Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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CBLP-1 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(GL,P-I) and the related peptide glucagon are produced
via differential processing of proglucagon and have opposing biological
activities.
Proglucagon itself is produced in a-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-1 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, Mol. Endocrinol. 9:267-277 and Bell et al., 1983,
Nature
304:368-371). By convention, the numbering of the amino acids of GLP-1 is
based on
the GLP-1 (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-1 (7-37)-OH and GLP-1 (7-36)-NH2. Both GLP-1 (7-37)-OH
(or
simply GL,P-1 (7-37)) and GLP-1 (7-3 6)-NH2 have the same activities. For
convenience,
the term "GL,P-1", 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-1 (1-31) is the same as GLP-1(7-37), and GLP-1(1-30) is the same as GL,P-1
(7-36).
Glucagon is secreted from the a-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 riet result of these actions is
to
significantly increase the release of glucose to the liver. GLP-1, in
contrast, lowers
glucagon secretion, while stimulating insulin secretion, glucose uptake and
cyclic-AMP
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(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).
GL,P-1 has a number of other important activities. For instance, GL,P-1 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-1 can promote cell differentiation and
replication,
which in turn aids in the preservation of pancreatic islet cells and an
increase in (3-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-1 can
increase
satiety and decrease food intake (see, e.g., Toft-Nielsen et al., 1999,
Diabetes Care
22:1137-1143; Flint et al., 1998, J. Clin. Invest. 101:515-520; Gutswiller et
al., 1999 Gut
44:81-86).
Other research indicates that GL,P-1 induces (3-cell-specific genes, including
GLUT-1 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-1 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 GL,P-l 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.
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A key hurdle for effective treatment with GLP- 1, however, has been the very
short half-life of the peptide, which typically is only a few minutes (see,
e.g., Holst, 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 al., 2002,
Diabetologia 45:195-202).
Accordingly, there thus remains a need for improved molecules that have GLP-1
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-1 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-1 compound linked to the antibody that binds glucagon, wherein the GLP-1
compound has a GLP-1 activity. In certain compositions, the antibody comprises
(i) a
heavy chain variable region and (ii) a light chain variable region; and the
GLP-1
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-1 compound is
linked to
the amino terminus of the light chain variable region, and/or the carboxy
terminus of the
GLP-1 compound is linked to the amino terminus of the heavy chain variable
region.
A variety of GLP-1 compounds can be attached to the anti-glucagon antibody. In
one aspect, a GLP-1 compound in a composition as provided herein comprises a
C'1LP-1
peptide that has at least 90% sequence identity to SEQ ID NO: 1 and has a GLP-
1
activity.
In certain compositions, the GLP-1 compound comprises the amino acid sequence
of formula I (SEQ ID NO: 92):
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Xaal -XaaZ-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-XaaB-Xaa9-Xaalo-Xaa> >-Xaa i Z_Xaa]3-
Xaa 14-Xaa l 5-Xaa l 6-Xaa i 7-Xaal 8-Xaal9-Xaa20-Xaa21 _Xaa22-Xaa23-
Xaa24.Xaa25-
Xaa26-Xaa27_Xaa28_Xaa29-Xaa30 -Xaa3 I - Xaa32-Xaa33-Xaa34-Xaa35-Xaa36-Xaa37-
C(O)-RI (Forlnula I, SEQ ID NO: 92)
wherein,
R, 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, a-fluoromethyl-histidine or a-
methyl-histidine;
Xaa at position 2 is Gly, bAla (2-aminopropionic acid), Asp, Ala, 1-amino-
cylcopentanecarboxylic acid, 2-aminoisobutryic acid or alpha-alpha-
disubstituted amino
acids;
Xaa at position 3 is Glu, Asp, or Lys;
Xaa at position 4 is Gly, Thr or His;
Xaa at position 5 is Tlu=, Ala, Gly, Ser, Leu, Ile, Val, Glu, Asp, or Lys;
Xaa at position 6 is: His, Trp, Phe, or Tyr;
Xaa at position 7 is Thr or Gly;
Xaa at position 8 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu, Asp, or Lys;
Xaa at position 9 is Asp, Asn or Glu;
Xaa at position 10 is Val, Ala, Gly, Ser, Thr, Leu, Ile, Tyr, Glu, Asp, Trp,
or Lys;
Xaa at position 11 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu, Asp, or Lys;
Xaa at position 12 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu, 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, Glu, Asp, Gln, Lys, Homolysine,
Ornithine,
4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, or
homoglutamic acid;
Xaa at position 14 is Leu, Ala, Gly, Ser, Thr, Ile, Val, Glu, Asp, Met, Trp,
Tyr,
Asn, Gln, Lys, Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic
acid, or homoglutamic acid;
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Xaa at position 15 is Glu, Asp, Lys, Homolysine, Ornithine, 4-carboxy-
phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, or homoglutamic
acid;
Xaa at position 16 is Gly, Ala, Ser, Thr, Leu, Ile, Val, Glu, Asp, Asn, Lys,
Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-
Homoglutamic acid, or homoglutamic acid;
Xaa at position 17 is Gln, Asn, Arg, Glu, Asp, Lys, Ornithine, 4-carboxy-
phenylalanine, beta-glutamic acid, beta-I-Iomoglutamic acid, or homoglutamic
acid;
Xaa at position 18 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Arg, Glu, Asp, Asn,
Lys,
Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-
Homoglutarnic acid, or homoglutamic acid;
Xaa at position 19 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Glu, Asp, Asn, Lys,
Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-
Homoghatamic acid, or homoglutamic acid;
Xaa at position 20 is Lys, Homolysine, Arg, Gln, Glu, Asp, Thr, His,
Ornithine,
4-carboxy-phenylalanine, beta-glutamic acid, or homoglutamic acid;
Xaa at position 21 is Leu, Glu, 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, Glu, Lys, Homolysine, Ornithine, 4-
carboxy-
phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, or homoglutamic
acid;
Xaa at position 23 is Ile, Leu, Val, Ala, Phe, Asp, Glu, Lys, Homolysine,
Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic
acid,
or homoglutamic acid;
Xaa at position 24 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Glu, Asp, Lys,
Homolysine,
Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic
acid,
or homoglutamic acid;
Xaa at position 25 is Trp, Phe, Tyr, Glu, Asp, Asn, or Lys;
Xaa at position 26 is Leu, Gly, Ala, Ser, Thr, Ile, Val, Glu, Asp, or Lys;
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Xaa at position 27 is Val, Gly, Ala, Ser, Thr, Leu, Ile, Glu, Asp, Asn, or
Lys;
Xaa at position 28 is Asn, Lys, Arg, Glu, Asp, or His;
Xaa at position 29 is Gly, Ala, Ser, Thr, Leu, Ile, Val, Glu, Asp, or Lys;
Xaa at position 30 is Gly, Arg, Lys, Glu, Asp, Thr, Asn, or His;
Xaa at position 31 is Pro, Gly, Ala, Ser, Thr, Leu, Ile, Val, Glu, Asp, or
Lys;
Xaa at position 32 is Thr, Gly, Asn, Ser, Lys, or is omitted;
Xaa at position 33 is Gly, Asn, Ala, Ser, Thr, Ile, Val, Leu, Phe, Pro, or is
omitted;
Xaa at position 34 is Gly, Thr, or is omitted;
Xaa at position 35 is Thr, Asn, Gly or is omitted;
Xaa at position 36 is Gly or is omitted;
Xaa at position 37 is Gly 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-1 activity.
The GLP-1 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) CDR.s selected from the group consisting of:
(i) a
LC CDR 1 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 CDR1 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 CDR.3 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).
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In certain compositions, the antibody that is part of the composition includes
an
antibody that comprises:
(a) one or more L,C CDRs selected from the group consisting of: (i) a LC CDRI
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 CDR1
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 cornpositions 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-1 analog. Certain such
polypeptides have the
amino acid sequence of any of SEQ ID NO: 41-74. The invention further provides
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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 GL,P-1 peptide. In certain aspects,
the
polypeptide is a fiision protein that comprises the amino acid sequence of SEQ
ID NO:
83. In certain such fiision proteins, the GLP-l 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 adrninistering
to the
subject an effective amount of the pliarrnaceutical 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-1 compound linked thereto, wherein the GLP-1
compound has GLP-1 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-1 compound linked thereto, wherein the GLP-1 compound has
GLP-1 activity.
Specific embodiments will become evident from the following more detailed
description of certain preferred embodiments and the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
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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-1
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-1 receptor
agonist activity of GLP(A2G)-AG159L,C:AG159 IgG2 (GL,P(A2G)-AG159) in the
presence of glucagon. Also depicted on the graph is the dose response curve of
the
activation of the GLP-1 receptor by glucagon alone (without GL,P(A2G)-AG159) (-
---- ).
Figure 3 depicts a graph showing that the presence of GL,P(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)-AG159L,C:AG159 IgG2
(a construct in which GLP(A2G) was fused to the light chain of the AG159
antibody) and
various other antibody fusions. The antibody fusions included the following
GLP-1
peptides fused to the light chain (LC) of AG159: A2G/K28N/R30T (SEQ ID NO:
28),
A2G/Q17N/A19T (SEQ ID NO: 23), A2G/VIOQ/V27Q (SEQ ID NO: 9), and
A2G/W25Q/V27Q (SEQ ID NO: 12). These LC fusions were paired with AG159 IgG2
heavy chains to give the following antibodies, which were tested:
GLP(A2G/K28N/R30T)-AG159L,C:AG159 IgG2, GL,P(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 liours after a single injection. The antibody fusions
tested included the
following GL,P-1 peptides fused to the light chain (LC) of AG159: 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 AG159 IgG2 heavy chains to give the following antibodies,
which were
tested: GLP(A2G)-AG 159LC:AG 159 IgG2, GLP(A2G/G31 N/+G32/+T3 3)-
AG159LC:AG159 IgG2, GLP(A2G/G29N/G31/T)-AG159LC:AG159 IgG2, and
GLP(A2G/K28N/R3 0T)-AG 159LC:AG 159 IgG2.
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Figure 6 depicts a graph showing that GL,P(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 AG159 and paired with the heavy chain of AG159 to
give the
antibody fusion GL,P(A2G/R30G)-AG 159L,C:AG 159 IgG2.
Figure 8 depicts a graph showing the differences in activity between the
attaclunent of GLP(A2G) to either the LC or HC of AG159, such that the
resulting
antibody was respectively GLP(A2G)-AG159LC:AG159 IgG2 (referred to as
GL,P(A2G)-AG159 LC in FIG. 8) and AG159LC:GL,P(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 GL,P(A2G/R30G)-AG 159LC:AG 159 IgG2.
Figure 11 depicts a graph showing AG159 neutralization of glucagon stimulated
reporter activity.
Figure 12 depicts a graph showing that AG159 disrupts 125i-glucagon binding to
the human glucagon receptor.
Figure 13 depicts a graph showing that AG159 reduces blood glucose in ob/ob
mice.
DETAILED DESCRIPTION
The section headings used herein are for organizational purposes only and are
not
to be coristrued as limiting the subject matter described. All references
cited in this
application are expressly incorporated by reference herein for any purpose.
1. Definitions
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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 TECHNOLOGY (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 marmer. 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 experirnents that measure GLP-1 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 examiriing 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 polyriucleotides, 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
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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 fragnients, 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 embodirnents, 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 ftinctional 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
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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 terrn 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, VL, and a constant region
domain, CL. 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, VH, and three constant region
domains,
CHl, CH2, and CIi3. The VH domain is at the amino-terrninus of the
polypeptide, and the
C domains are at the carboxyl-terminus, with the CI-13 being closest to the -
COOH end.
Heavy chains according to the invention may be of any isotype, including IgG
(including
IgGI, IgG2, IgG3 and IgG4 subtypes), IgA (including IgA, 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
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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,
rnouse,
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 CH1 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 CH 1 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 CH1 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
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chain, which forms an antigen-binding region. Single chain antibodies are
discussed in
detail in International Patent Application Publication No. WO 88/01649 and
IJ.S. Patent
Nos. 4,946,778 and 5,260,203, the disclosures of which are incorporated by
reference.
A "domain antibody" is an immunologically fiinctional immunoglobulin fragment
containing only the variable region of a heavy chain or the variable region of
a light
chain. In sorne instances, two or more VH regions are covalently joined with a
peptide
linker to create a bivalent domain antibody. The two VH 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. Immunoh 79:315-321; Kostelny et crl. (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
CA 02649751 2008-10-17
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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"7 or 10"8 M. In some
embodiments,
the equilibrium dissociation constant may be < 10-9 M or < 10"10 M.
The term "nati.irally-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
natural ly-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 whicli 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 ernbodiments, 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
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WO 2007/124463 PCT/US2007/067152
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 transcriptiori 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 terrn "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 aligmnents (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
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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 exarnple, 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 COMPIJTATIONAL MOLECULAR BIOLOGY, (Lesk, A.M., ed.), 1988,
Oxford iJriiversity Press, New York; BIOCOMPIJTING: INFORMATICS AND
GENOME PROJECTS, (Smith, D.W., ed.), 1993, Academic Press, New York;
COMPUTER. ANALYSIS OF SEQTJENCE DATA, Part 1, (Griffin, A.M., and Griffin,
H.G., eds.), 1994, Humana Press, New Jersey; von Heinje, G., SEQtJENCE
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 IJniversity 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, iricluding GAP (Devereux et al., 1984, Nucl. Acid. Res., 12:387;
Genetics
Computer Group, University of Wisconsin, Madison, WI), BLASTP, BLASTN, and
FASTA (Altschul et al., 1990, J. Mol. 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 el al., 1990, supra). The well-known Smith Waterman algorithm may
also be
used to determine identity.
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Certain aligrunent 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,
IJniversity 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 certairr
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 perralty), 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 tJSA, 89:10915-10919 for the BLOSUM 62 comparison matrix) is also used by
the
algorithm.
In certain embodirnents, the parameters for a polypeptide sequence comparison
include the following:
Algorithm: Needleman et al,, 1970, J. Mol. Biol., 48:443-453;
Comparison matrix: BLOStJM 62 from Henikoff et al., 1992, supra;
Gap Penalty: 12
Gap Length Penalty: 4
Threshold of Similarity: 0
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The GAP program may be useftil with the above parameters. For nucleotide
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 a-
, a-
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, y-
carboxyglutamate,
s-N,N,N-trimethyllysine, E-N-acetyllysine, 0-phosphoserine, N-acetylserine, N-
formylmethionine, 3-methylhistidine, 5-hydroxylysine, cr-N-methylarginine, and
other
similar amino acids and imino acids (e.g., 4-hydroxyproline). In the
polypeptide notation
used herein, the left-liand 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-1 compound
means positions that are located toward the carboxyl end of the polypeptide
relative to the
position beitig referenced, i.e., to the right of the position being
referenced. The term
"upstrearn" when used in reference to a GLP-1 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.
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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 hereiri, 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 attachrnent 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 Icnown 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,
355, 90Y,
99mTc, r11In, "SI, 13'1), fluorescent labels (e.g., fluorescein isothiocyanate
or FITC,
rhodamine, or lanthariide phosphors), enzymatic labels (e.g., horseradish
peroxidase, (~-
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 (CHZ)n, where n < about 20) of various
lengths to reduce
potential steric hindrance.
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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
Cornpositions comprising an anti-glucagon antibody (i.e., an antibody that
binds
glucagon) that is linked to a GLP-1 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-1 is still able to bind to the
GLP-1
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-1
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-1 compound that is linked to the anti-glucagon
antibody
can be native GLP-1, any of the GLP-1 analogs that are known in the art, or
one of the
GLP-1 analogs disclosed herein that have an activity of native GLP-1. In some
compositions, the anti-glucagon antibody and the GLP-1 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-1 compound are not part of a fusion protein
and
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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-1 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 cornpounds 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-1 Compound/Antibody Compositions and Polypeptides
The compositions that are provided generally comprise an antibody that binds
glucagon and one or more GLP-1 compounds that are linked to the antibody. When
used
to describe the relationship between the anti-glucagon antibody and the GLP-1
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-1 compound maintains a C'iLP-1
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-1 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-1 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-1
compound
and the antibody. Further details regarding options for linking the antibody
and GLP-1
compound(s) are listed below. The compound(s) can be linked to the N- or C-
terminus of
the antibody or both.
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In certain embodiments, the GL,P-1 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-1
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
(Ka) for glucagon of at least 104 or 105/M x seconds. Other antibodies have a
ka 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 Koff of 1 x 10"4s-
1, 1 x 10"5s"'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
cornpositions
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
underivatized or unmodified antibody.
Also provided are polypeptides that contain glucagon binding sites optionally
fused with one or more GLP-1 compounds.
In certain embodiments, a GLP-1/antibody composition comprises at least one
anti-glucagon antibody, at least one linker and at least one GLP-1 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 GL,P-1 compound in some compositions is linked to the N-
terminus of the light and/or heavy chain of the antibody, whereas in other
compositions
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WO 2007/124463 PCT/US2007/067152
the N-terminus of the GLP-1 cornpound is linked to the C-terminus of the light
and/or
heavy chain. In some compositions, the GLP-1 compound is linked via its N- or
C-
terminus to another molecule via its N- or C- terminus.
Some GLP-1/antibody compositions comprise one anti-glucagon antibody and
one GLP-1 compourid. Other cornpositions comprise one antibody and two
compounds.
Still other compositions comprise one antibody and more than two GLP-1
compounds.
Certain compositions comprise more than one antibody and one GLP-1 compound.
Other compositions comprise more than one antibody and more than one compound.
In certain compositions, a first GLP-1 compound is linked to the heavy chain
of a
anti-glucagon antibody and a second GLP-1 compourid having the same or
different
amino acid sequence as the first compound is linked to the light chain of the
aritibody.
The antibody and compound in some compositions of this type are linked via a
linker.
In other compositions, at least one GLP-1 compound is fused to the heavy chain
of an anti-glucagon antibody. In still other compositions, the GLP-1 compound
is fused
to the light chain of a anti-glucagon antibody. In some compositions, a first
GLP-1
compound is fused to the heavy chain of an anti-glucagon antibody and a second
GLP-1
compound having the same or different amino acid sequence as the first
compound is
fused to the liglit chain of the antibody. In certain embodiments, the heavy
chain of an
anti-glucagon antibody is fused to at least two GLP-1 compounds having the
same or
different sequence. In certain embodiments, the light chain of the antibody is
fused to at
least two GLP-1 compounds having the same or different sequence. In certain
embodiments, the heavy chain of the antibody is fused to at least two first
GLP-1
compounds having the same or different sequence and the light chain of the
antibody is
fused to at least two second GLP-1 compounds having the same or different
sequence.
Certain compositions have a ratio of two GLP-1 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-1
compound liriked to a second heavy chain of the antibody. In certain
embodiments, such
a composition comprises a first GLP-1 compound linked to a first light chain
of a
glucagon antibody and a second GLP-1 compound linked to a second light chain
of the
CA 02649751 2008-10-17
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antibody. Other compositions comprising a ratio of two GLP-1 compounds per one
glucagon antibody will be apparent to those of ordinary skill in the art.
Some compositions comprise four GLP-1 compounds per anti-glucagon antibody.
Thus, in some embodiments, a composition comprises a first GLP-1 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-1 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 linlced 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 cornpositions, a first GL,P-1
compound and
a second GLP-1 compound are linked to the N-terminus of a first heavy chain of
an anti-
glucagon antibody, and a third GL,P-1 compound and a fourth GLP-1 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 iri the art can design
additional
compositions that comprise a ratio of four GL,P-1 compounds per one anti-
glucagon
antibody.
Still other compositions comprise eight GLP-1 compounds per one anti-glucagon
antibody. For example, some compositions comprise a first GLP-1 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-
terrninus of the second light chain of the antibody. Other compositions
comprise a first
and second GL,P-1 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
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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-1 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')2, Fv, single chain Fv fragment, a domain
antibody or an
immunoadhesion). The composition may also include a polypeptide that has the
capacity
to bind glucagorr (e.g., a polypeptide that includes antibody antigen-binding
sites).
One exemplary antibody that binds glucagon that is useful in sorne of the
compositions that are provided is referred to as AG159. 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 IJS
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 fixll-
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
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chairi 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 defirie the antibody's
isotype as IgM,
IgD, IgG, IgA, and IgE, respectively. IgG has several subtypes, including, but
not
limited to, IgG1, IgG2, IgG3, and IgG4. IgM subtypes include IgM1 and IgM2.
IgA
subtypes include IgAl 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 CH 1, CH2 and Ci-13. 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-terrninal, both light and heavy chain
variable
regions typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and
FR4.
The assignment of amino acids to each domain is typically in accordance with
the
defrnitioris 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.);
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see also Chothia & Lesk, 1987, J. Mol. 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. Mol. 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.
Bioteclmol. 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. CDR 1 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) (wliere "Xxx" is any amino acid). The
residue following the heavy chain CDR1 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 CDR 1 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.
CDR 1 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 CDRI and the first residue of CDR2,
and CDR2
is almost always 7 residues long. CDR2 is typically preceded by Ile-Tyr, Val-
Tyr, Ile-
Lys, or Ile-Phe. There are almost always 32 residues between the light chain
CDR2 and
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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 rneant herein, the length of heavy chain framework
regions fall
within the following ranges: FR1, 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: FRI, 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 CDR.3s, can result in altered
antibodies that
bind to the same antigen, but with greater affinity. See e.g. Schier et al.,
1996, J. Mol.
Biol. 263:551-67; Yang et al., 1995, J, Mol. 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.
88:11120-
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. Mal. Biol.
310:591-601; Desiderio et al., 2001, J. Mol. Biol. 310:603-15; Yang et al.,
1995, J. Mol.
Biol. 254:392-403; Schier et al., 1996, J. Mol. 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.
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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
AG159.
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
Internal Abbrev Amino Acid Sequence SEQ ID
Ref. No. Name/ NO:
Chain
Type
AG159 IgGl Hl/ QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAP 82
mature form Heavy GKGLEWVAVIYYDGSNKYYADSVK.GRFTISRDITKNTLYLQ
MNSLRAEDTAVYYCARASR.GFDYWGQGTLVTVSSASTKGPS
VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS
GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP
SNTKVDKKVEPKSCDKTHTCPPCPAPEL.LGGPSVFLFPPKP
KUTLMISRTPEVTCVVZTDVSHEDPEVKFNWYVDGVEVHNAK
TKPR.EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP
APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK
GFYPSDIAVEWESNGQPENNYK.TTPPVLDSDGSFFLYSKLT
VDK.SR.WQQGNVFSCSVMHEALHNHYTQK.SLSLSPG(K)
AG159 IgG2 H2/ QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVR.QAP 89
Heavy GKGLEWVAVIYYDGSNKYYADSVKGR.FTISRDITKNTLYLQ
mature form MNSLRAEDTAVYYCAR.ASR.GFDYWGQGTLVTVSSASTK.GPS
VFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTS
GVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKP
SNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTL
MISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPR.
EEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIE
KTISK.TK.GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP
SDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDK.S
R.WQQGNVFSCSVMHEALHNHYTQKSLSLSPG(K)
AG159 Kappa L1/ 40
Light
EIVLTQSPATLSLSPGDRATLSCRASQSVSSYLAWYQQKPG
QAPRLLISDASNRATGIPARFSGSGSGTDFTLTISSLEPED
FAVYYCQQRSNWITFGQGTRLEIKR.TVAAPSVFIFPPSDEQ
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE
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QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT
KSFNRGEC
Table 2
INTERNAL SEQUENCE SEQ
REF. NO. ID
NO:
AG159 mature QVQLVESGGGWQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIYYDGSNK. 39
IgG2 YYADSVKGRFTISRDITKNTLYLQMNSLRAEDTAVYYCARASRGFDYWGQGTLVTVSS
(sequence ASTKGPSVFPLAPCSR.STSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
without C- SSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVER.K.CCVECPPCPAPPVAG
terminal PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQ
lysine, FNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPP
e.g., as SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLT
expressed in VDKSR.WQQGNVFSCSVMHEALHNHYTQK.SLSLSPG
CHO cells)
AG159 Light EIVLTQSPATLSLSPGDRATLSCRASQSVSSYLAWYQQKPGQAPRLLISDASNRATGI 75
Chain mature PARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWITFGQGTRLEIKRTVAAPSVFI
form FPPSDEQLKSGTASVVCLLNNFYPR.EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL
SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
human kappa RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE 80
constant QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
region
AG159 light MDMRVPAQLLGLLLLWLR.GAR.CEIVLTQSPATLSLSPGDRATLSCRASQSVSSYLAWY 81
chain QQKPGQAPRLLISDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNW
including ITFGQGTR.LEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNA
signal LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR.
peptide GEC
human IgG1 ASTKGPSVFPLAPSSKSTSGGTAALGC.LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ 87
constant SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE
region LLGGPSVFLFPPKPKDTLMISR.TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP
REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY
TLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
AG159 IgG1 MDMRVPAQLLGLLLLWLRGARCQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHW 88
HC including VR.QAPGKGLEWVAVIYYDGSNKYYADSVKGRFTISRDITKNTLYLQMNSLRAEDTAVY
signal YCARASRGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP
peptide VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKV
DKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPK.PKDTLMISRTPEVTCVVVDVSHE
DPEVKFNWYVDGVEVHNAKTK.PREEQYNSTYRWSVLTVLHQDWLNGKEYKCK.VSNKA
LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNG
QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL
SPG(K)
human IgG2 ASTKGPSVFPLAPCSR.STSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ 90
constant SSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTK.VDK.TVERKCCVECPPCPAPPVAG
region PSVFLFPPKPKDTLMISR.TPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAK.TKPR.EEQ
FNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPR.EPQVYTLPP
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SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLT
VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(K)
AG159 IgG2 MDMRVPAQLLGLLLLWLR.GAR.CQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHW 91
HC including VRQAPGKGLEWVAVIYYDGSNKYYADSVKGRFTISRDITKNTLYLQMNSLRAEDTAVY
signal YCARASR.GFDYWGQGTLVTVSSASTKGPSVFPLAPCSR.STSESTAALGCLVKDYFPEP
peptide VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKV
DKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV
QFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGK.EYKCKVSNKGLPAP
IEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN
NYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK.SLSLSPG(
K)
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 H1 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 L l 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-1 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, arid 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.
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Table 3
INTERNAL SEQUENCE SEQ
REF. NO. ID
NO:
variable EIVLTQSPATLSLSPGDRATLSCRASQSVSSYLAWYQQKPGQAPRLLISDASNRATGI 79
region of PARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWITFGQGTRLEIK.
the AG159
light chain
AG159 heavy QVQLVESGGGVVQPGRSLR.LSCAASGFTFSSYGMHWVR.QAPGKGLEWVAVIYYDGSNK 83
chain YYADSVKGRFTISRDITKNTLYLQMNSLRAEDTAVYYCARASRGFDYWGQGTLVTVSS
variable
region
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, 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 light chain variable region in some antibodies comprises a
sequence
of amino acids that lias 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 sequerice 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 (CDR.s) and framework regions (FR) of a
given antibody may be identified using the system described by Kabat et al. in
Sequences
of Proteins of Immunological Interest, 5th Ed., US Dept. of Health and Human
Services,
PHS, NIH, NIH Publication no. 91-3242, 1991. Certain antibodies of the
composition
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WO 2007/124463 PCT/US2007/067152
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
Chain CDR AA Sequence
Light CDR1 RASQSVSSYLA
(SEQ ID NO:76)
Light CDR2 DASNRAT
(SEQ ID NO:77)
Light CDR3 QQRSNWIT
(SEQ ID NO:78)
Heavy CDR1 SYGMH
(SEQ ID NO:84)
Heavy CDR2 VIYYDGSNKYYADSVK.G
(SEQ ID NO:85)
Heavy CDR3 ASRGFDY
(SEQ ID NO:86)
The antibodies of certain GLP-1 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 CDR.s 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).
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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-1 compound/antibody cornpositions 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 rnyeloma cells to produce hybridornas. 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, NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U, MPC-11,
MPC 11-X45-GTG 1.7 and S194/5XXO Bul; 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 imnrunized anirnal; 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.
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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-1 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 cliain 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 chirneric antibodies, see, for example, IJ.S. Patent No.
4,816,567; and
Morrison et al., Proc. Natl. Acad. Sci. 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.
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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-
hurnan 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 AG159 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 immuriized 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
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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 a.inino acid sequences, including
the
variable regions. For further details of such methods, see, for example,
W096/33735 and
W094/02602, which are hereby incorporated by reference. Additional methods
relating
to transgenic mice for malcirig 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 W091/10741, W090/04036, 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, refeiTed to herein as "HuMab" mice,
contain a human immunoglobulin gene minilocus that encodes unrearranged human
heavy ( and y) and ic light chain immunoglobulin sequences, together with
targeted
mutations that inactivate the endogenous and ic 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 ah, 1994, Nature
368:
856-859; Lonberg, 1994, Handbook of Exp. Pharmacology 113: 49-101; Taylor et
al.,
1994, International Immunology 6: 579-591; L,onberg 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
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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. Tecln-iologies utilized for producing human antibodies in these
transgenic mice
are disclosed also in WO 98/24893, and Mendez et al., 1997, Nature Genetics
15: 146-
156, which are hereby incorporated by reference. For example, the HCo7 and
HCo12
transgenic mice strains can be used to generate human anti-glucagon
antibodies.
tJsing hybridorna teclulology, 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 al., 1991, J. Mol. Biol. 227:381; and Marks et al.,
1991, J.
Mol, Biol. 222:581). Phage display techniques rnimic imrnune 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. W099/10494 (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 liybridomas or linking of Fab' fragments. See,
e.g.,
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Songsivilai & Lachmann, 1990, Clin. Exp. Immunol. 79: 315-321; Kostelny et
al., 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, Val, Leu, Ile;
2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;
3) acidic: Asp, Glu;
4) basic: His, Lys, Arg;
5) residues that influence chain orientation: Gly, 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.
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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 el al.,
1982, J. Moh
Biol, 157:105-131). It is known that certain amino acids rnay 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 substitutiori 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
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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 prirnary 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
Original Exemplary
Residues Substitutions
Ala Val, Leu, Ile
Arg Lys, Gln, Asn
Asn Gln
Asp Glu
Cys Ser, Ala
Gln Asn
Glu Asp
Gly Pro, Ala
His Asn, Gln, Lys, Arg
Ile Leu, Val, Met, Ala,
Phe, Norleucine
Leu Norleucine, Ile,
Val, Met, Ala, Phe
Arg, Gln, Asn,
Lys 1,4 Diamine-butyric
Acid
Met Leu, Phe, Ile
Phe Leu, Val, Ile, Ala,
Tyr
Pro Ala
Ser Thr, Ala, Cys
Thr Ser
Trp Tyr, Phe
Tyr Trp, Phe, Thr, Ser
Val Ile, Met, Leu, Phe,
Ala, Norleucine
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
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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 irnportant amino acid residues.
One skilled in the art cari 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
clioose 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
experimerits, 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 113:211-222;
Chou et
al., 1978, Adv. Enzymal. Relat. Areas Mol. Biol. 47:45-148; Chou et al., 1979,
Ann. Rev.
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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 embodirnents,
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
CA 02649751 2008-10-17
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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 51: 1-84; Ravetch and Bolland, 2001,
Anrru.
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 el 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
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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 iminunoadhesin to bind specifically to a particular antigen
of interest
(e.g., glucagon or an epitope thereof).
Mimetics (e.g., peptide mimetics" or "peptidomirnetics") 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
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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 ai-t (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-lifflced dirners, 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-1 Compounds
A variety of GLP-1 compounds can be linlced to the anti-glucagon antibody of
the
composition, including GLP-1 itself and a wide variety of GLP-1 analogs.
As used herein, the term "GL,P-1" refers to glucagon-like peptide 1 as
described
in the Background above. The carboxyl terminus of GLP-1 (1-31)-OH can be
cleaved to
produce GLP-1 (1-30)-NH2. As discussed above, both GLP-1 (1-31)-OH, also
referred to
as GLP-1 (1-31), and GLP-1 (1-30)-NH2, have the same activities. For
convenience, the
terms "GLP-1" and "native GLP-1" 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 wliich the N-
terminal
histidine of GLP-1 is considered as residue number one. Thus, native GLP-1
(i.e., GLP-
1(1-31)-OH) has the following amino acid sequence:
'His-2 Ala-3Glu-4Gly-SThr-'Phe-'Thr-BSer-9Asp-10Val-" Ser-1ZSerj 3Tyr-' 4Leu-
IsGlu-16C'rly-"Gln-"Ala-19A1a-20Lys?IGIu-22Phe 23I1e?4Ala-2STrp-26Leu?'Val-
28Lys?9Gly-3 Arg 31Gly (SEQ ID NO: 1).
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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 Gly at position
16 has been
substituted with Ala. If amino acids are added at the amino terminus of GLP-1
(1-31),
the positions are consecutively numbered in decreasing order, such that the
amino acid
immediately upstream of position I is amino acid -1, and the next upstream
amino acid is
at position -2 and so on. If arnino acids are added at the carboxyl terminus
of GLP-1, 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-1
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-1 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-1 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-1 compound" as used herein refers to a molecule that comprises a GLP-1
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-1 peptide" as used herein refers to native GLP-1 or a peptide
with
one or more alterations in the amino acid sequence of native GLP-1 (1-31)-QH
or GLP-1
(1-30)-NH2 but that retains at least one activity of native GLP-1. The term
also includes
members of the exendin family such as exendin-3 and exendin-4 (see, e.g.,
IJ.S. Patent
No. 5,424,286) or peptides with one or more alterations in the amino acid
sequence of the
exendin, provided the peptide retairis at least one GLP-1 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).
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Exendin-3 has the following amino acid sequence:
His Ser Asp Gly Thr Phe Thr Ser Asp Leu Ser Lys Glrr 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
The phrase "GLP-1 activity" or grammatical equivalents thereof refers broadly
to
any activity associated with GLP-1 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 (3-cell proliferation and
replication, increase
in (3-cell mass, increase in satiety and decrease in food intake when
admirristered to a
subject.
The term "GLP-1 peptide" also includes variants, fragments and derivatives of
the
GLP-1 peptides that are functional equivalents to one of the GLP-1 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-1 peptide.
"GLP-1 variants" include peptides that are "substantially identical" (see
definition
supra) to the GLP-1 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-1 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-1 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-1 peptide as described herein, provided the variant still has a GLP-
1 activity.
A"GLP-1 derivative" as used herein refers to one of the GLP-1 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.
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A"GL,P-1 fragment" refers to truncated forins of the GLP-1 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-1 peptides set forth herein. Truncation can be
at either
the amino and/or carboxyl termimis.
Numerous examples of GLP-1 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-1 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;
IJS 2003/0221201; IJS 2003/0226155; US 2004/0023334; US 2004/0 1 43 1 04;
US 2005/0107318; US 2004/0106547; IJS 2004/0176307; IJS 2004/0052862;
US 2004/0082507; US 2004/0146985; US 2004/0053370; IJS 2003/0199672; and
IJS 2001/0011071, each of which is incorporated herein by reference in its
entirety. Still
other GL,P-1 peptides that can be used in certain compositions are described
for example
in the following published PCT applications: WO 00/34331; WO 0034332;
W002/46227; W003/060071; W02005/003296; WO 03/018516; WO 01/98331;
WO 03/059934; WO 2004/078777; WO 99/30731; WO 98/43658; WO 00/16797;
WO 00/15224; W003/103572; W003/087139; 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 91/11457; 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;
W02005/027978; WO 04/074315; W02005/028516; and W02005/046716.
Additional GLP-1 peptides that can be used in some compositions are described
in
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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-1 peptides that are in some compositions comprise the amino acid
sequence of formi.ila I (SEQ ID NO: 92):
Xaar-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaaio-XaaI I-Xaa12_Xaa13-
Xaa ]4-Xaa l5-Xaal6-Xaal 7-Xaa,8-Xaa19-XaaZO-Xaa2 l_Xaa22-Xaa23-Xaa24_Xaa25-
Xaa26-Xaa27_Xaa28_Xaa29-Xaa3O -Xaa3 I - Xaa32-Xaa3:3-Xaa34-Xaa35-Xaa36-Xaa37-
C(O)-R, (Formula I, SEQ ID NO:92)
wherein,
R, is OR2 or NRZR3;
R.2 and R3 are independently hydrogen or (Cr-C8)alkyl;
Xaa at position 1 is: L-histidine, D-histidine, desamino-histidine, 2-amino-
histidine, 3-hydroxy-histidine, homohistidine, a-fluoromethyl-histidine or a-
methyl-histidine;
Xaa at position 2 is Gly, bAla (2-aminopropionic acid), Asp, Ala, 1-amino-
cylcopentanecarboxylic acid, 2-aminoisobutryic acid or alpha-alpha-
disubstituted amino
acids;
Xaa at position 3 is Glu, Asp, or Lys;
Xaa at position 4 is Gly, Thr or His;
Xaa at position 5 is Thr, Ala, Gly, Ser, Leu, Ile, Val, Glu, Asp, or Lys;
Xaa at position 6 is: His, Trp, Phe, or Tyr;
Xaa at position 7 is Thr or Gly;
Xaa at position 8 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu, Asp, or Lys;
Xaa at position 9 is Asp, Asn or Glu;
Xaa at position 10 is Val, Ala, Gly, Ser, Thr, Leu, Ile, Tyr, Glu, Asp, Trp,
or Lys;
Xaa at position 11 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu, Asp, or Lys;
Xaa at position 12 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu, Asp, Trp, Tyr,
Asn,
Lys, Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-
Homoglutamic acid, or homoglutamic acid;
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Xaa at position 13 is Tyr, Phe, Trp, Glu, Asp, Gln, Lys, Homolysine,
Ornithine,
4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, or
homoglutamic acid;
Xaa at position 14 is Leu, Ala, Gly, Ser, Thr, Ile, Val, Glu, Asp, Met, Trp,
Tyr,
Asn, Gln, Lys, Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic
acid, or homoglutamic acid;
Xaa at position 15 is Glu, Asp, Lys, Homolysine, Ornithine, 4-carboxy-
phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, or homoglutarnic
acid;
Xaa at position 16 is Gly, Ala, Ser, Thr, Leu, Ile, Val, Glu, Asp, Asn, Lys,
Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-
Homoglutamic acid, or homoglutamic acid;
Xaa at position 17 is Gln, Asn, Arg, Glu, Asp, Lys, Ornithine, 4-carboxy-
phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, or homoglutamic
acid;
Xaa at position 18 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Arg, Ghz, Asp, Asn,
Lys,
Homolysine, Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-
Homoglutamic acid, or homoglutamic acid;
Xaa at position 19 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Glu, 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, Gln, Glu, Asp, Thr, His,
Ornithine,
4-carboxy-phenylalanine, beta-glutamic acid, or homoglutamic acid;
Xaa at position 21 is Leu, Glu, 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, Glu, Lys, Homolysine, Ornithine, 4-
carboxy-
phenylalanine, beta-glutamic acid, beta-Homoglutamic acid, or homoglutarnic
acid;
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Xaa at position 23 is Ile, Leu, Val, Ala, Phe, Asp, Glu, Lys, Homolysine,
Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic
acid,
or homoglutamic acid;
Xaa at position 24 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Glu, Asp, Lys,
Homolysine,
Ornithine, 4-carboxy-phenylalanine, beta-glutamic acid, beta-Homoglutamic
acid,
or homoglutamic acid;
Xaa at position 25 is Trp, Phe, Tyr, Glu, Asp, Asn, or Lys;
Xaa at position 26 is Leu, Gly, Ala, Ser, Thr, Ile, Val, Glu, Asp, or Lys;
Xaa at position 27 is Val, Gly, Ala, Ser, Thr, Leu, Ile, Glu, Asp, Asn, or
Lys;
Xaa at position 28 is Asn, Lys, Arg, Glu, Asp, or His;
Xaa at position 29 is Gly, Ala, Ser, Thr, Leu, Ile, Val, Glu, Asp, or Lys;
Xaa at position 30 is Gly, Arg, Lys, Glu, Asp, Thr, Asn, or His;
Xaa at position 31 is Pro, Gly, Ala, Ser, Thr, Leu, Ile, Val, Glu, Asp, or
Lys;
Xaa at position 32 is Thr, Gly, Asn, Ser, Lys, or is omitted;
Xaa at position 33 is Gly, Asn, Ala, Ser, Thr, Ile, Val, Leu, Phe, Pro, or is
omitted;
Xaa at position 34 is Gly, Thr, or is omitted;
Xaa at position 35 is Thr, Asn, Gly or is omitted;
Xaa at position 36 is Gly or is omitted;
Xaa at position 37 is Gly 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-1 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 cornpositions, the GLP-1 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-1 peptide in some other compositions
comprises SEQ ID NO: 1-35, SEQ ID NO: 126, or SEQ ID NO: 127 with no more than
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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-1 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
GLP-1 Peptide Identifier GLP-1 Peptide SEQ ID NO:
GLP(A2G) HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRG 126
GLP1(A2G/V27E/K28N/R.30G) HGEGTFTSDVSSYLEGQAAKEFIAWLENGGG 2
GLP1(A2G/ V27K/K28N/R30G) HGEGTFTSDVSSYLEGQAAKEFIAWLKNGGG 3
GLP1(A2G/R30G) HGEGTFTSDVSSYLEGQAAKEFIAWLVK.GGG 4
GLP1(A2G delta C-term) HGEGTFTSDVSSYLEGQAAKEFIAWL 5
HGEGTFTSDVSSYLEGQAAKEFIAWLKNGGP
GLP1(A2G chimera) SSGAPPPS 6
GLP(A2G/VlOQ) HGEGTFTSDQSSYLEGQAAKEFIAWLVK.GRG 7
GLP(A2G/VlOQ/L14Q) HGEGTFTSDQSSYQEGQAAKEFIAWLVKGRG 8
GLP(A2G/V10Q/V27Q) HGEGTFTSDQSSYLEGQAAK.EFIAWLQKGR.G 9
GLP(A2G/L14Q) HGEGTFTSDVSSYQEGQAAKEFIAWLVKGRG 10
GLP(A2G/W25Q) HGEGTFTSDVSSYLEGQAAKEFIAQLVKGRG 11
GLP(A2G/W25Q/V27Q) HGEGTFTSDVSSYLEGQAAKEFIAQLQKGRG 12
GLP(A2G/V27Q) HGEGTFTSDVSSYLEGQAAKEFIAWLQKGRG 13
GLP(A2N/G4T) HNETTFTSDVSSYLEGQAAKEFIAWLVK.GRG 14
GLP(A2G/E3N) HGNGTFTSDVSSYLEGQAAKEFIAWLVKGR.G 15
GLP(E3N) HANGTFTSDVSSYLEGQAAK.EFIAWLVKGR.G 16
GLP(A2G/T5N) HGEGNFTSDVSSYLEGQAAKEFIAWLVKGR.G 17
GLP(A2G/D9N/S11T) HGEGTFTSNVTSYLEGQAAKEFIAWLVKGR.G 18
GLP(A2G/V10N/S12T) HGEGTFTSDNSTYLEGQAAKEFIAWLVKGRG 19
GLP(A2G/S12N/L14T) HGEGTFTSDVSNYTEGQAAKEFIAWLVKGRG 20
GLP(A2G/Ll4N/G16T) HGEGTFTSDVSSYNETQAAKEFIAWLVKGRG 21
GLP(A2G/G16N/A18T) HGEGTFTSDVSSYLENQTAKEFIAWLVKGRG 22
GLP(A2G/Q17N/A19T) HGEGTFTSDVSSYLEGNATKEFIAWLVKGRG 23
GLP(A2G/A18N/K20T) HGEGTFTSDVSSYLEGQNATEFIAWLVKGRG 24
GLP(A2G/A19N/E21T) HGEGTFTSDVSSYLEGQANKTFIAWLVKGRG 25
GLP(A2G/W25N/V27T) HGEGTFTSDVSSYLEGQAAKEFIANLTKGRG 26
GLP(A2G/V27N/G29T) HGEGTFTSDVSSYLEGQAAKEFIAWLNK.TRG 27
GLP(A2G/K28N/R30T) HGEGTFTSDVSSYLEGQAAKEFIAWLVNGTG 28
GLP(A2G/G29N/G31T) HGEGTFTSDVSSYLEGQAAKEFIAWLVKNRT 29
HGEGTFTSDVSSYLEGQAAKEFIAWLVKGNG
GLP(A2G/R30N/+T32) T 30
HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRN
GLP(A2G/G31N/+G32/+T33) GT 31
HGEGTFTSDVSSYLEGQAAK.EFIAWLVKGRG
GLP(A2G/+N32/+G33/+T34) NGT 32
GLP(A2G/+G32/+N33/+G34/+T HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRG 33
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35) GNGT
GLP(A2G/+G32/+T33/+G34/+N HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRG
35/+G36/+T37) GTGNGT 34
GLP(A2G/+G32/+S33/+G34/+N HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRG
35/+G36/+T36)/ GSGNGT 35
C. Linking Antibody and GLP-1 Compound
As noted above, when the term "linked" is used in reference to the anti-
glucagon
antibody and the GLP-1 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
linlced together
by peptide bonds, i.e., a linker comprises a peptide. Thus, in certain
embodiments, a
linker is a peptide having between I 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.,
(G1y)4, (Gly)5), a poly(Gly-Ala), and a polyalanine. Certain exemplary linkers
include,
but are not limited to:
(G1Y)3LYs(G1Y)4 (SEQ ID NO:);
(Gly).3AsnGlySer(Gly)2 (SEQ ID NO:);
(Gly)3Cys(Gly)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 Gly-
Gly-Gly-I,ys-Gly-Gly-Gly-Gly. In certain embodiments, a linker comprises a
combination of Gly and Ala residues. In certain embodiments, a linker
comprises 10 or
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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
liiikers 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-
(CH2)S-
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., C1-C6) lower acyl, halogen (e.g., Cl, Br), CN, NH2,
phenyl, etc. A
non-limiting exemplary non-peptide litiker is a PEG lil-ilcer,
O
O O
N O
H
wherein n is a number sucli that the linker has a molecular weight of 100 to
5000 kD. In
certain embodiments, n is a nurnber 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 frorn a chemical and/or enzymatic
process used to corinect 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,).
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D. Specific E',xamples of GLP-1 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 AG159 (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-1
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-1 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-1 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-1 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-1 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-1 peptides disclosed herein.
As already described at length above, these GLP-1 peptides can be linked to
the
AG159 antibody or fragments in a variety of different ways, including, for
example, such
that multiple GLP-1 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 forrns. 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-1
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-1 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
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location of the addition. If the GLP-1 peptide is attached to the L,C, this is
indicated as
GLP-AG159LC:AG15, with the GLP-1 peptide being listed to the left of the
colon. If the
GLP-1 peptide is attached to the HC, this is indicated as AG159LC:GL,P-AG159,
i.e.,
with the GLP-1 peptide being listed to the right of the colon. The GLP-1
peptide and the
light or heavy chain of the AG159 antibody are fused together via a linker
(e.g. SEQ ID
Nos: 36-38).
In these specific compositions, the AG159 antibody is shown to be of either
the
IgGI or IgG2 isotype, but could be of any of the other immunoglobulin
isotypes. In these
fusions, the carboxy terminus of the GLP-1 peptide is fused to the amino
terminus of the
AG159 antibody via a linker (e.g., SEQ ID Nos:36-38). However, as noted above,
the
GLP-1 peptides can be attached at other locations and in other orientations.
Certain compositions comprise a light chain polypeptide ffixsion 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 AG159 (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 AG159 to form an
antibody with
tetrameric structure.
Other cornpositions are similar except that the GLP-1 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 AG159 (SEQ ID NO: 82 or 89) instead of the light chain. Such heavy chain
polypeptide fusions can be paired with the light chain of AG159 (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 AG159 to form an antibody with
tetrameric structure.
As described in detail above, the GLP-1 compound/antibody compositions
provided herein include those in which the antibody and/or the GLP-1 peptide
are
variants of those listed in the tables herein. The alterations can be in the
antibody and/or
the GLP-1 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.
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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
Inter'nal Ref Amino Acid Sequence of the GLP-1 Heavy or Light Chain SEQ
## Fusion (includes linker sequence) ID
NO:
AG159LC:GLP(A HGEGTFTSDVSSYLEGQAAK.EFIAWLVK.GR.GGSGSATGGSGSTASSGSGSATGGGGGG
128
2G)-AG159IgG2 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIYYDGSNK
YYADSVKGRFTISRDITKNTLYLQMNSLRAEDTAVYYCARASR.GFDYWGQGTLVTVSS
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV.LQ
SSGLYSLSSWTVPSSNFGTQTYTCNVDHK.PSNTKVDKTVERKCCVECPPCPAPPVAG
PSVFLFPPK.PKDTLMISR.TPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQ
FNSTFR.VVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTK.GQPREPQVYTLPP
SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLT
VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
GLP1(A2G)- HGEGTFTSDVSSYLEGQAAKEFIAWLVKGR.GGSGSATGGSGSTASSGSGSATGGGGGG 129
AG159LC:AG159 EIVLTQSPATLSLSPGDRATLSCRASQSVSSYLAWYQQKPGQAPRLLISDASNRATGI
IgG2 PARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWITFGQGTRLEIK.RTVAAPSVFI
FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL
SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR.GEC
GLP1(A2G/K28E HGEGTFTSDVSSYLEGQAAKEFIAWLENGGGGSGSATGGSGSTASSGSGSATGGGGG 41
/G29N/R30G)- GEIVLTQSPATLSLSPGDRATLSCRASQSVSSYLAWYQQKPGQAPRLLISDASNRAT
AG159LC:AG159 GIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWITFGQGTRLEIKRTVAAPS
IgG2 VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS
TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
GLP1(A2G/G29N HGEGTFTSDVSSYLEGQAAKEFIAWLKNGGGGSGSATGGSGSTASSGSGSATGGGGG 42
/R30G)- GEIVLTQSPATLSLSPGDRATLSCRASQSVSSYLAWYQQKPGQAPRLLISDASNRAT
AG159LC:AG159 GIPAR.FSGSGSGTDFTLTISSLEPEDFAVYYCQQR.SNWITFGQGTR.LEIKRTVAAPS
IgG2 VFIFPPSDEQLKSGTASVVCLLNNFYPR.EAK.VQWKVDNALQSGNSQESVTEQDSKDS
TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
GLP1(A2G/R.30G HGEGTFTSDVSSYLEGQAAKEFIAWLVKGGGGSGSATGGSGSTASSGSGSATGGGGG 43
GEIVLTQSPATLSLSPGDRATLSCRASQSVSSYLAWYQQKPGQAPR.LLISDASNRAT
AG159LC:AG159 GIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWITFGQGTR.LEIKRTVAAPS
IgG2 VFIFPPSDEQLKSGTASVVCLLNNFYPREAK.VQWKVDNALQSGNSQESVTEQDSKDS
TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
GLP1-AG159LC HGEGTFTSDVSSYLEGQAAKEFIAWLGSGSATGGSGSTASSGSGSATGGGGGGEIVL 44
deltaC- TQSPATLSLSPGDRATLSCRASQSVSSYLAWYQQKPGQAPRLLISDASNRATGIPAR
term:AG159 FSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWITFGQGTRLEIKRTVAAPSVFIFP
IgG2 PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS
STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR.GEC
GLPl-AG159 LC HGEGTFTSDVSSYLEGQAAKEFIAWLKNGGPSSGAPPPSGSGSATGGSGSTASSGSG 45
chime.ra:AG159 SATGGGGGGEIVLTQSPATLSLSPGDRATLSCRASQSVSSYLAWYQQKPGQAPRLLI
IgG2 SDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWITFGQGTRLEI
KR.TVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESV
TEQDSKDSTYSLSSTLTLSKADYEKHK.VYACEVTHQGLSSPVTKSFNRGEC
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GLP(A2G/V10Q) HGEGTFTSDQSSYLEGQAAKEFIAWLVKGRGSGGGGSGGGGSGGGGSGGGGSGGGGG 46
EIVLTQSPATLSLSPGDRATLSCRASQSVSSYLAWYQQKPGQAPRLLISDASNRATG
AG159LC:AG159 IPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWITFGQGTRLEIKRTVAAPSV
IgG2 FIFPPSDEQLKSGTASVVCLLNNFYPR.EAKVQWKVDNALQSGNSQESVTEQDSKDST
YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK.SFNRGEC
GLP(A2G/V10Q/ HGEGTFTSDQSSYQEGQAAKEFIAWLVKGRGSGGGGSGGGGSGGGGSGGGGSGGGGG 47
L14Q)-AG159 EIVLTQSPATLSLSPGDRATLSCRASQSVSSYLAWYQQKPGQAPRLLISDASNRATG
LC:AG159 IgG2 IPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWITFGQGTRLEIKRTVAAPSV
FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST
YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
GLP(A2G/VlOQ/ HGEGTFTSDQSSYLEGQAAKEFIAWLQKGRGSGGGGSGGGGSGGGGSGGGGSGGGGG 48
V27Q)-AG159 EIVLTQSPATLSLSPGDRATLSCRASQSVSSYLAWYQQKPGQAPRLLISDASNRATG
LC:AG159 IgG2 IPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQR.SNWITFGQGTRLEIKRTVAAPSV
FIFPPSDEQLKSGTASWCLLNNFYPR.EAKVQWKVDNALQSGNSQESVTEQDSKDST
YSLSSTLTLSKADYEKHK.VYACEVTHQGLSSPVTK.SFNRGEC
GLP(A2G/L14Q) HGEGTFTSDVSSYQEGQAAKEFIAWLVKGRGSGGGGSGGGGSGGGGSGGGGSGGGGG 49
-AG159 EIVLTQSPATLSLSPGDRATLSCRASQSVSSYLAWYQQKPGQAPRLLISDASNRATG
LC:AG159 IgG2 IPAR.FSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWITFGQGTRLEIKRTVAAPSV
FIFPPSDEQLKSGTASWCLLNNFYPR.EAKVQWKVDNALQSGNSQESVTEQDSKDST
YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
LP(A2G/W25Q)- HGEGTFTSDVSSYLEGQAAKEFIAQLVKGRGSGGGGSGGGGSGGGGSGGGGSGGGGG 50
AG159 EIVLTQSPATLSLSPGDRATLSCRASQSVSSYLAWYQQKPGQAPRLLISDASNRATG
LC:AG159 IgG2 IPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWITFGQGTRLEIKRTVAAPSV
FIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST
YSLSSTLTLSKADYEKHK.VYACEVTHQGLSSPVTKSFNRGEC
GLP(A2G/W25Q/ HGEGTFTSDVSSYLEGQAAKEFIAQLQKGRGSGGGGSGGGGSGGGGSGGGGSGGGGG 51
V27Q)-AG159 EIVLTQSPATLSLSPGDRATLSCRASQSVSSYLAWYQQKPGQAPRLLISDASNRATG
LC:AG159 IgG2 IPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWITFGQGTRLEIKRTVAAPSV
FIFPPSDEQLKSGTASWCLLNNFYPR.EAKVQWKVDNALQSGNSQESVTEQDSKDST
YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK.SFNRGEC
GLP(A2G/V27Q) HGEGTFTSDVSSYLEGQAAKEFIAWLQKGRGSGGGGSGGGGSGGGGSGGGGSGGGGG 52
-AG159 EIVLTQSPATLSLSPGDRATLSCRASQSVSSYLAWYQQKPGQAPRLLISDASNRATGI
LC:AG159 IgG2 PARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWITFGQGTRLEIKRTVAAPSVFI
FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL
SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
GLP(A2N/G4T)- HNETTFTSDVSSYLEGQAAKEFIAWLVKGRGSGGGGSGGGGSGGGGSGGGGSGGGGGE 53
AG159 IVLTQSPATLSLSPGDRATLSCRASQSVSSYLAWYQQK.PGQAPRLLISDASNRATGIP
LC:AG159 IgG2 ARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWITFGQGTRLEIKRTVAAPSVFIF
PPSDEQLKSGTASWC.L.LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS
STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
GLP(A2G/E3N)- HGNGTFTSDVSSYLEGQAAKEFIAWLVKGRGSGGGGSGGGGSGGGGSGGGGSGGGGGE 54
AG159 IVLTQSPATLSLSPGDRATLSCRASQSVSSYLAWYQQKPGQAPRLLISDASNRATGIP
LC:AG159 IgG2 ARFSGSGSGTDFTLTISSLEPEDFAVYYCQQR.SNWITFGQGTRLEIKR.TVAAPSVFIF
PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS
STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR.GEC
GLP(E3N)- HANGTFTSDVSSYLEGQAAKEFIAWLVKGRGSGGGGSGGGGSGGGGSGGGGSGGGGGE 55
AG159 IVLTQSPATLSLSPGDRATLSCRASQSVSSYLAWYQQKPGQAPRLLISDASNRATGIP
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LC:AG159 IgG2 ARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWITFGQGTRLEIKRTVAAPSVFIF
PPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS
STLTLSKA.DYEK.HKVYACEVTHQGLSSPVTKSFNRGEC
GLP(A2G/T5N)- HGEGNFTSDVSSYLEGQAAKEFIAWLVKGR.GSGGGGSGGGGSGGGGSGGGGSGGGGGE 56
AG159 IVLTQSPATLSLSPGDRATLSCRASQSVSSYLAWYQQKPGQAPRLLISDASNRATGIP
LC:AG159 IgG2 ARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWITFGQGTRLEIKRTVAAPSVFIF
PPSDEQLKSGTASVVCLLNNFYPREAKVQWK.VDNALQSGNSQESVTEQDSKDSTYSLS
STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
GLP(A2G/D9N/S HGEGTFTSNVTSYLEGQAAKEFIAWLVKGRGSGGGGSGGGGSGGGGSGGGGSGGGGGE 57
11T)-AG159 IVLTQSPATLSLSPGDRATLSCRASQSVSSYLAWYQQKPGQAPRLLISDASNRATGIP
LC:AG159 IgG2 ARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWITFGQGTR.LEIKRTVAAPSVFIF
PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS
STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
GLP(A2G/V10N/ HGEGTFTSDNSTYLEGQAAKEFIAWLVKGR.GSGGGGSGGGGSGGGGSGGGGSGGGGGE 58
S12T)-AG159 IVLTQSPATLSLSPGDRATLSCRASQSVSSYLAWYQQKPGQAPRLLISDASNRATGIP
LC:AG159 IgG2 ARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWITFGQGTRLEIKR.TVAAPSVFIF
PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS
STLTLSKADYEK.HKVYACEVTHQGLSSPVTKSFNRGEC
GLP(A2G/S12N/ HGEGTFTSDVSNYTEGQAAKEFIAWLVKGRGSGGGGSGGGGSGGGGSGGGGSGGGGGE 59
L14T)-AG159 IVLTQSPATLSLSPGDRATLSCRASQSVSSYLAWYQQKPGQAPRLLISDASNRATGIP
LC:AG159 IgG2 ARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWITFGQGTRLEIKR.TVAAPSVFIF
PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS
STLTLSKADYEKHKVYACEVTHQGLSSPVTK.SFNRGEC
GLP(A2G/L14N/ HGEGTFTSDVSSYNETQAAKEFIAWLVKGRGSGGGGSGGGGSGGGGSGGGGSGGGGGE 60
G16T)-AG159 IVLTQSPATLSLSPGDRATLSCRASQSVSSYLAWYQQKPGQAPRLLISDASNRATGIP
LC:AG159 IgG2 ARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWITFGQGTRLEIKRTVAAPSVFIF
PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS
STLTLSKADYEK.HK.VYACEVTHQGLSSPVTKSFNRGEC
GLP(A2G/G16N/ HGEGTFTSDVSSYLENQTAKEFIAWLVKGRGSGGGGSGGGGSGGGGSGGGGSGGGGGE 61
A18T)-AG159 IVLTQSPATLSLSPGDRATLSCRASQSVSSYLAWYQQKPGQAPRLLISDASNRATGIP
LC:AG159 IgG2 ARFSGSGSGTDFTLTISS.LEPEDFAVYYCQQRSNWITFGQGTR.LEIKR.TVAAPSVFIF
PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS
STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
GLP(A2G/Q17N/ HGEGTFTSDVSSYLEGNATK.EFIAWLVK.GRGSGGGGSGGGGSGGGGSGGGGSGGGGGE 62
A19T)-AG159 IVLTQSPATLSLSPGDR.ATLSCRASQSVSSYLAWYQQKPGQAPRLLISDASNRATGIP
LC:AG159 IgG2 ARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWITFGQGTRLEIKRTVAAPSVFIF
PPSDEQLKSGTASVVCLLNNFYPR.EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS
STI.,TI,SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
GLP(A2G/A18N/ HGEGTFTSDVSSYLEGQNATEFIAWLVKGRGSGGGGSGGGGSGGGGSGGGGSGGGGGE 63
K.20T)-AG159 IVLTQSPATLSLSPGDRATLSCRASQSVSSYLAWYQQKPGQAPRLLISDASNRATGIP
LC:AG159 IgG2 ARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWITFGQGTRLEIKRTVAAPSVFIF
PPSDEQLKSGTASVVCLLNNFYPREAK.VQWKVDNALQSGNSQESVTEQDSKDSTYSLS
STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
GLP(A2G/A19N/ HGEGTFTSDVSSYLEGQANKTFIAWLVKGRGSGGGGSGGGGSGGGGSGGGGSGGGGGE 64
E21T)-AG159 IVLTQSPATLSLSPGDRATLSCRASQSVSSYLAWYQQKPGQAPRLLISDASNRATGIP
LC:AG159 IgG2 ARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWITFGQGTR.LEIKRTVAAPSVFIF
PPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS
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STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
GLP(A2G/W25N/ HGEGTFTSDVSSYLEGQAAKEFIANLTKGRGSGGGGSGGGGSGGGGSGGGGSGGGGGE 65
V27T)-AG159 IVLTQSPATLSLSPGDRATLSCRASQSVSSYLAWYQQKPGQAPRLLISDASNRATGIP
LC:AG159 IgG2 ARFSGSGSGTDFTLTISSLEPEDFAVYYCQQR.SNWITFGQGTR.LEIKR.TVAAPSVFIF
PPSDEQLKSGTASWCLLNNFYPR.EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS
STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
GLP(A2G/V27N/ HGEGTFTSDVSSYLEGQAAKEFIAWLNKTR.GSGGGGSGGGGSGGGGSGGGGSGGGGGE 66
G29T)-AG159 IVLTQSPATLSLSPGDRATLSCRASQSVSSYLAWYQQKPGQAPRLLISDASNRATGIP
LC:AG159 IgG2 ARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWITFGQGTRLEIKRTVAAPSVFIF
PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS
STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR.GEC
GLP(A2G/K28N/ HGEGTFTSDVSSYLEGQAAKEFIAWLVNGTGSGGGGSGGGGSGGGGSGGGGSGGGGGE 67
R30T)-AG159 IVLTQSPATLSLSPGDRATLSCRASQSVSSYLAWYQQKPGQAPRLLISDASNRATGIP
LC:AG159 IgG2 ARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWITFGQGTR.LEIKRTVAAPSVFIF
PPSDEQLKSGTASVVCLLNNFYPR.EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS
STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
GLP(A2G/G29N/ HGEGTFTSDVSSYLEGQAAKEFIAWLVKNRTSGGGGSGGGGSGGGGSGGGGSGGGGGE 68
G31T)-AG159 IVLTQSPATLSLSPGDRATLSCRASQSVSSYLAWYQQKPGQAPRLLISDASNRATGIP
LC:AG159 IgG2 ARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWITFGQGTRLEIKRTVAAPSVFIF
PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS
STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR.GEC
GLP(A2G/R.30N/ HGEGTFTSDVSSYLEGQAAKEFIAWLVKGNGTSGGGGSGGGGSGGGGSGGGGSGGGGG 69
+T32)-AG159 EIVLTQSPATLSLSPGDRATLSCRASQSVSSYLAWYQQKPGQAPRLLISDASNRATGI
LC:AG159 IgG2 PAR.FSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWITFGQGTRLEIKRTVAAPSVFI
FPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL
SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
GLP(A2G/G31N/ HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRNGTSGGGGSGGGGSGGGGSGGGGSGGGG 70
+G32/+T33)- GEIVLTQSPATLSLSPGDRATLSCRASQSVSSYLAWYQQKPGQAPRLLISDASNRATG
AG159 IPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWITFGQGTRLEIKRTVAAPSVF
LC:AG159 IgG2 IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS
LSSTLTLSKADYEK.HKVYACEVTHQGLSSPVTKSFNRGEC
GLP(A2G/+N32/ HGEGTFTSDVSSYLEGQAAK.EFIAWLVKGR.GNGTSGGGGSGGGGSGGGGSGGGGSGGG 71
+G33/+T34)- GGEIVLTQSPATLSLSPGDRATLSCRASQSVSSYLAWYQQKPGQAPRLLISDASNRAT
AG159 GIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWITFGQGTRLEIKRTVAAPSV
LC:AG159 IgG2 FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY
SLSSTLTLSKADYEKHK.VYACEVTHQGLSSPVTKSFNRGEC
GLP(A2G/+G32/ HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRGGNGTSGGGGSGGGGSGGGGSGGGGSGG 72
+N33/+G34/+T3 GGGEIVLTQSPATLSLSPGDRATLSCRASQSVSSYLAWYQQKPGQAPRLLISDASNRA
5)-AG159 TGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWITFGQGTRLEIKRTVAAPS
LC:AG159 IgG2 VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST
YSLSSTLTLSKADYEK.HKVYACEVTHQGLSSPVTKSFNR.GEC
GLP(A2G/+G32/ HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRGGTGNGTSGGGGSGGGGSGGGGSGGGGS 73
+T33/+G34/+N3 GGGGGEIVLTQSPATLSLSPGDRATLSCRASQSVSSYLAWYQQKPGQAPRLLISDASN
5/+G36/+T37)- RATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWITFGQGTRLEIKRTVAA
AG159 PSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD
LC:AG159 IgG2 STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
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GLP(A2G/+G32/ HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRGGSGNGTSGGGGSGGGGSGGGGSGGGGS 74
+S33/+G34/+N3 GGGGGEIVLTQSPATLSLSPGDRATLSCRASQSVSSYLAWYQQKPGQAPRLLISDASN
5/+G36/+T36)/ RATGIPAR.FSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWITFGQGTRLEIKR.TVAA
-AG159 PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK.VQWK.VDNALQSGNSQESVTEQDSKD
LC:AG159 IgG2 STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
E. Optional Components
In some compositions, the GLP-1 compound and/or the anti-glucagon antibody
are modified to include additional components. For instance, the GLP-1
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 0-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-(C1-C10), 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-1 compound is complexed with suitable
divalent
metal cations. Divalent metal complexes of GLP-1 compounds can be administered
subcutaneously as suspensions, and have a decreased rate of release in vivo,
because such
complexes of GLP-1 compounds are generally insoluble in aqueous solutions of
about
physiological pH. Non-limiting examples of divalent metal cations suitable for
complexing with a GLP-1 compound include Zn++, Mn++, Fe++, Ca++, Co++, Cd++,
Ni++,
and the like. Divalent metal complexes of GLP-1 compounds can be obtained, for
example, using techniques as described in WO 01/98331, which is incorporated
herein by
reference.
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IV. Nucleic Acids
Nucleic acids that encode one or both chains of an antibody or the fusion of a
GL,P-1 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) rnay 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 kriown 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
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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 et al., 1986, Trends Biochem. Sci. 11:287, Maniatis et
al., 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
inverition 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. coli) 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 coriventional 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 integrarrts, 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.
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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-hurnari 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 isolatiori 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 irnmunized 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) rnay be prepared by fusing DNA encoding a peptide
linker
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between DNAs encoding the two variable domain polypeptides (VL and Vi-I). 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 VL
and VH-
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 Mol
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 riew 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
Mol. Bio1.178:303-16. 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 beerr used to prepare high affinity antibodies to
the hapten
2-phenyloxazol-5-one, as described by Marks et al., 1992, BioTechnology,
10:779.
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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 nonnative 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
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exemplified by U.S. Pat. Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455
(which
patents are hereby incoiporated 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-
rnediated
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 liglit
chain variable region; one or rnore 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 dihydrofolate 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 liereby 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 liost 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
prornoter, one or more enhancer sequences, an origin of replication, a
transcriptional
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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 affrnity purification of the antibody
from the
host cell. Affinity purifrcation 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 purifred 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 organisrn, 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 usefizl in the vectors of this invention rnay be obtained
by any
of several methods well known in the art. Typically, flanking sequences useful
herein
will have been previously identified by mappirig 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 knowri. Here, the flanking sequence may be synthesized using
the
rnethods described herein for nucleic acid synthesis or cloning.
Where all or only a portion of the flanking sequence is known, it rnay be
obtained
using PCR and/or by screening a genomic library with a suitable
oligonucleotide and/or
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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 frorn 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 lcnown. A suitable promoter is operably linked
to the DNA
encoding anti-glucagon antibody by removing the promoter from the source DNA
by
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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 laiown and include, but are not limited to,
those obtained
from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus
(such as
Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus,
retroviruses, hepatitis-B virus and most preferably Simian Virus 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. Nath
Acad. Sci.
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. Sci. (J S.A., 75:
3727-3 1); or the
tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. 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: 115-22); 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. 1: 268-76); the alpha-feto-protein gene control region that
is active in
liver (Krumlauf et al., 1985, Mol. 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
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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
hypothalarnus (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; Adarnes et al., 1985, Nature 318: 533-38; Alexander et
al., 1987,
Mol. Cell Biol. 7: 1436-44).
An ei-fllancer sequence may be inserted into the vector to increase the
transcription
in higher eukaryotes of a nucleic acid encoding an ariti-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 terrnination 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 elernent encodes a protein necessary for the survival
and growth of a host cell grown in a selective culture mediuni. 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)
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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 arnplifiable selectable markers for
rnamrnalian
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. iJnder 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
rnRNA
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 soine 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
arnino acid of the
mature protein) one or more additional amino acids incident to expression,
which may
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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
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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-1 (see Background), the
compositions comprising GLP-1 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 (3-cell-specific genes
(e.g., GLUT-1
transporter, insulin receptor and hexokinase-1), 5) increase (3-cell mass by
inhibiting (3-
cell apoptosis and increasing P-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-1 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
syndrorne, and maturity-onset diabetes of the young (MODY). Thus, the
compositions
comprising GLP-1 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-1 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 cornpositions comprising GLP-1 compounds.
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Additional diseases that can be treated with the compositions comprising GLP-1
compounds include: obesity, irritable bowel syndrome, stroke, catabolic
changes after
surgery, myocardial infarction,), and hyperglycemia. The GL,P-1 compounds can
also be
used as a sedative.
The compositions comprising GLP-1 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-1 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 anirnal (e.g., mouse or rat).
VII. Pharmaceutical Compositions
A. Composition
The GLP-1 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-
1/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 GL,P-1 compound/antibody composition, pharmaceutical
cornpositions 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-1 compounds
have
utility. General classes of other therapeutic agents that can be combined with
certain
GLP-1 compound/antibody compositions include, but are not limited to, insulin
releasing
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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 inliibitors (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 inliibitors
(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, biguariides,
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-1 analog.
In some embodiments, the pharmaceutical compositions comprise an effective
amount of one or a plurality of the GLP-1 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-1
compound/antibody composition are provided.
In certain embodiments, acceptable formulation materials preferably are
nontoxic
to recipients at the dosages and concentrations employed.
In certain ernbodirnents, 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,
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adsorption or penetration of the composition. In such embodiments, suitable
formulation
materials include, but are not limited to, amino acids (such as glycine,
glutamine,
asparagine, arginine or lysine); antimicrobials; antioxidants (such as
ascorbic acid,
sodium sulfite or sodium hydrogen-sulfite); buffers (such as borate,
bicarbonate, Tris-
HCI, citrates, phosphates or other organic acids); bulking agents (such as
mannitol or
glycine); chelatiiig 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 rnetal
halides, preferably
sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents;
excipients
and/or pharmaceutical adjuvants. See REMINGTON'S PHARMACETJTICAL,
SCIENCES, 18"' Edition, (A.R. Gem-iaro, 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
adrninistration, delivery format and desired dosage. See, for example,
REMINGTON'S
PHARMACEIJTICAL 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
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cerebrospinal fluid, possibly supplemented with other materials common in
compositions
for parenteral administration. Neutral buffered saline or saline mixed with
serum
albumin are further exernplary 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 fiirther include sorbitol or a suitable substitute therefor. In
certain
ernbodiments, 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 PHARMACEIJTICAL, SCIENCES, supra) in the
form of a lyophilized cake or an aqueous solution. Further, in certain
embodiments, the
GL,P-1/antibody compositions may be formulated as a lyophilizate using
appropriate
excipierits 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 certairr embodiments, buffers are
used to
maintain the composition at physiological pH or at a slightly lower pH,
typically within a
pH range of frorn 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 sohztion comprising the desired composition comprising GLP-
1
compound in a pharmaceutically acceptable vehicle. A particularly suitable
vehicle for
parenteral injection is sterile distilled water in which the GLP-1/antibody
composition is
forrnulated as a sterile, isotonic solution, properly preserved. In certain
embodiments, the
preparation can irrvolve the formulation of the desired molecule with an
agent, such as
injectable microsplieres, 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
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duration in the circulation. In certain embodiments, implantable drug delivery
devices
may be used to introduce the desired GL,P-1/antibody composition.
Pharmaceutical compositions can be formulated for inhalation. In these
embodiments, pharmaceutical compositions comprising GLP-1 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. PCT/IJS94/001875, 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-1 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-systernic 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-1 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 rnay
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 rnagnesium
stearate, stearic acid, or talc.
Additional pharmaceutical compositions will be evident to those skilled in the
art,
including formulatiorrs involving GLP-1 compound/antibody compositions in
sustained-
or controlled-delivery formulations. Techniques for formulating a variety of
other
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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/IJS93/00829,
which is
incoiporated by reference and describes controlled release of porous polymeric
microparticles for delivery of pharmaceutical compositions. Sustained-release
preparations may include semiperrneable 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,
Biopolyiners 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. Sci. 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 througli
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 adininistration 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.
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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-1
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-1 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 LD50/ED50. 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
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and size of the subject to be treated and the particular disease itself. In
general, however,
the total amount of the GLP-1 compound/antibody composition itself that is
administered
typically ranges from 1 g/kg body weight/day to 100 mg/kg/day. In some
instances, the
dosage ranges from 10 g/kg /day to 10 mg/kg/day. In other treatment regimens,
the
GLP-1 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 rninimizes 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, transderrnal,
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,
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lactose, sucrose, mannitol, starcli, 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 non-
aqueous 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
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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 Aizainst 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.
12:811-820), 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), IJ.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
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04/035747. The antibody selected from a screen of the hybridomas was
designated
AG159. The antibody was selected in part because it could neutralize glucagon
in vitro
and in vivo.
Example 2: Cloning of the Anti-Glucagon Antibody Light and Heavy Chains
The hybridoma expressing glucagon binding monoclonal antibody AG 159 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 GeneRacerTM Kit
(Invitrogen).
For preparing complete light chain encoding cDNA, the forward primer was the
GeneRacerTM 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 GeneRacerTM 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-1 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 arnino 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 terrninus and termination codon as well as a SaII restriction
site (5'- CTT
GTC GAC TCA ACA CTC TCC CCT GTT GAA GCT C-3'; SEQ ID NO: 108). The
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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 Xhal 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: 110) and the same reverse
primer. The resulting PCR product was purified, digested with Xbal and SalI,
gel isolated
and ligated into the mammalian expression vector pDSRa19 (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-1 signal peptide sequence (MDMRVPAQL,L,
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:
111) and the 3' reverse primer encoded the carboxyl end of the variable
region, including
a naturally occurring sense strand BsmBI 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: 113) 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 XbaI
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: 114) and the previous reverse primer. The resulting PCR product was
purified, digested with XbaI and BsmBI, gel isolated and ligated into the
mammalian
expression vector pDSRa19 containing the human IgGI constant region.
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Constr asction of the GLP-1 (A2G) AGI59antibody chain fusion genes
A DNA sequence encoding the upstream Xbal site, optimized Kozak sequence,
GLP-1(A2G) peptide and a linker sequence and part of the AG159 LC or HC cDNA
containing a unique restrictiori site were synthesized by Picoscript (Houston,
TX)
(MDMRVPAQLLGLLLLWLRGARCHGEGTFTSDV SSYLEGQAAK.EFIAWLVKGR
GGSGSATGGSGSTASSGSGSATGGGGGG; SEQ ID NO: 115). iJtilizing a naturally
occurring unique Kpnl site for the kappa chain, the XbaI-Kpnl fragment from
the
synthesized gene was used to replace the synonymous fragment in the AG159
kappa
chain pDSRa19 construct, resulting in the GLP-1 (A2G)-AG159 LC fusion gene.
Similarly, utilizing a naturally occurring unique Pvull site in the AG159
heavy chain
DNA sequence, the synthesized GLP-1(A2G) DNA sequence was cut with Xbal and
PvulI and used to replace the synonymous fragment in the AG159 heavy chain
construct
to create the GLP- 1 (A2G)-AG 159 IgG1 heavy chain fusion gene.
The isotype of the heavy chain constant region of AG159 was switched from
IgGI 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.
Constr uction of AG159 Kappa Light Chain and GLP-1 (A2G)-AG159 Kappa Light
Chain
Expression Plasmids
The full length AG159 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
SaII restriction site, a optimized Kozak sequence and the amino terminus of
the VK-1
signal sequence (5'-AAC CTC GAG GTC GAC TAG ACC ACC ATG GAC ATG AGG
GTG CCC GCT-3'; SEQ ID NO: 116) 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: 117). The resulting
fragment was purified, digested with SaII and Notl and cloned into the
expression vectors
pDC323 and pDSRa24. The same process was performed utilizing the GLP-1(A2G)-
AG159 L,C construct as a template.
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Construction of AG159 IgGI, AG159 IgG2, GLP-1(A2G)-AG159 IgGl and GLP-
I A2G)-AG159 IgG2 Heavy Chain Expression Plasmids
The AG159 IgGl heavy chain variable region fragment, described above,
amplified by PCR using a 5' primer encoding a SaII 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: 118).
The resulting PCR fragment was purified, digested with SaII and Notl, gel
isolated and
cloned in the expression vectors pDC324 and pDSRa24. The same process was
perforrned utilizing the GLP-1(A2G)-AG159 IgGI, AG159 IgG2 and GLP-1(A2G)-
AG159 IgG2 constructs described above as templates.
Construction of different GLP-1(A2G)-AG159 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. IJsing the
GLP-
1(A2G)-AG159 kappa chain DNA sequence as a template, PCR was done to introduce
a
novel BamHI restriction site in the DNA sequence coding for the linker
peptide. A
forward primer containing a BamHI site (5'-GCT TGG CTG GTT AAA GGT CGT GGC
GGA TCC GGC AGC GCT-3'; SEQ ID NO: 119) 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 AG159 LC. A 5' primer that
annealed in the
vector promoter region upstream of the Xhal 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 BamHI 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
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CCA AGC GAT GAA TTC-3'; SEQ ID NO: 125). The separate PCR products were
purified, digested with tha appropriate restriction enzymes (Xbal and BamIII,
or with
BamHI and KpnI) and ligated into the expression vector pDSRa20 containing the
Xbat
and Kpnl digested AG159 LC DNA.
Example 3: In vitro assays
Glucagon Receptor Functional Reporter Assax
In order to identify the compound/antibody compositions with neutralizing
activity, reporter cell lines expressing human or rat ghzcagon receptors were
generated.
Increased cAMP levels were measured through enhanced expression of a
luciferase
reporter gene. Briefly, CHOK1 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 37 C, 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 37 C in medium containing 0.5% protease-free BSA and 100 M
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 "IC50" represents the
concentration of the compound/antibody composition at which the glucagon
stimulated
activity is reduced by 50%.
GLP-1 R Functional Reporter AsM
In order to compare the potency of the GLP-1 compound/antibody compositions,
reporter cell lines expressing human or mouse GLP-1 receptors were generated.
Increased cAMP levels were measured through enhanced expression of a
luciferase
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reporter gene. Briefly, CHOKI cells expressing the mouse or human GLP-1
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 37 C, 5% CO2. The
evening prior
to assay, the cells were washed, the medium replaced with serum-free medium
containing
0.5% protease-free bovirre serum albumin (BSA), and then cultured overnight.
Cells
were exposed to a range of concentrations of test composition or GLP-1 for a
period of 6
hours at 37 C in rnedium containing 0.5% protease-free BSA and 100 M IBMX.
Cell
lysates were assayed for luciferase activity using the Luciferase Assay System
(Promega
Corporation, Madison, WI). Luciferase activity was measured using a
L,uminoskan
Ascent (Therrno 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-lcompound/antibody composition at which 50 percent of the maximal
activity is
achieved.
Membrane Preparation
CHOK1 cells expressing either human or mouse GLP-1 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
mls of ice cold sucrose buffer (25 mM Tris-HCI, 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 40 C for 10 minutes, resuspended in 25 ml assay
buffer (50
mM Tris-HCI, 5 mM MgC1Z, 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 hornogenized 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/rnl.
Ligand Binding Assays
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Binding assays were performed in 96-well U-bottom plates. Membranes (200 g
tissue) were incubated at room temperature for 2 hours in assay buffer
containing 0.2 nM
1251-GLP-1 (or 0.2 nM t25I-Glucagon) (PerkinElmer Life Sciences, Boston, MA)
and with
a range of concentrations of test composition or GLP-1 (or glucagon) in a
total volume of
100 l.d. In addition, non-specific binding was assessed in the presence of 1
M unlabeled
GL,P-1. The reaction was terminated by rapid filtration through iJnfilter-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 l of cold
50 mM
Tris-HC1, 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
"IC50"
represents the concentration of compound which reduces the maximal specific
"SI-GL,P-1
(or 125I-glucagon) binding by 50 percent.
Example 4: Glucagon Neutralizing Activity of AG159 Antibody
The antibody variable regions were cloned frorn cDNA and confirmed by mass
spectrophotometric analysis of antibody purified from the hybridoma. The
cloned
AG159 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 11, AG159 neutralization of glucagon stimulated
reporter
activity is shown. Cells expressing recombinant glucagon receptor were
incubated witli
0.1 nM glucagon in addition to a range of concentrations of AG159 or control
IgG. The
IC50 for AG159 in this assay is 4 nM (Figure 11). 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 AG159 or control antibody. The IC50
for
AG159 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 AG159.
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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 JAXO GEMMO
Strain - Spontaneous Mutation Congenic Mice, and are homozygous for the
diabetes
sporitaneous mutation (Lepr`ib). 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 1-
2wk
study) to about 10-1lweeks 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-1 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 1 hr, 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 norrnal. 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 rnice in a cage without food. After 4-5hrs, an
intraperitoneal glucose
tolerance test (glucose tolerance test rneasures 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-1 action in utilizing glucose can be seen as opposed to (-) control PBS.
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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 AG159
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 AG159 (see Example 2 above). Plasmids were co-
transfected
into CHO cells to produce full-length fusion proteins. The resulting antibody
structure
was AG159L,C: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 (IC50 5-10 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-1 receptor.
These in vitro
data demonstrated that the fusion GLP-1 analogue was bi-functional.
More specifically, GLP(A26)-AG159LC:AG159 IgG2 (GL,P(A2G)-AG159) was
assayed to determine if the construct would maintain GLP-1 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, GL,P(A2G)-
AG159
competes for 125I-GLP-1 binding to the human GLP-1 receptor in the presence of
glucagon.
In another experiment, GLP(A2G)-AG159 was evaluated for GLP-1 receptor
agonist activity in the presence of glucagon. The GLP-1R reporter assay was
performed
as described above in Example 3, with the addition of a range of glucagon
concentrations.
As sliown in Figure 2, GLP(A2G)-AG159 activates the human GL,P-1 receptor in
the
presence of glucagon. Also depicted on the graph is the dose response curve of
the
activation of the GLP-1 receptor by glucagon alone (without GL,P(A2G)-AG159) (-
---- ).
The data presented in Figure 2 is shown iri a different form in Figure 3. In
figure
3, the activity attributable to a specific GLP(A2G)-AG159 concentration
(without
glucagon) was subtracted from the total activity for all doses of glucagon
with the
respective GLP(A2G)-AG 159 concentration, such that the remaining activity was
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attributable to glucagon. As shown in figure 3, the presence of GL,P(A2G)-AG
159 dose-
dependently decreases the activity induced by glucagon.
Example 7: Results with Antibody Fusions Including Different GLP-1 Analogs
A variety of fiisions between a GLP-1 compound and AG159 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-1
had been modified (sequences for the fusions are listed in Tables 1 and 7).
Table 8
LC:HC pair Sequence of GLP-1 analog ICso ECso ECsO
Identifier human human mouse
(Bindi
ng)
AG159LC:GLP(A HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRG 12 nM -0.1 nM -1 nM
2G)-AG159IgG2 (SEQ ID NO: 126)
GLP (A2G)- HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRG 8.5 nM 0.13 nM 7.5
AG159Kappa:AG (SEQ ID NO: 126) nM
159 IgG1
GLP1 (A2G)- HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRG 4.5 nM 0.14 nM 4.9
AG159LC:AG159 (SEQ ID NO: 126) nM
IgG2
GLP1(A2G/V27E HGEGTFTSDVSSYLEGQAAKEFIAWLENGGG 55 nM 1.7 nM >100
/ K28N/R30G)- (SEQ ID NO: 2) nM
AG159LC:AG159
IgG2
GLP1(A2G/V27K HGEGTFTSDVSSYLEGQAAKEFIAWLKNGGG 9.1 nM 0.33 nM >10
/K28N/R.30G)- (SEQ ID NO: 3) nM
AG159LC:AG159
IgG2
GLP1(A2G/R30G HGEGTFTSDVSSYLEGQAAKEFIAWLVKGGG 4.8 nM 0.08 nM 6 nM
(SEQ ID NO: 4)
AG159LC:AG159
IgG2
GLPl-AG159LC HGEGTFTSDVSSYLEGQAAKEFIAWL >1 uM >10 nM >100
deltaC- (SEQ ID NO: 5) nM
term:AG159
IgG2
GLP1-AG159 LC HGEGTFTSDVSSYLEGQAAKEFIAWLKNGGP - 0.19 nM 1.7
chimera:AG159 SSGAPPPS (SEQ ID NO: 6) nM
IgG2
GLPl-SR-1 LC: HGEGTFTSDVSSYLEGQAAKEFIAWLVKGR.G 12 nM -0.1 nM 24 nM
SR-1 hIgGl HC (SEQ ID NO: 126)
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= not determined
To reduce proteolysis, a set of substitution mutant GLP-1 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-1 analogs were fused to the light chain of
AG159
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-1 Compound/Ab fusions
LC : HC pair EC50 ECs0
identifier GLP 1 peptide analog human mouse
GLP(A2G/VlOQ)- HGEGTFTSDQSSYLEGQAAKEFIAWLVKGRG 5 nM >100 nM
AG159LC:AG159 IgG2 (SEQ ID NO: 7)
GLP(A2G/V10Q/L14Q) HGEGTFTSDQSSYQEGQAAKEFIAWLVKGRG >100 nM >100 nM
-AG159 LC:AG159 (SEQ ID NO: 8)
IgG2
GLP(A2G/VlOQ/V27Q) HGEGTFTSDQSSYLEGQAAKEFIAWLQKGRG 18 nM >100 nM
-AG159 LC:AG159 (SEQ ID NO: 9)
IgG2
GLP(A2G/L14Q)- HGEGTFTSDVSSYQEGQAAKEFIAWLVKGRG 1 nM >50 nM
AG159 LC:AG159 (SEQ ID NO: 10)
IgG2
LP(A2G/W25Q)-AG159 HGEGTFTSDVSSYLEGQAAKEFIAQLVKGRG 2 nM >50 nM
LC:AG159 IgG2 (SEQ ID NO: 11)
GLP(A2G/W25Q/V27Q) HGEGTFTSDVSSYLEGQAAKEFIAQLQKGRG 11 nM >100 nM
-AG159 LC:AG159 (SEQ ID NO: 12)
IgG2
GLP(A2G/V27Q)- HGEGTFTSDVSSYLEGQAAKEFIAWLQKGRG 2 nM >50 nM
AG159 LC:AG159 (SEQ ID NO: 13)
IgG2
GLP(A2N/G4T)-AG159 HNETTFTSDVSSYLEGQAAKEFIAWLVKGRG 5.5 nM >50 nM
LC:AG159 IgG2 (SEQ ID NO: 14)
GLP(A2G/E3N)-AG159 HGNGTFTSDVSSYLEGQAAKEFIAWLVKGRG >40 nM >100 nM
LC:AG159 IgG2 (SEQ ID NO: 15)
GLP(E3N)-AG159 HANGTFTSDVSSYLEGQAAKEFIAWLVKGRG >50 nM >100 nM
LC:AG159 IgG2 (SEQ ID NO: 16)
GLP(A2G/T5N)-AG159 HGEGNFTSDVSSYLEGQAAKEFIAWLVKGRG undet'd undeY,'d
LC:AG159 IgG2 (SEQ ID NO: 17)
GLP(A2G/D9N/S11T)- HGEGTFTSNVTSYLEGQAAKEFIAWLVKGRG >100 nM undet'd
AG159 LC:AG159 (SEQ ID NO: 18)
IgG2
GLP(A2G/V10N/S12T) HGEGTFTSDNSTYLEGQAAKEFIAWLVKGRG undet'd undet'd
-AG159 LC:AG159 (SEQ ID NO: 19)
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IgG2
GLP(A2G/S12N/L14T) HGEGTFTSDVSNYTEGQAAK.EFIAWLVKGR.G >100 nM undet'd
-AG159 LC:AG159 (SEQ ID NO: 20)
IgG2
GLP(A2G/L14N/G16T) HGEGTFTSDVSSYNETQAAKEFIAWLVKGRG >100 nM undet'd
-AG159 LC:AG159 (SEQ ID NO: 21)
IgG2
GLP(A2G/G16N/A18T) HGEGTFTSDVSSYLENQTAK.EFIAWLVKGRG 1 nM >10 nM
-AG159 LC:AG159 (SEQ ID NO: 22)
IgG2
GLP(A2G/Q17N/A19T) HGEGTFTSDVSSYLEGNATKEFIAWLVKGRG 10 nM >100 nM
-AG159 LC:AG159 (SEQ ID NO: 23)
IgG2
GLP(A2G/A18N/K20T) HGEGTFTSDVSSYLEGQNATEFIAWLVKGRG >100 nM undet'd
-AG159 LC:AG159 (SEQ ID NO: 24)
IgG2
GLP(A2G/A19N/E21T) HGEGTFTSDVSSYLEGQANKTFIAWLVKGRG undet'd undet'd
-AG159 LC:AG159 (SEQ ID NO: 25)
IgG2
GLP(A2G/W25N/V27T) HGEGTFTSDVSSYLEGQAAKEFIANLTKGRG >100 nM undet'd
-AG159 LC:AG159 (SEQ ID NO: 26)
IgG2
GLP(A2G/V27N/G29T) HGEGTFTSDVSSYLEGQAAKEFIAWLNKTRG undet'd undet'd
-AG159 LC:AG159 (SEQ ID NO: 27)
IgG2
GLP(A2G/K28N/R30T) HGEGTFTSDVSSYLEGQAAKEFIAWLVNGTG 4 nM >50 nM
-AG159 LC:AG159 (SEQ ID NO: 28)
IgG2
GLP(A2G/G29N/G31T) HGEGTFTSDVSSYLEGQAAKEFIAWLVKNRT 4 nM >50 nM
-AG159 LC:AG159 (SEQ ID NO: 29)
IgG2
GLP(A2G/R30N/+T32) HGEGTFTSDVSSYLEGQAAKEFIAWLVKGNGT >100 nM undet'd
-AG159 LC:AG159 (SEQ ID NO: 30)
IgG2
GLP(A2G/G31N/+G32/ HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRNGT 0.9 nM >10 nM
+T33)-AG159 (SEQ ID NO: 31)
LC:AG159 IgG2
GLP(A2G/+N32/+G33/ HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRGNG >100 nM >100 nM
+T34)-AG159 T (SEQ ID NO: 32)
LC:AG159 IgG2
GLP(A2G/+G32/+N33/ HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRGGN undet'd undet'd
+G34/+T35)-AG159 GT (SEQ ID NO: 33)
LC:AG159 IgG2
GLP(A2G/+G32/+T33/ HGEGTFTSDVSSYLEGQAAK.EFIAWLVKGRGGT 1.8 nM >50 nM
+G34/+N35/+G36/+T3 GNGT (SEQ ID NO: 34)
7)-AG159 LC:AG159
IgG2
GLP(A2G/+G32/+S33/ HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRGGS 2.6 nM >50 nM
+G34/+N35/+G36/+T3 GNGT (SEQ ID NO: 35)
6)/-AG159 LC:AG159
IgG2
Example 8: In vivo Results
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Experiments were conducted with a number of different fusion molecules that
included AG159 and different GLP-1 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-1 compound/AG159 fusions. The
antibody fusions included either GLP(A2G) or one of the following GLP-1
peptides
fused to the light chain (LC) of AG159: A2G/K28N/R30T (SEQ ID NO: 28),
A2G/Ql7N/A19T (SEQ ID NO: 23), A2G/V10Q/V27Q (SEQ ID NO: 9), and
A2G/W25Q/V27Q (SEQ ID NO: 12). These L,C fusions were paired with AG159 IgG2
heavy chains to give the following antibodies, which were tested:
GLP(A2G/K28N/R30T)-AG159LC:AG159 IgG2, GL,P(A2G/Q17N/A19T)-
AG159LC:AG159 IgG2, GLP(A2G/V 10Q/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-1 peptides
fused to AG159LC. The antibody fusions tested included the following GLP-1
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 AG159 IgG2 heavy chains to give the
following
antibodies, which were tested: GLP(A2G)-AG159LC:AG159 IgG2,
GL,P(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
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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-1(A2G) was fused to the LC of AG159, 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
rnechanism 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 AG159 LC to give the
antibody GL,P(A2G/R30G)AG159LC:AG159 HC IgG2. 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-1 fused to LC v>FIC
This experiment was designed to determine whether the attaclunent of a GLP-1
compound to the LC or HC of AG159 resulted in differences in activity. The
constructs
tested were ones in which GLP(A2G) was attached to either the LC or HC of AG
159 to
give the following two antibody constructs GLP(A2G) AG159LC:AG159 IgG2
(GLP(A2G)-AG159 LC) and AG159LC:GLP-1-AG159 IgG2 (GL,P(A2G)-AG159 HC).
As can be seen in Figure 8, both constructs were essentially equally effective
in lowering
blood glucose levels.
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Example 10: Lean GTT Experiments
Another experiment was conducted with the fusion protein in which the GLP-1
analog A2G/R30G was fused to AG159 to give the antibody construct
GL,P(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-1 compounds is that they may loose efficacy after
multiple injections (determination of tachyphylaxis). Another experiment was
conducted
with the GLP(A2G/R.30G)AG159LC:AG159 HC IgG2 antibody, or simply R30G, to
determine if this was the case with this molecule. R30G was injected on day I
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
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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.
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