgG2 (GLP(A2G)-AG159 HC).

As can be seen in Figure 8, both constructs were essentially equally effective in lowering blood glucose levels. Example 10: Lean GTT Experiments

Another experiment was conducted with the fusion protein in which the GLP-I analog A2G/R30G was fused to AGl 59 to give the antibody construct GLP(A2G/R30G)AG159LC:AG159 HC IgG2, or simply R30G (see Tables 1 and 7 for sequences). This experiment was performed to determine if R30G could lower blood glucose for a long period in normal mice. A glucose tolerance test was added to the experimental design to allow us to increase our read out on the window of efficacy.

C57B16 mice were randomized on body weight. 9 am bleeds were performed to determine baseline blood glucose levels when either R30G or PBS was administered. After a 4-5 hr fast, an intraperitoneal glucose tolerance test was performed using a 2g/kg dose. Blood glucose levels were measured 30 min and 90 minutes after the glucose load and every 24 hours until blood glucose levels returned to the original values. As shown in Figure 9, treatment with the R30G composition improved glucose tolerance.

Example 12: Multi Dose Experiments

A problem with certain GLP-I compounds is that they may loose efficacy after multiple injections (determination of tachyphylaxis). Another experiment was conducted with the GLP(A2G/R30G)AG159LC:AG159 HC IgG2 antibody, or simply R30G, to determine if this was the case with this molecule. R30G was injected on day 1 in lean mice, as described in example 5. Four hours after the first injection, a glucose tolerance test was performed to demonstrate maximal efficacy. The second and third day vehicle or compound was injected. On the fourth day, 4 hours after the injection of vehicle or compound, a second glucose tolerance test was performed. As shown in Figure 10, it was evident that no noticeable tachyphylaxis was observed. Thus, R30G was as efficacious at lowering blood glucose during the second glucose tolerance test as it was during the first glucose tolerance test

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent or patent application were specifically and individually indicated to be so incorporated by reference.

Claims

We claim:

1. A composition comprising:

(a) an antibody that binds glucagon; and

(b) a GLP-I compound linked to the antibody that binds glucagon, wherein the GLP-I compound has a GLP-I activity.

2. The composition of claim 1, wherein the antibody is a neutralizing antibody.

3. The composition of claim 1 , wherein (a) the antibody comprises (i) a heavy chain variable region and (ii) a light chain variable region; and

(b) the GLP-I compound is linked to either the heavy chain variable region or the light chain variable region.

4. The composition of claim 3, wherein the GLP-I compound is linked to the light chain variable region.

5. The composition of claim 4, wherein the carboxy terminus of the GLP-I compound is linked to the amino terminus of the light chain variable region.

6. The composition of claim 3, wherein the GLP-I compound and the light chain variable region are linked by a linker.

7. The composition of claim 3, wherein the GLP-I compound is linked to the heavy chain variable region.

8. The composition of claim 7, wherein the carboxy terminus of the GLP-I compound is linked to the amino terminus of the heavy chain variable region.

9. The composition of claim 8, wherein the GLP-I compound and the heavy chain variable region are linked by a linker.

10. The composition of claim 1, wherein the antibody and the GLP-I compound are linked by chemical conjugation.

11. The composition of claim 1, wherein the antibody and the GLP-I compound are linked via a synthetic linker.

12. The composition of claim 1, wherein the antibody and the GLP-I compound are linked via a peptide linker.

13. The composition of claim 1, wherein the antibody and the GLP-I compound form a fusion protein.

14. The composition of claim 1, wherein the antibody is linked to a plurality of GLP- 1 compounds.

15. The composition of claim 1, wherein the antibody is linked to multiple different GLP-I compounds.

16. The composition of claim 1, wherein the GLP-I compound comprises a GLP-I peptide that has at least 90% sequence identity to SEQ ID NO: 1 and has a GLP-I activity.

17. The composition of claim 16, wherein the GLP-I compound has the amino acid sequence of SEQ ID NO: 1 with no more than 5 conservative amino acid substitutions.

18. The composition of claim 1, wherein the GLP-I compound comprises an exendin molecule.

19. The composition of claim 1, wherein the GLP-I compound comprises exendin 3 or exendin 4.

20. The composition of claim 1, wherein the GLP-I compound has at least 90% sequence identity to exendin-3 or exendin-4.

21. The composition of claim, wherein the GLP-I compound comprises a GLP-I peptide that comprises the amino acid sequence of formula I (SEQ ID NO: 92)

Xaa i -Xaa₂-Xaa₃-Xaa₄-Xaa₅ -Xaa₆-Xaa₇-Xaa₈-XaacrXaa i _O-Xaa π -Xaa _12-Xaa ₁₃ - Xaaπ-Xaai ₅-Xaai ₆-Xaai ₇-Xaai ₈-Xaai ₉-Xaa₂o-Xaa₂₁.Xaa2₂-Xaa₂₃-Xaa_24-Xaa₂₅-

Xaa₂₆-Xaa_27-Xaa_28-Xaa₂₉-Xaa₃₀ -Xaa₃i- Xaa₃₂-Xaa₃₃-Xaa₃₄-Xaa₃₅-Xaa₃₆-Xaa₃₇-

C(O)-Ri (Formula I, SEQ ID NO: 92) wherein,

Ri is OR₂ or NR₂R₃; R₂ and R₃ are independently hydrogen or (C]-C₈)alkyl;

Xaa at position 1 is: L-histidine, D-histidine, desamino-histidine, 2-amino- histidine, 3-hydroxy-histidine, homohistidine, α-fluoromethyl-histidine or α- methyl-histidine;

Xaa at position 2 is GIy, bAla (2-aminopropionic acid), Asp, Ala, 1-amino- cylcopentanecarboxylic acid, 2-aminoisobutryic acid or alpha-alpha-disubstituted amino acids;

Xaa at position 3 is GIu, Asp, or Lys;

Xaa at position 4 is GIy, Thr or His;

Xaa at position 5 is Thr, Ala, GIy, Ser, Leu, He, VaI, GIu, Asp, or Lys; Xaa at position 6 is: His, Tip, Phe, or Tyr;

Xaa at position 7 is Thr or GIy;

Xaa at position 8 is Ser, Ala, GIy, Thr, Leu, He, VaI, GIu, Asp, or Lys;

Xaa at position 9 is Asp, Asn or GIu;

Xaa at position 10 is VaI, Ala, GIy, Ser, Thr, Leu, He, Tyr, GIu, Asp, Trp, or Lys; Xaa at position 11 is Ser, Ala, GIy, Thr, Leu, He, VaI, GIu, Asp, or Lys; Xaa at position 12 is Ser, Ala, GIy, Thr, Leu, He, VaI, GIu, Asp, Tip, Tyr, Asn, Lys, Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta- Homoglutamic acid, or homoglutamic acid;

Xaa at position 13 is Tyr, Phe, Trp, GIu, Asp, GIn, Lys, Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, or homoglutamic acid;

Xaa at position 14 is Leu, Ala, GIy, Ser, Thr, He, VaI, GIu, Asp, Met, Trp, Tyr, Asn, GIn, Lys, Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, or homoglutamic acid; Xaa at position 15 is GIu, Asp, Lys, Homolysine, Ornithine, 4-carboxy- phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, or homoglutamic acid;

Xaa at position 16 is GIy, Ala, Ser, Thr, Leu, He, VaI, GIu, Asp, Asn, Lys, Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta- Homoglutamic acid, or homoglutamic acid;

Xaa at position 17 is GIn, Asn, Arg, GIu, Asp, Lys, Ornithine, 4-carboxy- phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, or homoglutamic acid; Xaa at position 18 is Ala, GIy, Ser, Thr, Leu, He, VaI, Arg, GIu, Asp, Asn, Lys, Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-

Homoglutamic acid, or homoglutamic acid;

Xaa at position 19 is Ala, GIy, Ser, Thr, Leu, He, VaI, GIu, Asp, Asn, Lys, Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta- Homoglutamic acid, or homoglutamic acid; Xaa at position 20 is Lys, Homolysine, Arg, GIn, GIu, Asp, Thr, His, Ornithine,

4-carboxy-phenylalanine, beta-glutamic acid, or homoglutamic acid; Xaa at position 21 is Leu, GIu, Asp, Thr, Lys, Homolysine, Ornithine, 4-carboxy- phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, or homoglutamic acid; Xaa at position 22 is Phe, Trp, Asp, GIu, Lys, Homolysine, Ornithine, 4-carboxy- phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, or homoglutamic acid;

Xaa at position 23 is He, Leu, VaI, Ala, Phe, Asp, GIu, Lys, Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, or homoglutamic acid;

Xaa at position 24 is Ala, GIy, Ser, Thr, Leu, He, VaI, GIu, Asp, Lys, Homolysine,

Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, or homoglutamic acid; Xaa at position 25 is Trp, Phe, Tyr, GIu, Asp, Asn, or Lys;

Xaa at position 26 is Leu, GIy, Ala, Ser, Thr, He, VaI, GIu, Asp, or Lys;

Xaa at position 27 is VaI, GIy, Ala, Ser, Thr, Leu, He, GIu, Asp, Asn, or Lys;

Xaa at position 28 is Asn, Lys, Arg, GIu, Asp, or His;

Xaa at position 29 is GIy, Ala, Ser, Thr, Leu, He, VaI, GIu, Asp, or Lys; Xaa at position 30 is GIy, Arg, Lys, GIu, Asp, Thr, Asn, or His;

Xaa at position 31 is Pro, GIy, Ala, Ser, Thr, Leu, He, VaI, GIu, Asp, or Lys;

Xaa at position 32 is Thr, GIy, Asn, Ser, Lys, or is omitted;

Xaa at position 33 is GIy, Asn, Ala, Ser, Thr, He, VaI, Leu, Phe, Pro, or is omitted; Xaa at position 34 is GIy, Thr, or is omitted;

Xaa at position 35 is Thr, Asn, GIy or is omitted;

Xaa at position 36 is GIy or is omitted;

Xaa at position 37 is GIy or is omitted; provided that when the amino acid at position 32, 33, 34, 35, 36 or 37 is omitted, then each amino acid downstream of that amino acid is also omitted, and wherein the compound has a GLP-I activity.

22. The composition of claim 21, wherein the GLP-I compound comprises a GLP-I peptide that comprises the amino acid sequence of any of SEQ ID NO: 1-35, SEQ ID NO: 126, or SEQ ID NO: 127, with no more than 5 conservative amino acid substitutions.

23. The composition of claim 21, wherein the GLP-I compound comprises a GLP-I peptide that comprises the amino acid sequence of any of SEQ ID NO: 1-35, SEQ ID NO: 126, or SEQ ID NO: 127.

24. The composition of claim 1, wherein the antibody comprises:

(a) a light chain variable region (VL) having at least 90% sequence identity with SEQ ID NO: 79; or

(b) heavy chain variable region (VH) having at least 90% sequence identity with SEQ ID NO: 83; or

(c) a VL of (a) and a VH of (b).

25. The composition of claim 24, wherein the antibody consists of two identical VH and two identical VL.

26. The composition of claim 24, wherein the antibody comprises a VL that has at least 95% sequence identity with SEQ ID NO: 79; and a VH that has at least 95% sequence identity with SEQ ID NO: 83.

27. The composition of claim 26, wherein the antibody consists of two identical VH and two identical VL.

28. The composition of claim 24, wherein the antibody comprises a VL that has the amino acid sequence of SEQ ID NO: 79; and a VH that has the amino acid sequence of SEQ ID NO: 83.

29. The composition of claim 28, wherein the antibody consists of two identical VH and two identical VL.

30. The composition of claim 1, wherein the antibody comprises: (a) a light chain comprising the amino acid sequence of any of SEQ ID NOs: 40- 81;

(b) a heavy chain comprising the amino acid sequence of any of SEQ ID NOs: 39 or 82-91 ; or (c) a light chain comprising the amino acid sequence of any of SEQ ID NOs: 40-

81 and a heavy chain comprising the amino acid sequence of any of SEQ ID NOs: 39 or 82-91.

31. The composition of claim 30, wherein the antibody consists of two identical light chains and two identical heavy chains.

32. The composition of claim 1, 24, or 30, wherein the antibody is a monoclonal antibody.

33. The composition of claim 1 , 24, or 30, wherein the antibody is a scFv, a Fab, a Fab' or a (Fab')₂.

34. The composition of claim 1, 24, or 30, wherein the antibody is a human or humanized antibody.

35. A polypeptide comprising a glucagon binding antibody light chain variable region linked to a GLP-I peptide.

36. The polypeptide of claim 35 comprising the amino acid sequence of any of SEQ ID NO: 41-74 or 129.

37. An antibody that binds glucagon comprising the polypeptide of claim 36.

38. A polypeptide comprising a glucagon binding antibody heavy chain variable region linked to a GLP-I compound.

39. The polypeptide of claim 38 comprising the amino acid sequence of SEQ ID NO: 128.

40. An antibody that binds glucagon comprising the polypeptide of claim 39.

41. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of the composition of claim 1 , 24, or 30.

42. A method for treating a subject with a metabolic disorder, comprising administering to the subject an effective amount of the pharmaceutical composition of claim 41, wherein the metabolic disorder is selected from the group of diabetes, obesity and metabolic syndrome.

43. A method for enhancing insulin expression in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition of claim 41.

44. A method for promoting insulin secretion in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition of claim 41.

45. A method for treating a subject with a metabolic disorder, comprising administering to the subject an effective amount of the composition of claim 1, 24, or 30, wherein the metabolic disorder is selected from the group of diabetes, obesity and metabolic syndrome.

46. A method for enhancing insulin expression in a subject, comprising administering to the subject an effective amount of the composition of claim 1, 24, or 30.

I l l

7. A method for promoting insulin secretion in a subject, comprising administering the subject an effective amount of the composition of claim 1, 24, or 30.