Note: Descriptions are shown in the official language in which they were submitted.
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HUMAN GLP-1 MIMETIBODIES AND COMPOSITIONS FOR TREATING
OBESITY AND RELATED DISORDERS, METHODS AND USES
PRIORITY
This application claims priority to US provisional application No. 60/752,826,
filed
December 22, 2005, which is entirely incorporated herein by reference.
BACKGROUND OF THE INVENTION
FIELD OF THE INVENTION
[1] The present invention relates to mammalian GLP-1 mimetibodies, specified
portions
and variants specific for biologically active proteins, fragment or ligands,
GLP-I
mimetibody encoding and complementary nucleic acids, host cells, and methods
of
making and using thereof, including therapeutic formulations, administration
and
devices for treating obesity related disorders.
RELATED ART
[2] Recombinant proteins are an emerging class of therapeutic agents. Such
recombinant
therapeutics have cngcndcrcd advances in protcin formulation and chemical
modification. Such modifications can potentially enhance the therapeutic
utility of
therapeutic proteins, such as by increasing half lives (e.g., by blocking
their exposure to
proteolytic enzymes), enhancing biological activity, or reducing unwanted side
effects.
One such modification is the use of immunoglobulin fragments fused to receptor
proteins, such as enteracept. Therapeu.tic proteins have also been constructed
using the
Fc domain to attempt to provide a longer half-life or to incorporate functions
such as Fc
receptor binding, protein A binding, and complement fixation.
[3] Obesity is a chronic disease manifested by the excess of fat mass in
proportion to
body size. Approximately one third of Americans are over-weight based on Body
Mass
Index (BMI >25 kg/m2), and obesity is considered to be reaching epidemic
proportions.
The importance of obesity's threat to health is emphasized by the fact that
obesity is an
underlying cause or a risk factor for developing other diseases such as Type 2
Diabetes,
cardiovascular disease, osteoartritis, and sleep apnea. Even a modest decrease
in body
weight (5-10% of initial body weight) may significantly decrease rislc factors
for
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2
developing obesity-associated diseases and improve metabolic syndrome
conditions
characterized by obesity, atherogenic dyslipidemia, raised blood pressure and
insulin
resistance.
[4] The need to treat obesity is widely recognized and efforts are being made
by all
major pharmaceutical companies to devclop a successful thcrapy. Obesity is
currcntly
treated, by: 1) life style changes, 2) three drugs currently on the market
(Phentramine,
Orlisata and Sibutramine) that have a modest effect on weight loss and are
associated
with significant side effects and 3) by surgery.
[5] Glucagon-like peptide-1 (GLP-1) is a 30 amino acid hormone secreted from
gut
endocrine cells in response to nutrient ingestion. GLP-1 travels through the
circulation
and binds to the GLP-1 receptor on the pancreas, resulting in an increase in
insulin
secretion. In addition, it has been shown that GLP-1 reduces gastric emptying
which
decreases the amount of glucose that is released into the circulation. These
actions
combine to lower blood glucose levels. Thus, the mechanism of biological
activity of
GLP-1 suggests that it could be an attractive therapeutic for the treatment of
type 2
diabetes. GLP-1 also has the potential to treat obesity. Several studies have
shown that
GLP-1 administered either peripherally or intracerebroventricularly (ICV)
decreases
food intake in animal models. A study in humans delivering GLP-1 continuously
for
five days in obese, diabetic patients resulted in a reduction in food intake
and a
reduction in body weight. However, GLP-1 is not being developed as a
therapeutic
because of its exceptionally short half-life (T1/2 - 1-2 min). It is rapidly
degraded by
dipeptidyl protease (DPP-IV), thus reducing the length of the peptide by 2
amino acids
at the N-terminus and inactivating it.
[6] There are several GLP-1 analogues that are currently in development or on
the
market. BycttaTM is a recently markctcd GLP-1 analogue dcvclopcd by Amylin and
Eli
Lilly. It was first identified in the saliva of the gila monster lizard, and
is 53% identical
to GLP- 1. ByettaTM is resistant to DPP-IV, yet it still requires twice daily
pre-prandial
dosing partially due to its short irz vivo half-life (less than 30 minutes).
During clinical
trials where Byetta TM was evaluated as a therapy for Type 2 Diabetes, HbAlc
levels
3 0 were lowered approximately 1% following 82 weeks of treatment.
Interestingly,
patients taking ByettaTM had a sustained decrease in body weight (5-10 pounds)
during
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the course of the study, supporting the theory that GLP-1 analogues have
potential for
the treatment of obesity. Liraglutide is a lipidated GLP-1 analogue being
developed by
Novo Nordisk, and it is currently in clinical trials. Based upon the
pharmacokinetics of
the molecule, it is anticipated that liraglutide will be dosed once daily. A
GLP-1 therapy
that has an increased half-life such that it could be dosed once weekly or
monthly would
have a significant advantage over other GLP-1 peptides in development.
[7] Accordingly, there is a need to provide improved and/or modified versions
of GLP-l
therapeutic proteins, which overcome one more of these and other problems
known in
the art. The mimetibody technology provides a novel delivery platform for
peptide
therapeutics. A GLP-1 mimetibody may provide a means of delivering the GLP-1
peptide in a sustained manner, providing an improvement over GLP-1 peptides
currently
in development. Furthermore, based upon its dimeric structure and its tissue
distribution
characteristics, a GLP-1 mimetibody could have differentiable features with
regard to
insulin secretion, (3-cell preservation, and food intake.
SUMMARY OF THE INVENTION
[8] The prescnt invention provides human GLP-1 mimctibodics, including
modificd
immunoglobulins, cleavage products and other specified portions and variants
thereof,
as well as GLP-1 mimetibody compositions, encoding or complementary nucleic
acids,
vectors, host cells, compositions, formulations, devices, transgenic animals,
transgenic
plants, and methods of making and using thereof, as described and/or enabled
herein, in
combination with what is known in the art.
[9] The Mimetibod.yTM technology provides a novel delivery platform for
peptide
therapeutics. A GLP-1 MimetibodyTM may provide a means of delivering the GLP-1
peptide in a sustained manner, providing an improvement over GLP-1 peptides
currently
in development. ByettaTM shows sustained weight loss even though the half-life
of the
molecule is relatively short. A GLP-1 analogue with a sustained
pharmacokinetic
profile has the potential to reduce food intake and body weight to a greater
extent.
Furthermore, the GLP-1 MimetibodyTM is quite different from other GLP-1
analogues in
development or on the market. It is a large protein rather than a peptide, and
it has two
peptides per molecule. Based upon its size and dimeric structure, a GLP-1
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MimetibodyT"" could have differentiable features relative to other molecules.
For
example, it is possible that activation of the GLP-1 receptor by a dimeric
molecule is
different from a monomeric molecule, resulting in differences in the signaling
pathway.
In addition, it is possible that the size of the molecule results in a very
different tissue
distribution profile, which may result in different pharmacodynamic
properties.
[10] A MimetibodyTM construct incorporating a GLP-1 peptide provides a novel
therapy for the reduction of body weight in obese individuals. In animal
models that are
known to correlate with therapeutic efficacy, CNTO 736, a GLP-1 MimetibodyTM,
decreascs food intake and body weight, due to the reduction in fat mass.
[11] The present invention also provides at least one isolated GLP-1
mimetibody or
specified portion or variant as described herein and/or as known in the art.
The GLP-1
mimetibod.y can optionally comprise at least one CH3 region directly linked,
with at least
one CH2 region directly linked with at least one portion of at least one hinge
region or
fragment thereof (H), directly linked with at least one partial variable
region (V),
directly linked with an optional linker sequence (L), directly linked to at
least one GLP-
1 therapeutic peptide (P).
[12] In a preferred embodiment a pair of a CH3-CH2-hinge-partial V region
sequence-linker-therapeutic peptide sequence, the pair optionally linked by
association
or covalent linkage, such as, but not limited to, at least one Cys-Cys
disulfide bond or at
least one CH4 or other immunglobulin sequence. In one embodiment, a GLP-1
mimetibody comprises formula (I):
a. (P(n)-L(o)-V(p)-H(q)-CH2(r)-CH3(s))(t),
[13] wherein P is at least one bioactive GLP-1 peptide, variant or derivative,
L is at least
one linker sequence, which can be a polypeptide that provides structural
flexibility by allowing
the mimetibody to have alternative orientations and binding properties, V is
at least one portion
of a C-terminus of an immunoglobulin variable regioii, H is at least one
portion of an
immunoglobulin variable hinge region, CH2 is at least a portion of an
immunoglobulin CH2
constant region, CH3 is at least a portion of an immunoglobulin CH3 constant
region, n is an
integer from 1 to 10, and o, p, q, r, s, and t can be independently an integer
from 0 to 10,
mimicing different types of immunoglobulin molecules, e.g., but not limited to
IgGI, IgG2,
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IgG3, IgG4, IgAl, IgA2, IgM, IgD, IgE, or any subclass thereof, and the like,
or any
combination thereof.
[14] The variable region of the antibody sequence can be, but not limited to,
at least
one portion of at least one of SEQ ID NOS:47-55, or fragment thereof as
described in
5 Table 1, further optionally comprising at least one substitution, insertion
or deletion as
further described in Figures 1-9 of PCT publication WO 05/05604 (PCT
USO4/19898)
filed June 24, 2004 and published January 20, 2005, with corresponding SEQ ID
NOS: 1-9. The CH2, CH3 and hinge region can be, but not limited to, at least
one
portion of at least one of SEQ ID NOS:56-64, or fragment thereof as described
in Table
1, further optionally comprising at least one substitution, insertion or
deletion as as
further described in Figures 32-40 of PCT publication WO 05/05604 (PCT
USO4/19898) filed June 24, 2004 and published January 20, 2005, with
corresponding
SEQ ID NOS:32-40.
[15] Thus, a GLP-1 rnimetibody of the present invention mimics at least a
portion of
an antibody or immnuoglobulin structure or function with its inherent
propcrtics and
functions, while provid.ing a GLP-1 therapeutic peptide and its inherent or
acquired. in
vitro, in vivo or in situ properties or activities. The various portions of
the antibody and
therapeutic peptide portions of GLP-1 mimetibody of the present invention can
vary as
described herein in combination with what is known in the art.
[16] The present invention also provides at least one isolated GLP-1
mimetibody or
specified portion or variant that has at least one activity, such as, but not
limitcd to
known biological activities of at least one bioactive GLP-1 peptide or
polypeptide
corresponding to the P portion of formula (I), as described herein or known in
the art.
[17] In one aspect, the present invention provides at least one isolated human
GI.P-1
mimetibody comprising at least one polypeptide sequence of SEQ ID NO: 1, or
optionally with one or more substitutions, deletions or insertions as
described herein or
as known in the art. In another aspect, at least one GLP-1 mimetibody or
specified
portion or variant of the invention mimics the binding of at least one GLP-1
peptide or
polypeptide corresponding to the P portion of the mimetibody in formula (I),
to at least
one epitope comprising at least 1-3, to the entire amino acid sequence of at
least one
ligand, e.g., but not limited to, a GLP-1 receptor, or fragment thereof,
wherein the ligand
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binds to at least a portion of SEQ ID NO: 1, or optionally with one or more
substitutions,
deletions or insertions as described herein or as known in the art. The at
least one GLP-
1 mimetibody can optionally bind GLP-1 receptor with an affinity of at least
10-9 M, at
least 10-10 M, at least 10-11 M. or at least 10"12 M. A GLP-1 mimetibody can
thus be
screened for a corresponding activity according to known methods, such as, but
not
limited to the binding activity towards a receptor or fragment thereof.
[18] The present invention further provides at least one anti-idiotype
antibody to at
least one GLP-1 mimetibody of the present invention. The anti-idiotype
antibody or
fragment specifically binds at least one GLP-l mimetibody of the present
invention.
The anti-id.iotype antibody includes any protein or peptide containing
molecule that
comprises at least a portion of an immunoglobulin molecule, such as but not
limited to
at least one complimetarity determing region (CDR) of a heavy or light chain
or a ligand
binding portion thereof, a heavy chain or light chain variable region, a
heavy.chain or
light chain constant region, a framework region, or any portion thereof, that
competitively binds a GLP-1 ligand binding region of at least one GLP- 1
mimetibody of
the present invention. Such idiotype antibodies of the invention can include
or be
derived from any mammal, such as but not limited to a human, a mouse, a
rabbit, a rat, a
rodent, a primate, and the like.
[19] The present invention provides, in one aspect, isolated nucleic acid
molecules
comprising, complementary, having significant identity or hybridizing to, a
polynucleotide encoding at least one GLP-1 mimetibody or GLP-1 mimetibody anti-
idiotype antibody, or specified portions or variants thereof, comprising at
least one
specified sequence, domain, portion or variant thereof. The present invention
further
provides recombinant vectors comprising at least one of said isolated GLP-1
mimetibody or GLP-1 mimetibody anti-idiotype antibody encoding nucleic acid
molecules, host cells containing such nucleic acids and/or recombinant
vectors, as well
as methods of making and/or using such GLP-1 mimetibody or GLP-1 mimetibody
anti-
idiotypc antibody nuclcic acids, vcctors and/or host cclis.
[20] Also provided is an isolated nucleic acid encoding at least one isolated
mammalian GLP-1 mimetibody or GLP-1 mimetibody anti-idiotype antibody; an
isolated nucleic acid vector comprising the isolated nucleic acid, and/or a
prokaryotic or
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eukaryotic host cell comprising the isolated nucleic acid. The host cell can
optionally be
at least one selected from COS-1, COS-7, HEK293, BHK21, CHO, BSC-1, Hep G2,
653, SP2/0, 293, HeLa, myeloma, or lymphoma cells, or any derivative,
immortalized or
transformed cell thereof.
[21] Thc present invention also provides at lcast one method for cxpressing at
least
one GLP-1 mimetibody or GLP-1 mimetibod.y anti-idiotype antibody, or specified
portion or variant in a host cell, comprising culturing a host cell as
described herein
and/or as known in the art under conditions wherein at least one GLP-1
mimetibody or
GLP- 1 mirnetibody anti-idiotype antibody, or specified portion or variant is
expressed in
detectable and/or recoverable amounts. Also provid.ed. is a method for
producing at least
one GLP-1 mimetibody or GLP-1 mimetibody anti-idiotype antibody, comprising
translating the GLP-1 mimetibody or GLP-1 niimetibody anti-idiotype antibody
encoding nucleic acid under conditions in vitro, in vivo or in situ, such that
the GLP- 1
mimetibody or GLP-1 mimetibody anti-idiotype antibody is expressed in
detectable or
recoverable amounts.
[22] Also provided is a method for producing at least one isolated human GLP-1
mimetibody or GLP-1 anti-idiotype antibody of the present invention,
comprising
providing a host cell or transgenic animal or transgenic plant capable of
expressing in
recoverable amounts the GLP-1 mimetibody or GLP-1 anti-idiotype antibody.
[23] Further provided in the present invention is at least one GLP-1
mimetibody
produced by the above methods.
[24] The present invention also provides at least one composition comprising
(a) an
isolated GLP-1 mimetibody or specified portion or variant encoding nucleic
acid and/or
GLP-1 mimetibody as described herein; and (b) a suitable carrier or diluent.
The carrier
or diluent can optionally be pharmaceutically acceptable, according to known
methods.
The composition can optionally further comprise at least one further compound,
protein
or composition.
[25] Also provided is a composition comprising at least one isolated human GLP-
1
mimetibody and at least one pharmaceutically acceptable carrier or diluent.
The
composition can optionally furthcr comprise an cffcctivc amount of at lcast
onc
compound or protein selected from at least one of a detectable label or
reporter, an anti-
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infective drug, a diabetes or insuling metabolism related drug, a
cardiovascular (CV)
system drug, a central nervous system (CNS) drug, an autonomic nervous system
(ANS)
drug, a respiratory tract drug, a gastrointestinal (GI) tract drug, a hormonal
drug, a drug
for fluid or electrolyte balance, a hematologic drug, an antineoplactic, an
immunomodulation drug, an ophthalmic, otic or nasal drug, a topical drug, a
nutritional
drug, a TNF antagonist, an antirheumatic, a muscle relaxant, a narcotic, a non-
steroid
anti-inflammatory drug (NTHE), an analgesic, an anesthetic, a sedative, a
local
anethetic, a neuromuscular blocker, an antimicrobial, an antipsoriatic, a
corticosteriod,
an anabolic steroid, an erythropoietin, an immunization, an immunoglobulin, an
immunosuppressive, a growth hormone, a hormone replacement drug, a
radiopharmaceutical, an antidepressant, an antipsychotic, a stimulant, an
asthma
medication, a beta agonist, an inhaled steroid, an epinephrine or analog, a
cytokine, or a
cytokine antagonist.
[26] The present invention also provides at least one composition, device
and/or
method of delivery of a therapeutically or prophylactically effective amount
of at least
one GLP-1 mimetibody or specified portion or variant, according to the present
invention.
[27] The present invention further provides at least one GLP-1 mimetibody
method
or composition, for administering a therapeutically effective amount to
modulate or treat
at least one GLP-1 related condition in a cell, tissue, organ, animal or
patient and/or,
prior to, subsequent to, or during a related condition, as known in the art
and/or as
described herein.
[28] The present invention further provides at least one GLP-1 mimetibody,
specified portion or variant in a method or composition, when administered in
a
therapeutically effective amount, for modulation, for treating or reducing the
symptoms
of, at least one metabolic, immunc, cardiovascular, infcctious, malignant,
and/or
neurologic disease in a cell, tissue; organ, animal or patient and/or, as
needed in many
different conditions, such as but not limited to, prior to, subsequent to, or
during a
related disease or treatment condition, as known in the art.
[29] The present invention further provides at least one GLP-1 mimetibody,
specified portion or variant in a method or composition, when administered in
a
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therapeutically effective amount, for modulation, for treating or reducing the
symptoms
of at least one of a diabetes or insuling metabolism related disorder, a bone
and joint
disorder, cardiovascular disoder, a dental or oral disorder, a dermatologic
disorder, an
ear, nose or throat disorder, an endocrine or metabolic disorder, a
gastrointestinal
disorder, a gynecologic disorder, a hepatic or biliary disorder, a an
obstetric disorder, a
hematologic disorder, an immunologic or allergic disorder, an infectious
disease, a
musculoskeletal disorder, a oncologic disorder, a neurologic disorder, a
nutritrional
disord.er, an opthalmologic disorder, a pediatric disorder, a poisoning
disorder, a
psychiatric disorder, a renal disorder, a pulmonary disorder, or any other
known
disorder, (See, e.g., The Merck Manual, 17th ed., Merck Research Laboratories,
Merck
and Co., Whitehouse Station, NJ (1999), entirely incoporated herein by
reference), as
needed in many different conditions, such as but not limited to, prior to,
subsequent to,
or during a related disease or treatment condition, as known in the art.
[30] The present invention also provides at least one composition, device
andlor
method of delivery, for diagnosing GLP-1 related conditions, of at least one
GLP-1
mimetibody, according to the present invention.
[31] The present invention further provides at least one GLP-1 mimetibody
method
or composition, for diagnosing at least one GLP-1 related condition in a cell,
tissue,
organ, animal or patient and/or, prior to, subsequent to, or during a related
condition, as
lrnown in the art and/or as described herein.
[32] Also provided is a method for diagnosing or treating a disease condition
in a
cell, tissue, organ or animal, comprising: (a) contacting or administering a
composition
comprising an effective amount of at least one isolated human GLP-1 mimetibody
of the
invention with, or to, the cell, tissue, organ or animal. The method can
optionally
further comprise using an effective amount of 0.001-50 mg/kilogram of the
cells, tissue,
organ or animal pcr 0-24 hours, 1-7 days, 1-52 weeks, 1-24 months, 1-30 ycars
or any
range or value therein. The method. can optionally further comprise using the
contacting
or the administrating by at least one mode selected from parenteral,
subcutaneous,
intramuscular, intravenous, intrarticular, intrabronchial, intraabdominal,
intracapsular,
intracartilaginous, intracavitary, intracelial, intracelebellar,
intracerebroventricular,
intracolic, intracervical, intragastric, intrahepatic, intramyocardial,
intraosteal,
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intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic,
intrapulmonary,
intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial,
intrathoracic, intrauterine,
intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, or
transdermal. The
method can optionally further comprise administering, prior, concurrently or
after the
5 (a) contacting or administering, at least one composition comprising an
effective amount
of at least one compound or protein selected from at least one of a detectable
label or
reporter, an anti-infective drug, a diabetes or insuling metabolism related
drug, a
cardiovascular (CV) system drug, a central nervous system (CNS) drug, an
autonomic
nervous system (ANS) drug, a respiratory tract drug, a gastrointestinal (GI)
tract drug, a
10 hormonal drug, a drug for fluid or electrolyte balance, a hematologic drug,
an
antineoplactic, an immunomodulation drug, an ophthalmic, otic or nasal drug, a
topical
drug, a nutritional drug, a TNF antagonist, an antirheumatic, a muscle
relaxant, a
narcotic, a non-steroid anti-inflammatory drug (NSAID), an analgesic, an
anesthetic, a
sedative, a local anethetic, a neuromuscular blocker, an antimicrobial, an
antipsoriatic, a
corticosteriod, an anabolic steroid, an erythropoietin, an immunization, an
immunoglobulin, an immunosuppressive, a growth hormone, a hormone replacement
drug, a radiopharmaceutical, an antidepressant, an antipsychotic, a stimulant,
an asthma
medication, a beta agonist, an inhaled steroid, an epinephrine or analog, a
cytokine, or a
cytokine antagonist.
[33] Also provided is a medical device, comprising at least one isolated human
GLP-
1 mimetibody of the invention, wherein the device is suitable to contacting or
administerting the at least one GLP-1 mimetibody by at least one mode sclcctcd
from
parenteral, subcutaneous, intramuscular, intravenous, intrarticular,
intrabronchial,
intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial,
intracelebellar, intracerebroventricular, intracolic, intracervical,
intragastric,
intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardiac,
intraperitoneal,
intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal,
intraretinal,
intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus,
vaginal, rectal,
buccal, sublingual, intranasal, or transdermal.
[34] Also provided is an article of manufacture for human pharmaceutical or
diagnostic use, comprising packaging material and a container comprising a
solution or
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a lyophilized form of at least one isolated human GLP-1 mimetibody of the
present
invention. The article of manufacture can optionally comprise having the
container as a
component of a parenteral, subcutaneous, intramuscular, intravenous,
intrarticular,
intrabronchial, intraabdominal, intracapsular, intracartilaginous,
intracavitary,
intracelial, intracelebellar, intracerebroventricular, intracolic,
intracervical, intragastric,
intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardiac,
intraperitoneal,
intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal,
intraretinal,
intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus,
vaginal, rectal,
buccal, sublingual, intranasal, or transdermal delivery device or system.
[35] The present invention further provides any invention d.escribed. herein.
DESCRIPTION OF THE INVENTION
[36] The present invention provides isolated, recombinant and/or synthetic
mimetibodies or specified portions or variants, as well as compositions and
encoding
nucleic acid molecules comprising at least one polynucleotide encoding at
least one
GLP-1 mimetibody. Such mimetibodies or specified portions or variants of the
present
invention comprise specific GLP-1 mimetibody sequences, domains, fragments and
specified variants thereof, and methods of making and using said nucleic acids
and
mimetibodies or specified portions or variants, including therapeutic
compositions,
methods and devices.
[37] The present invention also provides at least one isolated GLP-1
mimetibody or
specified portion or variant as described herein and/or as known in the art.
The GLP-1
mimetibody can optionally comprise at least one CH3 region directly linked
with at least
one CH2 region directly linked with at least one hinge region or fragment
thereof (H),
directly linked with at least one partial variable region (V), directly linked
with an
optional linker sequence (L), directly linked to at least one GLP-1
therapeutic peptide
(P).
[38] In a preferred embodiment a GLP-1 mimetibody comprises formula (I):
((P(n)-L(o)-V(p)-H(q)-CH2(r)-CH3 (s))(t),
[39] where P is at least one bioactive GLP-1 polypeptide, L is at least one
linker
scqucncc, which can be a polypcptidc that provides structural flcxablity by
allowing the
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mimietibody to have alternative orientations and binding properties, V is at
least one
portion of a C-terminus of an irnmunoglobulin variable region, H is at least
one portion
of an immunoglobulin variable hinge region, CH2 is at least a portion of an
immunoglobulin CH2 constant region, CH3 is at least a portion of an
immunoglobulin
CH3 constant region, m, n, o, p, q, r, s and t can be independently an integer
between
and including 0 and 10, mimicing different types of irnmunoglobulin molecules,
e.g.,
but not limited to IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgM, IgD, IgE, or any
subclass
thereof, and. the like, or any combination thereof.
[40] Thus, a GLP-1 mimetibody of the present invention mimics an antibody
structure with its inherent properties and. functions, while providing a
therapeutic
peptide and its inherent or acquired in vitro, in vivo or in situ properties
or activities. In
a preferred embodiment where t=1, the monomer CH3-CH2-hinge-partial J sequence-
linker-therapeutic peptide can be linked to other monomers by association or
covalent
linkage, such as, but not limited to, a Cys-Cys disulfide bond. The various
portions of
the antibody and the GLP-1 therapeutic peptide portions of at least one GLP-1
mimetibody of the present invention can vary as described herein in
combinatoin with
what is known in the art.
[41] The portion of CH3-CH2-hinge may be extensively modified to form a
variant in
accordance with this invention, provided binding to the salvage receptor is
maintained.
In such variants, one may remove one or more native sites that provide
structural
features or functional activity not required by the fusion molecules of this
invention.
One may remove these sites by, for example, substituting or deleting residues,
inserting
residues into the site, or truncating portions containing the site. The
inserted or
substituted residues may also be altered amino acids, such as peptidomimetics
or D-
2 5 amino acids. A variant of CH3-CH2-hinge may lack one or more native sites
or residues
that affect or are involved in (1) disulfide bond formation, (2)
incompatibility with a
selected host cell, (3) heterogeneity upon expression in a selected host cell,
(4)
glycosylation, (5) intcraction with complement, (6) binding to an Fc rcccptor
other than
a salvage receptor, or (7) antibod.y-dependent cellular cytotoxicity (ADCC).
Exemplary
CH3-CH2-hinge variants include molecules and sequences in which: 1. Sites
involved in
disulfide bond formation are removed. Such removal may avoid reaction with
other
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13
cysteine-containing proteins present in the host cell used to produce the
molecules of the
invention. For this purpose, the cysteine residues may be deleted or
substituted with
other amino acids (e.g., alanyl, seryl). Even when cysteine residues are
removed, the
single chain CH3-CH2-hinge domains can still form a dimeric CH3-CH2-hinge
domain
that is held together non-covalently; 2. The CH3-CH2-hinge region is modified
to make it
more compatible with a selected host cell. For example, when the molecule is
expressed
recombinantly in a bacterial cell such as E. coli, one may remove the PA
sequence in the
hinge, which may be recognized by a digestive enzyme in E. coli such as
proline
iminopeptidase; 3. A portion of the hinge region is deleted or substituted
with other
amino acids to prevent heterogeneity when expressed in a selected host cell;
4. One or
more glycosylation sites are removed. Residues that are typically glycosylated
(e.g.,
asparagine) may confer cytolytic response. Such residues may be deleted or
substituted
with unglycosylated residues (e.g., alanine); 5. Sites involved in interaction
with
complement, such as the C 1 q binding site, are removed. Complement
recruitment may
not be advantageous for the molecules of this invention and so may be avoided
with
such a variant; 6. Sites are removed that affect binding to Fc receptors other
than a
salvage receptor. The CH3-CH2-hinge region may have sites for interaction with
certain
white blood cells that are not required for the fusion molecules of the
present invention
and so may be removed; 7. The ADCC site is removed. ADCC sites are known in
the
art; see, for example, Molec. Immunol. 29 (5): 633-9 (1992) with regard to
ADCC sites
in IgGl. These sites, as well, are not required for the fusion molecules of
the present
invention and so may be removed.
[42] Linker polypeptide provides structural flexibility by allowing the
mimetibody to have
alternative orientations and binding properties. When present, its chernical
structure is not
critical. The linker is preferably made up of amino acids linked together by
peptide
bonds. Thus, in preferred embodiments, the linker is made up of from 1 to 20
amino
acids linked by peptide bonds, wherein the amino acids are selected from the
20
naturally occurring amino acids. Some of these amino acids may be
glycosylated, as is
well understood by those in the art. In a more preferred embodiment, the 1 to
20 amino
acids are selected from glycine, alanine, serine, proline, asparagine,
glutamine, and
lysine. Even more preferably, a linker is made up of a majority of amino acids
that are
sterically unhindered, such as glycine and alanine. Thus, preferred linkers
are poly(Gly-
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14
Ser), polyglycines (particularly (Gly)4, (Gly)5), poly(Gly-Ala), and
polyalanines. Other
specific examples of linkers are: (Gly)3Lys(Gly)4 (SEQ ID NO:65),
(Gly)3AsnGlySer(Gly)2 (SEQ ID NO:66), (Gly)3Cys(Gly)4 (SEQ ID NO:67), and
GlyProAsnGlyGly (SEQ ID NO:68).
[43] To explain the abovc nomcnclaturc, for example, (Gly)3Lys(Gly)4 mcans Gly-
Gly-Gly-Lys-Gly-Gly-Gly-Gly. Combinations of Gly and. Ala are also preferred.
The
linkers shown here are exemplary; linkers within the scope of this invention
may be
much longer and may include other residues.
[44] Non-peptide linkers are also possible. For example, alkyl linkers such as
NH-
(CH2)s-C(O)-, wherein s=2-20 could be used. These alkyl linkers may further be
substituted by any non-sterically hindering group such as lower allkyl (e.g.,
C1- C6)
lower acyl, halogen (e.g., Cl, Br), CN, NH2, phenyl, etc. An exemplary non-
peptide
linker is a PEG linker which has a molecular weight of 100 to 5000 kD,
preferably 100
to 500 kD. The peptide linkers may be altered to form derivatives in the same
manner as
described above.
[45] As used herein, a "GLP-1 peptide," or "GLP-1 peptide, variant, or
derivative"
can be at least one GLP-1 peptide, GLP-1 fragment, GLP-1 homolog, GLP-1
analog, or
GLP-1 derivative. A GLP-1 peptide has from about twenty-five to about forty-
five
naturally occurring or non-naturally occurring amino acids that have
sufficient
homology to native GLP-1 (7-37) such that they exhibit insulinotropic activity
by
binding to the GLP-1 receptor on [3-cells in the pancreas. GLP-1 (7-37) has
the amino
acid sequence of SEQ ID NO:15:
[46] His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-
Ala-Lys-Glu-Phe-lle-Ala-Trp-Leu-V al-Lys-Gly-Arg-Gly.
[47] A GLP-1 fragment is a polypeptide obtained after truncation of one or
more
amino acids from the N-terminus and/or C-terminus of GLP-1 (7-37) or an analog
or
derivative thereof. A GLP-1 homolog is a peptide in which one or more amino
acids
have been added to the N-terr.ninus and/or C-terminus of GLP-1 (7-37), or
fragments or
analogs thereof. A GLP-1 analog is a peptide in which one or more amino acids
of
GLP-1 (7-37) have been modificd and/or substitutcd. A GLP-1 analog has
sufficient
homology to GLP-l (7-37) or a fragment of GLP-1 (7-37) such that the analog
has
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insulinotropic activity. A GLP-1 derivative is defined as a molecule having
the amino
acid sequence of a GLP-1 peptide, a GLP-1 homolog or a GLP-1 analog, but
additionally having chemical modification of one or more of its amino acid
side groups,
a-carbon atoms, terminal amino group, or terminal carboxylic acid group.
5 [48] Numcrous active GLP-1 fragmcnts, analogs and derivatives arc known in
the art
and, any of these analogs and derivatives can also be part of the GLP-1
mimetibody of
the present invention. Some GLP-1 analogs and GLP-1 fragments known in the art
are
disclosed in U.S. Pat. Nos. 5,118,666, 5,977,071, and 5,545,618, and
Adelhorst, et al., J.
Biol. Chern. 269:6275 (1994). Examples include, but not limited to, GLP-1 (7-
34), GLP-
10 1 (7-35), GLP-1 (7-36), Gln9-GLP-1(7-37), D-Gln9-GLP-1(7-37), Thr16-Lysl8-
GLP-1
(7-37), and Lys18-GLP-1 (7-37).
[49] A "GLP-1 mimetibody," "GLP-1 mimetibody portion," or "GLP-1 mimetibody
fragment" and/or "GLP-1 mimetibody variant" and the like has, mimics or
simulates at
least one biological activity, such as but not limited to ligand binding, in
vitro, in situ
15 and/or preferably in vivo, of at least one GLP-1 peptide, variant or
derivative, such as
but not limited to at least one of SEQ ID NO:1. For example, a suitable GLP-1
mimetibody, specified portion, or variant can also modulate, increase, modify,
activate,
at least one GLP-1 receptor signaling or other measurable or detectable
activity.
[50] GLP-1 mimetibodies useful in the methods and compositions of the present
invention are characterized by suitable affinity binding to protein ligands,
for example,
GLP-1 receptors, and optionally and preferably having low toxicity. In
particular, a
GLP-1 mimetibody, where the individual components, such as the portion of
variable
region, constant region (without a CH1 portion) and framework, or any portion
thereof
(e.g., a portion of the J, D or V rgions of the variable heavy or light chain;
at least a
portion of at least one hinge region, the constant heavy chain or light chain,
and the like)
individually and/or collcctivcly optionally and preferably posscss low
immunogenicity,
is useful in the present invention. The mimetibodies that can be used in the
invention
are optionally characterized by their ability to treat patients for extended
periods with
good to excellent alleviation of symptoms and low toxicity. Low immunogenicity
and/or high affinity, as well as other undefined properties, may contribute to
the
therapeutic results achieved. "Low immunogenicity" is defined herein as
raising
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significant HA_MA, HACA or HAHA responses in less than about 75%, or
preferably
less than about 50, 45, 40, 35, 30, 35, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2,
and/or 1% of the
patients treated and/or raising low titres in the patient treated (less than
about 300,
preferably less than about 100 measured with a double antigen enzyme
immunoassay)
(see, e.g., Elliott et al., Lancet 344:1125-1127 (1994)).
[51] Utility. The isolated, nu.cleic acids of the present invention can be
used for
production of at least one GLP-1 mimetibody, fragment or specified variant
thereof,
which can be used to effect in an cell, tissue, organ or animal (including
mammals and
humans), to modulate, treat, alleviate, help prevent the incidence of, or
reduce the
symptoms of, at least one obesity related condition, selected. from, but not
limited to, at
least one of a obesity related disorder, any overweight condition related to
excess body
fat, an insulin metabolism related disorder, an immune disorder or disease, a
cardiovascular disorder or disease, an infectious, malignant, and/or
neurologic disorder
or disease, as well as other known or specified protein related conditions.
[52] Such a method can comprise administering an effective amount of a
composition or a pharmaceutical composition comprising at least one GLP-1
mimetibody or specified portion or variant to a cell, tissue, organ, animal or
patient in
need of such modulation, treatment, alleviation, prevention, or reduction in
symptoms,
effects or mechanisms. The effective amount can comprise an amount of about
0.0001
to 500 mg/kg per single or multiple administration, or to achieve a serum
concentration
of 0.01-5000 g/mi serum concentration per single or multiple adminstration,
or any
effective range or value therein, as done and determined using known methods,
as
described herein or known in the relevant arts.
[53] Citations. All publications or patents cited herein are entirely
incorporated
herein by reference as they show the state of the art at the time of the
present invention
and/or to provide description and enablement of the present invention.
Publications refer
to any scientific or patent publications, or any other information available
in any media
format, including all recorded, electronic or printed formats. The following
references
are entirely incorporated lierein by reference: Ausubel, et al., ed., Current
Protocols in
Molecular Biology, John Wiley & Sons, Inc., NY, NY (1987-2003); Sambrook, et
al.,
Molecular Cloning: A Laboratory Manual, 2 d Edition, Cold Spring Harbor, NY
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(1989); Harlow and Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor,
NY
(1989); Colligan, et al., eds., Current Protocols in Immunology, John Wiley &
Sons,
Inc., NY (1994-2003); Colligan et at., Current Protocols in Protein Science,
John Wiley
& Sons, NY, NY, (1997-2003).
[54] Mimetibodies of the Present Invention. The GLP-1 mimetibody can
optionally comprise at least one CH3 region directly linked with at least one
CH2 region
directly linked with at least one portion of at lesat one hinge region
fragment (H), such
as comprising at least one core hinge region, directly linked with at least
one partial
variable region (V), directly linked with an optional linker sequence (L),
directly linked
to at least one GLP-1 therapeutic peptide (P). In a preferred. embodiment, a
pair of a
CH3-CH2-H-V-L-P can be linked by association or covalent linkage, such as, but
not
lirnited to, a Cys-Cys disulfide bond. Thus, a GLP-1 mimetibody of the present
invention mimics an antibody structure with its inherent properties and
functions, while
providing a therapeutic peptide and its inherent or acquired in vitro, in vivo
or in situ
properties or activities. The various portions of the antibody and therapeutic
peptide
portions of at least one GLP-1 mimetibody of the present invention can vary as
described herein in combinatoin with what is lcnown in the art.
[55] Mimetibodies of the present invention thus provide at least one suitable
property as compared to known proteins, such as, but not limited to, at least
one of
increased half-life, increased activity, more specific activity, increased
avidity, increased
or descrease off rate, a selected or more suitable subset of activities, less
immungenicity,
increased quality or duration of at least one desired therapeutic effect, less
side effects,
and the like.
[56] Fragments of mimetibodies according to Formula (I) can be produced by
enzymatic cleavage, synthetic or recombinant techniques, as known in the art
and/or as
described herein. Mimctibodics can also be produccd in a variety of truncated
forms
using antibody genes in which one or more stop codons have been introd.uced.
upstream
of the natural stop site. The various portions of mimetibodies can be joined
together
chemically by conventional techniques, or can be prepared as a contiguous
protein using
genetic engineering techniques. For example, a nucleic acid encoding at least
one of the
constant regions of a human antibody chain can be expressed to produce a
contiguous
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protein for use in mimetibodies of the present invention. See, e.g., Ladner et
al., U.S.
Patent No. 4,946,778 and Bird, R.E. et al., Science, 242: 423-426 (1988),
regarding
single chain antibodies.
[57] As used herein, the term "human mimetibody" refers to an antibody in
which
substantially cvcry part of the protein (e.g., GLP-1 pcptidc, CH domains
(c.g., CH2, CH3),
hinge, V) is expected to be substantially non-immunogenic in humans with only
minor
sequence changes or variations. Such changes or variations optionally and
preferably
retain or reduce the immunogenicity in humans relative to non-modified human
antibodies, or mimetibodies of the prsent invention. Thus, a human antibody
and
corresponding GLP-1 mimetibody of the present invention is distinct from a
chimeric or
humanized antibody. It is pointed out that the GLP-1 mimetibody can be
produced by a
non-human animal or cell that is capable of expressing human immunoglobulins
(e.g.,
heavy chain and/or light chain) genes.
[58] Human mimetibodies that are specific for at least one protein ligand
thereof can
be designed against an appropriate ligand, such as an isolated GLP-1 receptor,
or a
portion thereof (including synthetic molecules, such as synthetic peptides).
Preparation
of such mimetibodies are performed using known techniques to identify and
characterize ligand binding regions or sequences of at least one protein or
portion
thereof.
[59] In a preferred embodiment, at least one GLP-1 mimetibody or specified
portion
or variant of the present invention is produced by at least one cell line,
mixed cell line,
immortalized cell or clonal population of irnmortalized and/or cultured cells.
Immortalized protein producing cells can be produced using suitable methods.
Preferably, the at least one GLP-1 mimetibody or specified portion or variant
is
generated by providing nucleic acid or vectors comprising DNA derived or
having a
substantially similar scqucncc to, at lcast one human immunoglobulin locus
that is
functionally rearranged, or which can undergo functional rearrangement, and.
which
further comprises a mimetibody structure as described herein, e.g., but not
limited to
Formula (I), wherein portions of C- terminal variable regions can be used for
V, hinge
3 0 regions for H, CH2 for CH2 and CH3 for CH3, as known in the art.
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[60] The term "functionally rearranged," as used herein refers to a segment of
nucleic acid from an immunoglobulin locus that has undergone V(D)J
recombination,
thereby producing an immunoglobulin gene that encodes an immunoglobulin chain
(e.g., heavy chain), or any portion thereof. A functionally rearranged
immunoglobulin
gene can be directly or indirectly identified using suitable methods, such as,
for
example, nucleotide sequencing, hybridization (e.g., Southern blotting,
Northern
blotting) using probes that can anneal to coding joints between gene segments
or
enzymatic amplification of immunoglobu.lin genes (e.g., polymerase chain
reaction)
with primers that can anneal to coding joints between gene segments. Whether a
cell
produces a GLP-1 mimetibody or portion or variant comprising a particular
variable
region or a variable region comprising a particular sequence (e.g., at least
one P
sequence) can also be determined using suitable methods.
[61] Mimetibodies, specified portions and variants of the present invention
can also
be prepared using at least one GLP-1 rnimetibody or specified portion or
variant
encoding nucleic acid to provide transgenic animals or mammals, such as goats,
cows,
horses, sheep, and the like, that produce such mimetibodies or specified
portions or
variants in their milk. Such animals can be provided using known methods as
applied
for antibody encoding sequences. See, e.g., but not limited to, US patent nos.
5,827,690; 5,849,992; 4,873,316; 5,849,992; 5,994,616; 5,565,362; 5,304,489,
and the
like, each of which is entirely incorporated herein by reference.
[62] Mimetibodies, specified portions and variants of the present invention
can
additionally be prepared using at least one GLP-1 mimetibody or specified
portion or
variant encoding nucleic acid to provide transgenic plants and cultured plant
cells (e.g.,
but not limited to tobacco and maize) that produce such mimetibodies,
specified
portions or variants in the plant parts or in cells cultured therefrom. As a
non-limiting
example, transgenic tobacco leaves expressing recombinant proteins have been
successfully used to provide large amounts of recombinant proteins, e.g.,
using an
inducible promoter. Scc, e.g., Cramcr et at., Curr. Top. Microbol. Im.munol.
240:95-118
(1999) and. references cited, therein. Also, transgenic maize or corn have
been used. to
express mammalian proteins at commercial production levels, with biological
activities
equivalent to those produced in other recombinant systems or purified from
natural
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sources. See, e.g., Hood et al., Adv. Exp. Med. Biol. 464:127-147 (1999) and
references
cited therein. Antibodies have also been produced in large amounts from
transgenic
plant seeds including antibody fragments, such as single chain mimetibodies
(scFv's),
including tobacco seeds and potato tubers. See, e.g., Conrad et al., Plant
Mol. Biol.
5 38:101-109 (1998) and references cited therein. Thus, mimetibodies,
specified portions
and variants of the present invention can also be produced using transgenic
plants,
according to know methods. See also, e.g., Fischer et al., Biotechnol. Appl.
Biochem.
30:99-108 (Oct., 1999), Ma et al., Trends Biotechnol. 13:522-7 (1995); Ma et
al., Plant
Physiol. 109:341-6 (1995); Whitelam et al., Biochem. Soc. Trans. 22:940-944
(1994);
10 and references cited therein. The above references are entirely
incorporated herein by
reference.
[63] The mimetibodies of the invention can bind human protein ligands with a
wide
range of affinities (KD). In a preferred embodiment, at least one human GLP-1
mimetibody of the present invention can optionally bind at least one protein
ligand with
15 high affinity. For example, at least one GLP-1 mimetibody of the present
invention can
bind at least one protein ligand with a KD equal to or less than about 10-' M
or, more
preferably, with a KD equal to or less than about 0.1-9.9 (or any range or
value therein)
x 10-7, 10-11, 10-9, 10-10, 10-11, 10-12, or 10-13 M, or any range or value
therein.
[64] The affinity or avidity of a GLP-1 mimetibody for at least one protein
ligand
20 can be determined experimentally using any suitable method, e.g., as used
for determing
antibody-antigen binding affinity or avidity. (See, for example, Berzofsky, et
al.,
"Antibody-Antigen Interactions," In Fundatnental Irnmunology, Paul, W. E.,
Ed., Raven
Press: New York, NY (1984); Kuby, Janis Immunology, W. H. Freeman and Company:
New York, NY (1992); and methods described herein). The measured affmity of a
particular GLP-1 mimetibody-ligand interaction can vary if measured under
different
conditions (e.g., salt concentration, pH). Thus, measurements of affinity and
other
ligand-binding parameters (e.g., Kn, Ka, Kd) are preferably made with
standardized
solutions of GLP-1 mimctibody and ligand, and a standardized buffer, such as
the buffer
described, herein or known in the art.
[65] Nucleic Acid Molecules. Using the information provided herein, such as
the
nucleotide sequences encoding at least 90-100% of the contiguous amino acids
of at
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least one of SEQ ID NOS:1 and 6, as well as at least one portion of an
antibody,
wherein the above sequences are inserted as the P sequence of Formula (1) to
provide a
GLP-1 mimetibody of the present invention, further comprising specified
fragments,
variants or consensus sequences thereof, or a deposited vector comprising at
least one of
these sequences, a nucleic acid molecule of the present invention encoding at
least one
GLP-1 mimetibody or specified portion or variant can be obtained using methods
described herein or as known in the art.
[66] Nucleic acid molecules of the present invention can be in the form of
RNA,
such as mRNA, hnRNA, tRNA or any other form, or in the form of DNA, including,
but
not limited. to, eDNA and genomic DNA obtained by cloning or produced.
synthetically,
or any combination thereof. The DNA can be triple-stranded, double-stranded or
single-
stranded, or any combination thereof. Any portion of at least one strand of
the DNA or
RNA can be the coding strand, also known as the sense strand, or it can be the
non-
coding strand, also referred to as the anti-sense strand.
[67] Isolated nucleic acid molecules of the present invention can include
nucleic acid
molecules comprising an open reading frame (ORF), optionally with one or more
introns, nucleic acid molecules comprising the coding sequence for a GLP-1
mimetibody or specified portion or variant; and nucleic acid molecules which
comprise
a nucleotide sequence substantially different from those described above but
which, due
2 0 to the degeneracy of the genetic code, still encode at least one GLP-1
mimetibody as
described herein and/or as known in the art. Of course, the genetic code is
well known
in the art. Thus, it would be routine for one skilled in the art to generate
such
degenerate nucleic acid variants that code for specific GLP-1 mimetibody or
specified
portion or variants of the present invention. See, e.g., Ausubel, et al.,
supra, and such
nucleic acid variants are included in the present invention.
[68] As indicated herein, nuclcic acid rnoleculcs of the prescnt invcntion
which
comprise a nucleic acid. encoding a GLP-1 mimetibody or specified portion or
variant
can include, but are not limited to, those encoding the amino acid sequence of
a GLP-l
mimetibody fragment, by itself; the coding sequence for the entire GLP-1
mimetibody
3 0 or a portion thereof; the coding sequence for a GLP-1 mimetibody, fragment
or portion,
as well as additional sequences, such as the coding sequence of at least one
signal leader
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or fusion peptide, with or without the aforementioned additional coding
sequences, such
as at least one intron, together with additional, non-coding sequences,
including but not
limited to, non-coding 5' and 3' sequences, such as the transcribed, non-
translated
sequences that play a role in transcription, mRNA processing, including
splicing and
polyadenylation signals (for example - ribosome binding and stability of
mRNA); an
additional coding sequence that codes for additional amino acids, such as
those that
provide additional functionalities. Thus, the sequence encoding a GLP-1
mimetibody or
specified. portion or variant can be fused. to a marker sequence, such as a
sequ.ence
encoding a peptide that facilitates purification of the fused GLP-1 mimetibody
or
specified portion or variant comprising a GLP-1 mimetibody fragment or
portion.
[69] Polynucleotides Which Selectively Hybridize to a Polynucleotide as
Described Herein. The present invention provides isolated nucleic acids that
hybridize
under selective hybridization conditions to a polynucleotide disclosed herein,
or others
disclosed herein, including specified variants or portions thereof. Thus, the
polynucleotides of this embodiment can be used for isolating, detecting,
andlor
quantifying nucleic acids comprising such polynucleotides.
[70] Low or moderate stringency hybridization conditions are typically, but
not
exclusively, employed with sequences having a reduced sequence identity
relative to
complementary sequences. Moderate and high stringency conditions can
optionally be
employed for sequences of greater identity. Low stringency conditions allow
selective
hybridization of sequences having about 40-99% sequence identity and can be
employed
to identify orthologous or paralogous sequences.
[71] Optionally, polynucleotides of this invention will encode at least a
portion of a
GLP- 1 mimetibody or specified portion or variant encoded by the
polynucleotides
described herein. The polynucleotides of this invention embrace nucleic acid
sequences
that can be employed for sclcctive hybridization to a polynuclcotidc cncoding
a GLP-1
mimetibody or specified portion or variant of the present invention. See,
e.g., Ausubel,
supra; Colligan, supra, each entirely incorporated herein by reference.
[72] Construction of Nucleic Acids. The isolated nucleic acids of the present
3 0 invention can be made using (a) recombinant methods, (b) synthetic
techniques, (c)
purification techniques, or combinations thereof, as well-known in the art.
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[73] The nucleic acids can conveniently comprise sequences in addition to a
polynucleotide of the present invention. For example, a multi-cloning site
comprising
one or more endonuclease restriction sites can be inserted into the nucleic
acid to aid in
isolation of the polynucleotide. Also, translatable sequences can be inserted
to aid in the
isolation of the translated polynucleotide of the present invention. For
example, a hexa-
histidine marker sequence provides a convenient means to purify the proteins
of the
present invention. The nucleic acid of the present invention - excluding the
coding
sequence - is optionally a vector, adapter, or linker for cloning and/or
expression of a
polynucleotide of the present invention.
[74] Additional sequences can be added to such cloning and/or expression
sequences
to optimize their function in cloning and/or expression, to aid in isolation
of the
polynucleotide, or to improve the introduction of the polynucleotide into a
cell. Use of
cloning vectors, expression vectors, adapters, and linkers is well known in
the art. See,
e.g., Ausubel, supra; or Sambrook, supra.
[75] Recombinant Methods for Constructing Nucleic Acids. The isolated nucleic
acid compositions of this invention, such as RNA, eDNA, genomic DNA, or any
combination thereof, can be obtained from biological sources using any number
of
cloning methodologies known to those of skill in the art. In some embodiments,
oligonucleotide probes that selectively hybridize, under suitable stringency
conditions,
to the polynucleotides of the present invention are used to identify the
desired sequence
in a cDNA or genomic DNA library. The isolation of RNA, and construction of
cDNA
and genomic libraries, is well known to those of ordinary skill in the art.
(See, e.g.,
Ausubel, supra; or Sambrook, supra).
[76] Synthetic Methods for Constructing Nucleic Acids. The isolated nucleic
acids of the present invention can also be prepared by direct chemical
synthesis by
known methods (scc, e.g., Ausubel, et al., supra). Chemical synthesis
generally
produces a single-stranded, oligonucleotide, which can be converted into
double-
stranded DNA by hybridization with a complementary sequence, or by
polymerization
with a DNA polymerase using the single strand as a template. One of skill in
the art will
3 0 recognize that while chemical synthesis of DNA can be limited to sequences
of about
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24
100 or more bases, longer sequences can be obtained by the ligation of shorter
sequences.
[77] Recombinant Expression Cassettes. The present invention further provides
recombinant expression cassettes comprising a nucleic acid of the present
invention. A
nuclcic acid scqucncc of thc prescnt invcntion, for cxamplc a cDNA or a
gcnomic
sequence encoding a GLP-1 mimetibody or specified portion or variant of the
present
invention, can be used to construct a recombinant expression cassette that can
be
introduced into at least one desired host cell. A recombinant expression
cassette will
typically comprise a polynucleotide of the present invention operably linked
to
transcriptional initiation regulatory sequences that will direct the
transcription of the
polynucleotide in the intended host cell. Both heterologous and non-
heterologous (i.e.,
endogenous) promoters can be employed to direct expression of the nucleic
acids of the
present invention.
[78] In some embodiments, isolated nucleic acids that serve as promoter,
enhancer,
or other elements can be introduced in the appropriate position (upstream,
downstream
or in intron) of a non-heterologous form of a polynucleotide of the present
invention so
as to up or down regulate expression of a polynucleotide of the present
invention. For
example, endogenous promoters can be altered in vivo or in vitro by mutation,
deletion
and/or substitution, as known in the art. A polynucleotide of the present
invention can
be expressed in either sense or anti-sense orientation as desired. It will be
appreciated
that control of gene expression in either sense or anti-sense orientation can
have a direct
impact on the observable characteristics. Another method of suppression is
sense
suppression. Introduction of nucleic acid configured in the sense orientation
has been
shown to be an effective means by which to block the transcription of target
genes.
[79] Vectors And Host Cells. The present invention also relates to vectors
that
includc isolated nuclcic acid molecules of the prescnt invention, host cells
that are
genetically engineered with the recombinant vectors, and, the production of at
least one
GLP-1 mimetibody or specified portion or variant by recombinant techniques, as
is well
lcnown in the art. See, e.g., Sambrook, et al., supra; Ausubel, et al., supra,
each entirely
incorporated herein by reference.
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[80] The polynucleotides can optionally be joined to a vector containing a
selectable
marker for propagation in a host. Generally, a plasmid vector is introduced
into a cell
using suitable known methods, such as electroporation and the like, other
known
methods include the use of the vector as a precipitate, such as a calcium
phosphate
5 precipitate, or in a complex with a charged lipid. If the vector is a virus,
it can be
packaged in vitro using an appropriate packaging cell line and then transduced
into host
cells.
[81] The DNA insert should be operatively linked to an appropriate promoter.
The
expression constructs will further contain sites optionally for at least one
of transcription
10 initiation, termination and., in the transcribed region, a ribosome binding
site for
translation. The coding portion of the mature transcripts expressed by the
constructs
will preferably include a translation initiating at the beginning and a
termination codon
(e.g., UAA, UGA or UAG) appropriately positioned at the end of the mRNA to be
translated, with UAA and UAG preferred for mammalian or eukaryotic cell
expression.
15 [82] Expression vectors will preferably but optionally include at least one
selectable
marker. Such markers include, e.g., but not limited to, methotrexate (MTX),
dihydrofolate reductase (DHFR, US Pat.Nos. 4,399,216; 4,634,665; 4,656,134;
4,956,288; 5,149,636; 5,179,017, ampicillin, neomycin (G418), mycophenolic
acid, or
glutamine synthetase (GS, US Pat.Nos. 5,122,464; 5,770,359; 5,827,739)
resistance for
20 eukaryotic cell culture, and tetracycline or ampicillin resistance genes
for culturing in E.
coli and other bacteria or prokaryotics (the above patents are entirely
incorporated
hereby by reference). Appropriate culture mediums and conditions for the above-
described host cells are known in the art. Suitable vectors will be readily
apparent to the
skilled artisan. Introduction of a vector construct into a host cell can be
effected by
25 calcium phosphate transfection, DEAE-dextran mediated transfection,
cationic lipid-
mediated transfection, electroporation, transduction, infection or other known
methods.
Such methods are described in the art, such as Sambrook, supra, Chapters 1-4
and 16-
18; Ausubel, supra, Chapters 1, 9, 13, 15, 16, entirely incorporatcd herein by
reference.
[83] At least one GLP-1 mimetibody or specified portion or variant of the
present
invention can be expressed in a modified form, such as a fusion protein, and
can include
not only secretion signals, but also additional heterologous functional
regions. For
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instance, a region of additional amino acids, particularly charged amino
acids, can be
added to the N-terminus of a GLP-1 mimetibody or specified portion or variant
to
improve stability and persistence in the host cell, during purification, or
during
subsequent handling and storage. Also, peptide moieties can be added to a GLP-
1
mimetibody or specified portion or variant of the present invention to
facilitate
purification. Such regions can be removed prior to final preparation of a GLP-
1
mimetibody or at least one fragment thereof. Such methods are described in
many
standard laboratory manuals, such as Sambrook, supra, Chapters 17.29-17.42
and. 18.1-
18.74; Ausubel, supra, Chapters 16, 17 and 18, entirely incorporated herein by
reference.
[84] Those of ordinary skill in the art are knowledgeable in the numerous
expression
systems available for expression of a nucleic acid encoding a protein of the
present
invention.
[85] Illustrative of cell cultures useful for the production of the
mimetibodies,
specified portions or variants thereof, are mammalian cells. Mammalian cell
systems
often will be in the form of monolayers of cells although mammalian cell
suspensions or
bioreactors can also be used. A number of suitable host cell lines capable of
expressing
intact glycosylated proteins have been developed in the art, and include the
COS-1 (e.g.,
ATCC CRL 1650), COS-7 (e.g., ATCC CRL-1651), HEK293, BHK21 (e.g., ATCC
CRL-10), CHO (e.g., ATCC CRL 1610, DG-44) and BSC-1 (e.g., ATCC CRL-26) cell
lines, hepG2 cells, P3X63Ag8.653, SP2/0-Ag14, 293 cells, HeLa cells and the
like,
which are readily available from, for example, American Type Culture
Collection,
Manassas, Va (www.atcc.com). Preferred host cells include cells of lymphoid
origin
such as myeloma and lyxnphoma cells. Particularly preferred host cells are
P3X63Ag8.653 cells (ATCC Accession Number CRL-1580) and SP2/0-Ag14 cells
(ATCC Accession Number CRL-1851).
[86] Expression vectors for these cells can include one or more of the
following
expression control sequences, such as, but not limited to an origin of
replication; a
promoter (e.g., late or early SV40 promoters, the CMV promoter (e.g., US
Pat.Nos.
5,168,062; 5,385,839), an HSV tk promoter, a pgk (phosphoglycerate kinase)
promoter,
an EF-1 alpha promoter (e.g, US Pat.No. 5,266,491), at least one human
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immunoglobulin promoter; an enhancer, and/or processing information sites,
such as
ribosome binding sites, RNA splice sites, polyadenylation sites (e.g., an SV40
large T
Ag poly A addition site), and transcriptional terminator sequences. See, e.g.,
Ausubel et
al., supra; Sambrook, et al., supra. Other cells useful for production of
nucleic acids or
proteins of the present invention are known and/or available, for instance,
from the
American Type Culture Collection Catalogue of Cell Lines and Hybridomas
(www.atcc.org) or other known or commercial sources.
[87] When eukaryotic host cells are employed, polyadenlyation or transcription
terminator sequences are typically incorporated into the vector. An example of
a
termi.nator sequence is the polyadenlyation sequence from the bovine growth
hormone
gene. Sequences for accurate splicing of the transcript can also be included.
An
example of a splicing sequence is the VP1 intron from SV40 (Sprague, et al.,
J. Virol.
45:773-781 (1983)). Additionally, gene sequences to control replication in the
host cell
can be incorporated into the vector, as known in the art.
[88] Purification of a GLP-1 mimetibody or specified portion or variant
thereof.
A GLP-1 mimetibody or specified portion or variant can be recovered and
purified from
recambinant cell cultures by well-known methods including, but not limited to,
protein
A purification, ammonium sulfate or ethanol precipitation, acid extraction,
anion or
cation exchange chromatography, phosphocellulose chromatography, hydrophobic
interaction chromatography, affinity chromatography, hydroxylapatite
chromatography
and lectin chromatography. High performance liquid chromatography ("HPLC") can
also be employed for purification. See, e.g., Colligan, Current Protocols in
Immunology, or Current Protocols in Protein Science, John Wiley & Sons, NY,
NY,
(1997-2003), e.g., Chapters 1, 4, 6, 8, 9, 10, each entirely incorporated
herein by
reference.
[89] Mimctibodics or spccified portions or variants of the prescnt invcntion
include
naturally purified. products, products of chemical synthetic procedures, and.
products
produced by recombinant techniques from a eukaryotic host, including, for
example,
yeast, higher plant, insect and mammalian cells. Depending upon the host
employed in
a recombinant production procedure, the GLP-1 mimetibody or specified portion
or
variant of the present invention can be glycosylated or can be non-
glycosylated, with
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28
glycosylated preferred. Such methods are described in many standard laboratory
manuals, such as Sambrook, supra, Sections 17.37-17.42; Ausubel, supra,
Chapters 10,
12, 13, 16, 18 and 20, Colligan, Protein Science, supra, Chapters 12-14, all
entirely
incorporated herein by reference.
[90] MIMETIBODIES, SPECIFIED FRAGMENTS AND/OR VARIANTS.
The isolated mimetibodies of the present invention comprise a GLP-1 mimetibody
or
specified portion or variant encoded by any one of the polynucleotides of the
present
invention as discussed more fully herein, or any isolated or prepared GLP-1
mimetibody
or specified portion or variant thereof.
[91] Preferably, the GLP-1 mimetibody or ligand-binding portion or variant
binds at
least one GLP-1 protein ligand and thereby provides at least one GLP-1
biological
activity of the corresponding protein or a fragment thereof. Different
therapeutically or
diagnostically significant proteins are well known in the art and suitable
assays or
biological activities of such proteins are also well known in the art.
[92] Non-limiting examples of suitable GLP-1 peptides, variants and
derivatives for
this invention appear as SEQ ID NO:1: His-Xaa2-Xaa3-Gly-Xaa5-Xaa6-Xaa7-Xaa8-
Xaa9-Xaa 10-Xa a 11-Xaa 12-Xaa 13 -Xaa 14-Xaa l 5-Xaa l 6-Xa a 17 -X aa l 8-
Xaa l 9-Xaa2 0-
Xaa21-Phe-Xaa23-Xaa24-Xaa25-Xaa26-Xaa27-Xaa28-Xaa29-Xaa30-Xaa31, wherein:
Xaa2 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Glu, Asp, or Lys; Xaa3 is Glu, Asp,
or Lys;
Xaa5 is Thr, Ala, Gly, Ser, Leu, Ile, Val, Arg, His, Glu, Asp, or Lys; Xaa6 is
Phe, His,
Trp, or Tyr; Xaa7 is Thr or Asn; Xaa8 is Ser, Ala, Gly, Thr, Leu, Ile, Val,
Glu, Asp, or
Lys; Xaa9 is Asp or Glu; XaalO is Val, Ala, Gly, Ser, Thr, Leu, Ile, Met, Tyr,
Trp, His,
Phe, Glu, Asp, or Lys; Xaa11 is Ser, Val, Ala, Gly, Thr, Leu, Ile, Glu, Asp,
or Lys;
Xaa12 is Ser, Val, Ala, Gly, Thr, Leu, Ile, Glu, Asp or Lys; Xaa13 is Tyr,
Phe, Trp, Glu,
Asp or Lys; Xaal4 is Leu, Ala, Met, Gly, Ser, Thr, Leu, Ile, Val, Glu, Asp or
Lys;
Xaa15 is Glu, Ala, Thr, Scr, Gly, Gln, Asp or Lys; Xaa16 is Gly, Ala, Scr,
Thr, Lcu, Ilc,
Val, Gln, Asn, Arg, Cys, Glu., Asp or Lys; Xaa17 is Gln, Asn, Arg, His, Glu.,
Asp or
Lys; Xaa18 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Arg, Glu, Asp or Lys; Xaa19
is Ala,
Gly, Ser, Thr, Leu, Ile, Val, Met, Glu, Asp or Lys; Xaa2O is Lys, Arg, His,
Gln, Trp,
Tyr, Phe, Glu or Asp; Xaa21 is Glu, Leu, Ala, His, Phe, Tyr, Trp, Arg, Gln,
Thr, Ser,
Gly, Asp or Lys; Xaa23 is Ile, Ala, Val, Leu or Glu; Xaa24 is Ala, Gly, Ser,
Thr, Leu,
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Ile, Val, His, Glu, Asp or Lys; Xaa25 is Trp, Phe, Tyr, Glu, Asp or Lys; Xaa26
is Leu,
Gly, Ala, Ser, Thr, Ile, Val, Glu, Asp or Lys; Xaa27 is Val, Leu, Gly, Ala,
Ser, Thr, Ile,
Arg, Glu, Asp or Lys; Xaa28 is Lys, Asn, Arg, His, Glu or Asp; Xaa29 is Gly,
Ala, Ser,
Thr, Leu, Ile, Val, Arg, Trp, Tyr, Phe, Pro, His, Glu, Asp or Lys; Xaa30 is
Arg, His,
Thr, Ser, Trp, Tyr, Phe, Glu, Asp or Lys; and Xaa31 is Gly, Ala, Ser, Thr,
Leu, Ile, Val,
Arg, Trp, Tyr, Phe, His, Glu, Asp, Lys.
[93] Another preferred group of GLP-1 peptides, variants or derivatives are
exemplied in SEQ ID NO:6: His-Xaa2-Xaa3-Gly-Thr-Xaa6- Xaa7-Xaa8-Xaa9-Xaa10-
Ser-Xaal2-Tyr-Xaal4-Glu-Xaal6-Xaa17-Xaa18-Xaa19-Lys-Xaa21-Phe-Xaa23-Ala-
Trp-Leu-Xaa27-Xaa28-Gly-Xaa3O, wherein: Xaa2 is Ala, Gly, or Ser; Xaa3 is Glu.
or
Asp; Xaa6 is Phe or Tyr; Xaa7 is Thr or Asn; Xaa8 is Ser, Thr or Ala; Xaa9 is
Asp or
Glu; XaalO is Val, Leu, Met or Ile; Xaal2 is Ser or Lys; Xaal4 is Leu, Ala or
Met;
Xaal6 is Gly, Ala, Glu or Asp; Xaal7 is Gln or Glu; Xaa18 is Ala or Lys; Xaa19
is Ala,
Val, Tie, Leu or Met; Xaa2l is Glu or Leu; Xaa23 is Tle, Ala, Val, Leu or Glu;
Xaa27 is
Val or Lys; Xaa28 is Lys or Asn; and Xaa30 is Arg or Glu.
[94] These peptides can be prepared by methods disclosed and/or known in the
art.
The Xaas in the sequence (and throughout this specification, unless specified
otherwise
in a particular instance) include specified amino acid residues, derivatives
or modified
amino acids thereof. Because the enzyme, dipeptidyl-peptidase IV (DPP-IV), may
be
responsible for the observed rapid in vivo inactivation of administered GLP-1,
GLP-1
peptides, homologs, analogs and derivatives that are protected from the
activity of DPP-
IV in the context of mimetibody are preferred
[95] A GLP-1 mimetibody, or specified portion or variant thereof, that
partially or
preferably substantially provides at least one GLP-1 biological activity, can
bind the
GLP-1 ligand and thereby provide at least one activity that is otherwise
mediated
through the binding of GLP-1 to at least onc ligand, such as a GLP-1 rcccptor,
or
throu.gh other protein-dependent or med.iated. mechanisms. As used, herein,
the term
"GLP-1 mimetibody activity" refers to a GLP-1 mimetibody that can modulate or
cause
at least one GLP-1 dependent activity by about 20-10,000%, preferably by at
least about
60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140,
150, 160, 170,
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180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900, 1000,
2000, 3000,
4000, 5000, 6000, 7000, 8000, 9000 % or more, depending on the assay.
[96] The capacity of a GLP-1 mimetibody or specified portion or variant to
provide
at least one protein-dependent activity is preferably assessed by at least one
suitable
5 protein biological assay, as dcscribcd herein and/or as known in the art. A
human GLP-
1 mimetibody or specified. portion or variant of the invention can be similar
to any class
(IgG, IgA, IgM, etc.) or isotype and can comprise at least a portion of a
kappa or lambda
light chain. In one embodiment, the human GLP-1 mimetibody or specified
portion or
variant comprises IgG heavy chain variable fragments, hinge region, CH2 and
CH3 of,
10 at least one of isotypes, e.g., IgGl, IgG2, IgG3 or IgG4.
[97] At least one GLP-1 mimetibody or specified portion or variant of the
invention
binds at least one ligand, subunit, fragment, portion or any combination
thereof. The at
least one GLP-1 peptide, variant or derivative of at least one GLP-1
mimetibody,
specified portion or variant of the present invention can optionally bind at
least one
15 specified epitope of the ligand. The binding epitope can comprise any
combination of at
least one arnino acid sequence of at least 1-3 amino acids to the entire
specified portion
of contiguous amino acids of the sequences of a protein ligand, such as a GLP-
1
receptor or portion thereof.
[98] Such mimetibodies can be prepared by joining together the various
portions of
2 0 Formula (I) of the GLP-1 mimetibody using known techniques, by preparing
and
expressing at least one nucleic acid molecules that encode the GLP-1
mimetibody, using
known techniques of recombinant DNA technology or by using any other suitable
method, such as chemical synthesis.
[99] Mimetibodies that bind to human GLP-1 ligands, such as receptors, and
that
25 comprise a defined heavy or light chain variable region or portion thereof,
can be
prepared using suitable methods, such as phage display (Katsube, Y., et al.,
Int JMoI.
Med, 1(5):863-868 (1998), entirely incorporated herein by reference) or
methods that
cmploy transgenic animals, as known in the art. Thc GLP-1 mimetibody,
spccificd
portion or variant can be expressed using the encoding nucleic acid or portion
thereof in
30 a suitable host cell.
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[100] The invention also relates to mimetibodies, ligand-binding fragments and
immunoglobulin chains comprising amino acids in a sequence that is
substantially the
same as an amino acid sequence described herein. Preferably, such mimetibodies
or
ligand-binding fragments thereof can bind human GLP-1 ligands, such as
receptors,
with high affinity (e.g., KD less than or equal to about 10-7 M). Amino acid
sequences
that are substantially the same as the sequences described herein include
sequences
comprising conservative amino acid substitutions, as well as amino acid
deletions and/or
insertions. A conservative amino acid. su.bstitu.tion refers to the
replacement of a first
amino acid by a second amino acid that has chemical and/or physical properties
(e.g.,
charge, structure, polarity, hydrophobicity/ hydrophilicity) that are similar
to those of
the first amino acid. Conservative substitutions include replacement of one
amino acid
by another within the following groups: lysine (K), arginine (R) and histidine
(H);
aspartate (D) and glutamate (E); asparagine (N), glutamine (Q), serine (S),
threonine
(T), tyrosine (Y), K, R, H, D and E; alanine (A), valine (V), leucine (L),
isoleucine (1),
proline (P), phenylalanine (F), tryptophan (W), methionine (M), cysteine (C)
and
glycine (G); F, W and Y; C, S and T.
[101] Amino Acid Codes. The amino acids that make up mimetibodies or specified
portions or variants of the present invention are often abbreviated. The amino
acid
designations can be indicated by designating the amino acid by its single
letter code, its
three letter code, name, or three nucleotide codon(s) as is well understood in
the art (see
Alberts, B., et al., Molecular Biology of The Cell, Third Ed., Garland
Publishing, Inc.,
Ncw York, 1994).
SINGLE LETTER CODE THREE LETTER CODE NAME THREE NUCL
CODON(S)
A Ala Alanine GCA, GCC, GCG, GCU
C Cys Cysteine UGC, UGU
D Asp Aspartic acid GAC, GAU
E Glu Glutamic acid GAA, GAG
F Phe Phenylanine UUC, TJUU
G Gly Glycine GGA, GGC, GGG, GGU
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H His Histidine CAC, CAU
I lie Isoleucine AUA, AUC, AUU
K Lys Lysine AAA, AAG
L Leu Leucine UTJA, UUG, CUA, CUC, CUG, CUU
M Met Methionine AUG
N Asn Asparagine AAC, AAU
P Pro ProlineCCA, CCC, CCG, CCU
Q Gln Glutamine CAA, CAG
R Arg Arginine AGA, AGG, CGA, CGC, CGG, CGU
S Ser Serine AGC, AGU, UCA, UCC, UCG, UCU
T Thr Threonine ACA, ACC, ACG, ACU
V Val Valine GUA, GUC, GUG, GUU
W Trp Tryptophan UGG
Y Tyr Tyrosine UAC, UAU
[102] A GLP-1 mimetibody or specified portion or variant of the present
invention
can include one or more amino acid substitutions, deletions or additions,
either from
natural mutations or human manipulation, as specified herein. Such or other
sequences
that can be used in the present invention, include, but are not limited to the
following
sequences presented in Tablc 1, as shown corresponding to specified portions
of SEQ
ID NOS:47-64, where the partial variable region of the antibody sequence can
be, but is
not limited to, at least one portion of at least one of SEQ ID NOS:47-55, or
fragment
thereof as described in Table 1, further optionally comprising at least one
substitution,
insertion or delction as furthcr dcscribcd in Figures 1-9 of PCT publication
WO
05/05604 (PCT USO4/19898) filed June 24, 2004 and published January 20, 2005,
with
corresponding SEQ ID NOS:1-9. The CH2, CH3 and hinge region can be, but not
limited to, at least one portion of at least one of SEQ ID NOS:56-64, or
fragment thereof
as described in Table 1, further optionally comprising at least one
substitution, insertion
or deletion as as further described in Figures 32-40 of PCT publication WO
05/05604
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33
(PCT USO4/19898) filed June 24, 2004 and published January 20, 2005, with
corresponding SEQ ID NOS:32-40. Of course, the number of amino acid
substitutions a
skilled artisan would make depends on many factors, including those described
above.
Generally speaking, the number of amino acid substitutions, insertions or
deletions for
at least one of a GLP-1 mimetibody will not be more than 40, 30, 20,19, 18,
17, 16, 15,
14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 amino acids, such as 1-30 or any
range or value
therein, as specified herein.
[103] In formula I of the present invention ((P(n)-L(o)-V(p)-H(q)-CH2(r)-
CH3(s))(t),
the V, H, CH2, CH3 portions according to Formula I can be any suitable human
or
human compatable sequence, e.g., as presented in Table 1, where the partial
variable
region of the antibody sequence can be, but is not limited to, at least one
portion of at
least one of SEQ ID NOS:47-55, or fragment thereof as described in Table 1,
further
optionally comprising at least one substitution, insertion or deletion as
further described
in Figures 1-9 of PCT publication WO 05/05604 (PCT USO4/19898) filed June 24,
2004
and published January 20, 2005, with corresponding SEQ ID NOS:1-9; and where
the
CH2, CH3 and hinge region can be, but not limited to, at least one portion of
at least one
of SEQ ID NOS:56-64, or fragment thereof as described in Table 1, further
optionally
comprising at least one substitution, insertion or deletion as as further
described in
Figures 32-40 of PCT publication WO 05/05604 (PCT USO4/19898) filed June 24,
2004
and published January 20, 2005, with corresponding SEQ ID NOS:32-40, or as
known
in the art, or any combination or consensus sequence thereof, or any fusion
protein
thcrcof, prcfcrably of human origin or engineered to minimize immunogenicity
when
administered to humans.
[104] The P portion can comprise at least one GLP-1 therapeutic peptide known
in the
art or described herein, such as, but not limited to those presented in SEQ ID
NO: 1, or
any combination or consensus sequence thereof, or any fusion protein thereof.
In a
preferred embodiment, the P portion can comprise at least one GLP-1 peptide
having the
the scqucncc of at least one of SEQ ID NO:6, or any combination or consensus
sequence thereof, or any fu.sion protein thereof.
[105] The optional linker sequence can be any suitable peptide linker as known
in the
art. Preferred sequences include any combination of G and S, e.g., X] -X2-X3-
X4-...-
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Xn, where X can be G or S, and n can be 5-30. Non-limiting examples include
GS,
GGS, GGGS (SEQ ID NO:16), GSGGGS (SEQ ID NO:17), GGSGGGS (SEQ ID
NO:18), GGSGGGSGG (SEQ ID NO:19) and GGGSGGGSGG (SEQ ID NO:20); and
the like.
5[106] Amino acids in a GLP-1 mimetibody or spccificd portion or variant of
the
present invention that are essential for function can be identified by methods
known in
the art, such as site-directed mutagenesis or alanine-scanning mutagenesis
(e.g.,
Ausubel, supra, Chapters 8, 15; Cunningham and Wells, Science 244:1081-1085
(1989)). The latter procedure introduces single alanine mutations at every
residue in the
molecule. The resulting mutant molecules are then tested. for biological
activity, such
as, but not limited to at least one protein related activity, as specified
herein or as known
in the art. Sites that are critical for GLP-1 mimetibody or specified portion
or variant
binding can also be identified by structural analysis such as crystallization,
nuclear
magnetic resonance or photoaffinity labeling (Smith, et al., J. Mol. Biol.
224:899-904
(1992) and de Vos, et al., Science 255:306-312 (1992)).
[107] Mimetibodies or specified portions or variants of the present invention
can
comprise as the P portion of Formula (I), e.g. but not limited to, at least
one portion of at
least one of SEQ ID NOS:1 and 6. A GLP-1 mimetibody or specified portion or
variant
can further optionally comprise at least one functional portion of at least
one
polypeptide as P portion of Formula (1), at least 90-100% of at least on of
SEQ ID
NOS:1 and 6. Non-limiting variants that can enhance or maintain at least one
of the
listed activities above include, but are not limited to, any of the above
polypeptides,
further comprising at least one mutation corresponding to at least one
substitution,
insertion or deletion that does not significantly affect the suitable
biological activtities or
functions of said GLP-1 mimetibody.
[108] In onc cmbodimcnt, the P amino acid sequence, or portion thcrcof, has
about
90-100% identity (i.e., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or any
range or valu.e
therein) to the corresponding amino acid sequence of the corresponding portion
of at
least one of SEQ ID NOS:1 and 6. Preferably, 90-100% amino acid identity
(i.e., 90,
91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or any range or value therein) is
determined using
a suitable computer algorithm, as known in the art.
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[109] Mimetibodies or specified portions or variants of the present invention
can
comprise any number of contiguous amino acid residues from a GLP-1 mimetibody
or
specified portion or variant of the present invention, wherein that number is
selected
from the group of integers consisting of from 10-100% of the number of
contiguous
5 residues in a GLP-1 mimetibody. Optionally, this subsequence of contiguous
amino
acids is at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21,
22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120,
130, 140, 150,
160, 170, 180, 190, 200, 210, 220, 230, 240, 250 or more amino acids in
length, or any
range or value therein. Further, the number of such subsequences can be any
integer
10 selected from the group consisting of from I to 20, such as at least 2, 3,
4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more.
[110] As those of skill will appreciate, the present invention includes at
least one
biologically active GLP-1 mimetibody or specified portion or variant of the
present
invention. Biologically active mimetibodies or specified portions or variants
have a
15 specific activity at least 20%, 30%, or 40%, and preferably at least 50%,
60%, or 70%,
and most preferably at least 80%, 90%, or 95%-1000% of that of the native (non-
synthetic), endogenous or related and known inserted or fused protein or
specified
portion or variant. Methods of assaying and quantifying measures of enzymatic
activity
and substrate specificity are well known to those of skill in the art.
20 [111] In another aspect, the invention relates to human mimetibodies and
ligand-
binding fragments, as described herein, which are modified by the covalent
attachment
of an organic moiety_ Such modification can produce a GLP-1 mimetibody or
ligand-
binding fragment with improved pharmacokinetic properties (e.g., increased in
vivo
serum half-life). The organic moiety can be a linear or branched hydrophilic
polymeric
25 group, fatty acid group, or fatty acid ester group. In particular
embodiments, the
hydrophilic polymeric group can have a molecular weight of about 800 to about
120,000
Daltons and can be a polyalkane glycol (e.g., polyethylene glycol (PEG),
polypropylene
glycol (PPG)), carbohydrate polyrncr, amino acid polymcr or polyvinyl
pyrolidonc, and
the fatty acid or fatty acid. ester group can comprise from about eight to
about forty
30 carbon atoms.
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[112] The modified mimetibodies and ligand-binding fragments of the invention
can
comprise one or more organic moieties that are covalently bonded, directly or
indirectly,
to the GLP-1 mimetibody or specified portion or variant. Each organic moiety
that is
bonded to a GLP-1 mimetibody or ligand-binding fragment of the invention can
independently be a hydrophilic polymeric group, a fatty acid group or a fatty
acid ester
group. As used herein, the term "fatty acid" encompasses mono-carboxylic acids
and
di-carboxylic acids. A "hydrophilic polymeric group," as the term is used
herein, refers
to an organic polymer that is more soluble in water than in octane. For
example,
polylysine is more soluble in water than in octane. Thus, a GLP-1 mimetibody
modified
by the covalent attachment of polylysine is encompassed by the invention.
Hydrophilic
polymers suitable for modifying mimetibodies of the invention can be linear or
branched and include, for example, polyalkane glycols (e.g., PEG, monomethoxy-
polyethylene glycol (mPEG), PPG and the like), carbohydrates (e.g., dextran,
cellulose,
oligosaccharides, polysaccharides and the like), polymers of hydrophilic amino
acids
(e.g., polylysine, polyarginine, polyaspartate and the like), polyalkane
oxides (e.g.,
polyethylene oxide, polypropylene oxide and the like) and polyvinyl
pyrolidone.
Preferably, the hydrophilic polymer that modifies the GLP-1 mimetibody of the
invention has a molecular weight of about 800 to about 150,000 Daltons as a
separate
molecular entity. For example, PEG25oo~ PEG5000, PEG75009 PEG9oooa PEGioooo,
PEG1250N
PEG15000, and PEG20,000, wherein the subscript is the average molecular weight
of the
polymer in Daltons, can be used.
[113] The hydrophilic polymeric group can be substituted with one to about six
alkyl,
fatty acid or fatty acid ester groups. Hydrophilic polymers that are
substituted with a
fatty acid or fatty acid ester group can be prepared by employing suitable
methods. For
example, a polymer comprising an amine group can be coupled to a carboxylate
of the
fatty acid or fatty acid ester, and an activated carboxylate (e.g., activated
with N,N-
carbonyl diimidazole) on a fatty acid. or fatty acid ester can be coupled. to
a hydroxyl
group on a polymer.
[114] Fatty acids and fatty acid, esters suitable for modifying mimetibodies
of the
invention can be saturated or can contain one or more units of unsaturation.
Fatty acids
that are suitable for modifying mimetibodies of the invention include, for
example, n-
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dodecanoate (C12, laurate), n-tetradecanoate (C14, myristate), n-octadecanoate
(Cls,
stearate), n-eicosanoate (C2o, arachidate), n-docosanoate (C22a behenate), n-
triacontanoate (C30), n tetracontanoate (Cao), cis-09-octad.ecanoate (Clg,
oleate), all cis-
A5,8,11,14-eicosatetraenoate (C20, arachidonate), octanedioic acid,
tetradecanedioic
acid, octadecanedioic acid, docosanedioic acid, and the like. Suitable fatty
acid esters
include monoesters of dicarboxylic acids that comprise a linear or branched
lower alkyl
group. The lower alkyl group can comprise from one to about twelve, preferably
one to
about six, carbon atoms.
[115] The modified human mimetibodies and ligand-binding fragments can be
prepared using suitable methods, such as by reaction with one or more
modifying
agents. A "modifying agent" as the term is used herein, refers to a suitable
organic
group (e.g., hydrophilic polymer, a fatty acid, a fatty acid ester) that
comprises an
activating group. An "activating group" is a chemical moiety or functional
group that
can, under appropriate conditions, react with a second chemical group thereby
forming a
covalent bond between the modifying agent and the second chemical group. For
example, amine-reactive activating groups include electrophilic groups such as
tosylate,
mesylate, halo (chloro, bromo, fluoro, iodo), N-hydroxysuccinimidyl esters
(NHS), and
the like. Activating groups that can react with thiols include, for example,
maleimide,
iodoacctyl, acrylolyl, pyridyl disulfides, 5-thiol-2-nitrobcnzoic acid thiol
(TNB-thiol),
and the like. An aldehyde functional group can be coupled to amine- or
hydrazide-
containing molecules, and an azide group can react with a trivalent
phosphorous group
to form phosphorarnidate or phosphorimide linkages. Suitable methods to
introduce
activating groups into molcculcs arc known in the art (scc for example,
Hcrmanson, G.
T., Biocorrjugat.e Techniques, Academic Press: San Diego, CA (1996)). An
activating
group can be bonded directly to the organic group (e.g., hydrophilic polymer,
fatty acid,
fatty acid ester), or through a linker moiety, for example a divalent C1-ClZ
group
wherein one or more carbon atoms can be replaced by a heteroatom such as
oxygen,
nitrogen or sulfu.r. Suitable linker moieties include, for example,
tetraethylene glycol, -
(CH2)3-, -NH-(CH2)6-NH-, -(CH2)2-NH- and -CH2-O-CH2-CH2-O-CH2-CH2-O-CH-NH-
3 0 . Modifying agents that comprise a linker moiety can be produced, for
example, by
reacting a mono-Boc-alkyldiamine (e.g., mono-Boc-ethylenediamine, mono-Boc-
diaminohexane) with a fatty acid in the presence of 1-ethyl-3-(3-
dimethylaminopropyl)
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38
carbodiimide (EDC) to form an amide bond between the free amine and the fatty
acid
carboxylate. The Boc protecting group can be removed from the product by
treatment
with trifluoroacetic acid (TFA) to expose a primary amine that can be coupled
to
another carboxylate as described, or can be reacted with maleic anhydride and
the
resulting product cyclized to produce an activated maleimido derivative of the
fatty acid.
(See, for example, Thompson, et al., WO 92/16221 the entire teachings of which
are
incorporated herein by reference.)
[116] The modified mimetibodies of the invention can be produced by reacting
an
human GLP-1 mimetibody or ligand-binding fragment with a modifying agent. For
example, the organic moieties can be bonded to the GLP-1 mimetibody in a non-
site
specific manner by employing an amine-reactive modifying agent, for example,
an NHS
ester of PEG. Modified human mimetibodies or ligand-binding fragments can also
be
prepared by reducing disulfide bonds (e.g., intra-chain disulfide bonds) of a
GLP-1
mimetibody or ligand-binding fragment. The reduced GLP-1 mimetibody or ligand-
binding fragment can then be reacted with a thiol-reactive modifying agent to
produce
the modified GLP-1 mimetibody of the invention. Modified human mimetibodies
and
ligand-binding fragments comprising an organic moiety that is bonded to
specific sites
of a GLP-1 mimetibody or specified portion or variant of the present invention
can be
prepared using suitable methods, such as reverse proteolysis (Fisch et al.,
Bioconjugate
Chem., 3:147-153 (1992); Werlen et al., Bioconjugate Chem., 5:411-417 (1994);
Kurnaran et al., Protein Sci. 6(10):2233-2241 (1997); Itoh et al., Bioorg.
Chem., 24(1):
59-68 (1996); Capellas et al., Biotechnol. Bioeng., 56(4):456-463 (1997)), and
thc
methods described in Herrnanson, G. T., Bioconjugate Techniques, Academic
Press: San
Diego, CA (1996).
[117] GLP-1 MIMETIBODY COMPOSITIONS. The present invention also
provides at least one GLP-1 mimetibody or specified portion or variant
composition
comprising at least one, at least two, at least three, at least four, at least
five, at least six
or more mimctibodics or specified portions or variants thcrcof, as described
hcrcin
and/or as known in the art that are provided in a non-naturally occurring
composition,
mixture or form. Such composition percentages are by weight, volume,
concentration,
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39
molarity, or molality as liquid or dry solutions, mixtures, suspension,
emulsions or
colloids, as known in the art or as described herein.
[118] Such compositions can comprise 0.00001-99.9999 percent by weight,
=volume,
concentration, molarity, or molality as liquid, gas, or dry solutions,
mixtures,
suspcnsion, cmulsions or colloids, as known in the art or as dcscribcd hcrcin,
on any
range or value therein, such as but not limited. to 0.00001, 0.00003, 0.00005,
0.00009,
0.0001, 0.0003, 0.0005, 0.0009, 0.001, 0.003, 0.005, 0.009, 0.01, 0.02, 0.03,
0.05, 0.09,
0.1, 0.2, 0.3, 0.4., 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5,
1.6, 1.7, 1.8, 1.9, 2.0,
2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5,
3.6, 3.7, 3.8, 3.9, 4.0,
4.3, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45,
50, 55, 60, 65, 70,
71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,
90, 91, 92, 93,
94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9
%. Such
compositions of the present invention thus include but are not limited to
0.00001-100
mg/ml and/or 0.00001-100 mg/g.
[119] The composition can optionally further comprise an effective amount of
at least
one compound or protein selected from at least one of a obesity related drug,
an anti-
infective drug, a cardiovascular (CV) system drug, a central nervous system
(CNS)
drug, an autonomic nervous system (ANS) drug, a respiratory tract drug, a
gastrointestinal (GI) tract drug, a hormonal drug, a drug for fluid or
electrolyte balance,
a hematologic drug, an antineoplactic, an immunomodulation drug, an
ophthalmic, otic
or nasal drug, a topical drug, a nutritional drug or the like. Such drugs are
well known in
the art, including formulations, indications, dosing and administration for
each presented
herein (see e.g., Nursing 2001 Handbook of Drugs, 21st edition, Springhouse
Corp.,
Springhouse, PA, 2001; Health Professional's Drug Guide 2001, ed., Shannon,
Wilson,
Stang, Prentice-Hall, Inc, Upper Saddle River, NJ; Pharmcotherapy Handbook,
Wells et
al., ed., Appleton & Lange, Stamford, CT, each entirely incorporated herein by
reference).
[120] The obesity related drug can be at least one of a fat catabolic compound
or
treatment, caffeine, glitazones, insulin and derivatives, sulfonylureas,
meglitinides,
biguanides, alpha-glucosidase inhibitors, protein tyrosine phosphastase-1B,
glycogen
synthase kinase 3, gluconeogenesis inhibitors, pyruvate dehydrogenase kinase
(PDH)
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inhibitors, lipolysis inhibitors, fat oxidation inhibitors, carnitine
palmitoyltransferase I
and/or II inhibitors, beta-3 adrenoceptor agonists, sodium and glucose
cotransporter
(SGLT) inhibitors, or compounds that act on one or more of at least one of:
autoim.mune
suppression, immune regulation, activation, proliferation, migration and/or
suppressor
5 cell function of T-cells, inhibition of T cell receptor/peptide/MHC-II
interaction,
Induction of T cell anergy, deletion of autoreactive T cells, reduction of
trafficking
across blood brain barrier, alteration of balance of pro-inflammatory (Th1)
and
immunomodulatory (Th2) cytokines, inhibition of matrix metalloprotease
inhibitors,
neuroprotection, reduction of gliosis, promotion of re-myelination.
10 [121] The GI tract drug can be at least one selected. from antacids,
adsorbents,
antiflatulents, digestive enzymes, gallstone solubilizers, antidiarrheals,
laxatives,
antiemetics and antiulcer drugs. The hormonal drug can be at least one
selected from
corticosteroids, androgens, anabolic steroids, estrogens, progestins,
gonadotropins,
antidiabetic drugs, at least one glucagon, thyroid hormones, thyroid hormone
15 antagonists, pituitary hormones and parathyroid-like drugs. The drug for
fluid and
electrolyte balance can be at least one selected from diuretics, electrolytes,
replacement
solutions, acidifiers and alkalinizers. The hematologic drug can be at least
one selected
from hematinics, anticoagulants, blood derivatives and thrombolytic enzymes.
The
antineoplastics can be at least one selected from alkylating drugs,
antimetabolites,
20 antibiotic antineoplastics, antineoplastics that alter hormone balance and
miscellaneous
antineoplastics. The immunomodulation drug can be at least one selected from
immunosuppressants, vaccines, toxoids, antitoxins, antivenins, imrnunc serums
and
biological response modifiers. The ophthalmic, otic, and nasal drugs can be at
least one
selected from ophthalmic anti-infectives, ophthalmic anti-inflammatories,
miotics,
25 mydriatics, ophthalmic vasoconstrictors and miscellaneous ophthalmics,
otics, nasal
drugs. The topical drug can be at least one selected from local anti-
infectives,
scabicides, pediculicides and topical corticosteroids. The nutritional drug
can be at least
one selected from vitamins, minerals and calorics. See, e.g., contents of
Natrsing 2001
Df-ug Handbook, supra.
30 [122] The at least one antacid, adsorbents, or antiflatulents can be at
least one selected
from aluminum carbonate, aluminum hydroxide, calcium carbonate, magaldrate,
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41
magnesium hydroxide, magnesium oxide, simethicone, and sodium bicarbonate. The
at
least one digestive enymes or gallstone solubilizers can be at least one
selected from
pancreatin, pancrelipase, and ursodiol. The at least one antidiarrheal can be
at least one
selected from attapulgite, bismuth subsalicylate, calcium polycarbophil,
diphenoxylate
hydrochloride or atropine sulfate, loperamide, octreotide acetate, opium
tincture, opium
tincure (camphorated). The at least one laxative can be at least one selected
from
bisocodyl, calcium polycarbophil, cascara sagrada, cascara sagrada aromatic
fluidextract, cascara sagrada fluidextract, castor oil, docu.sate calcium,
docusate sodium,
glycerin, lactulose, magnesium citrate, magnesium hydroxide, magnesium
sulfate,
methylcellulose, mineral oil, polyethylene glycol or electrolyte solution,
psyllium,
senna, sodium phosphates. The at least one antiemetic can be at least one
selected from
chlorpromazine hydrochloride, dimenhydrinate, dolasetron mesylate, dronabinol,
granisetron hydrochloride, meclizine hydrochloride, metocloproamide
hydrochloride,
ondansetron hydrochloride, perphenazine, prochlorperazine, prochlorperazine
edisylate,
prochlorperazine maleate, promethazine hydrochloride, scopolamine,
thiethylperazine
maleate, trimethobenzamide hydrochloride. The at least one antiulcer drug can
be at
least one selected from cimetidine, cimetidine hydrochloride, famotidine,
lansoprazole,
misoprostol, nizatidine, omeprazole, rabeprozole sodium, rantidine bismuth
citrate,
ranitidine hydrochloride, sucralfate. (See, e.g., pp. 643-95 of Nuf=sing 2001
Drug
Handbook.) The at least one antidiabetic or glucaon can be at least one
selected from
acarbose, chlorpropamide, glimepiride, glipizide, glucagon, glyburide,
insulins,
metformin hydrochloride, miglitol, pioglitazone hydrochloride, repaglinide,
rosiglitazone maleate, troglitazone. The at least one thyroid hormone can be
at least one
sclcctcd from lcvothyroxinc sodium, liothyroninc sodium, liotrix, thyroid. The
at lcast
one thyroid hormone antagonist can be at least one selected. from methimazole,
potassium iodide, potassium iodide (saturated solution), propylthiouracil,
radioactive
iodine (sodium iodide 131I), strong iodine solution. The at least one
pituitary hormone
can be at least one selected from corticotropin, cosyntropin, desmophressin
acetate,
leuprolide acetate, rGLP-1 sitory corticotropin, somatrem, somatropin,
vasopressin. The
at least one parathyroid-like drug can be at least one selected from
calcifediol, calcitonin
(human), calcitonin (salmon), calcitriol, dihydrotachysterol, etidronate
disodium. (See,
e.g., pp. 696-796 of Nursing 2001 Drug Handbook.)
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42
[123] The at least one diuretic can be at least one selected from
acetazolamide,
acetazolamide sodium, amiloride hydrochloride, bumetanide, chlorthalidone,
ethacrynate sodium, ethacryn.ic acid, furosemide, hydrochlorothiazide,
indapamide,
mannitol, metolazone, spironolactone, torsemide, triamterene, urea. The at
least one
electrolyte or replacement solution can be at least one selected from calcium
acetate,
calcium carbonate, calcium chloride, calcium citrate, calcium glubionate,
calcium
gluceptate, calcium gluconate, calcium lactate, calcium phosphate (dibasic),
calcium
phosphate (tribasic), dextran (high-molecu.lar-weight), dextran (low-molecular-
weight),
hetastarch, magnesium chloride, magnesium sulfate, potassium acetate,
potassium
bicarbonate, potassium chloride, potassium gluconate, Ringer's injection,
Ringer's
injection (lactated), sodium chloride. The at least one acidifier or
alkalinizer can be at
least one selected from sodium bicarbonate, sodium lactate, tromethamine.
(See, e.g.,
pp. 797-533 of Nursing 2001 Drug Handbook.)
[124] The at least one hematinic can be at least one selected from ferrous
fumarate,
ferrous gluconate, ferrous sulfate, ferrous sulfate (dried), iron dextran,
iron sorbitol,
polysaccharide-iron complex, sodium ferric gluconate complex. The at least one
anticoagulant can be at least one selected from ardeparin sodium, dalteparin
sodium,
danaparoid sodium, enoxaparin sodium, heparin calcium, heparin sodium,
warfarin
sodium. The at least one blood derivative can be at least one selected from
albumin 5%,
albumin 25%, antihemophilic factor, anti-inhibitor coagulant complex,
antithrombin III
(human), factor IX (human), factor IX complex, plasma protein fractions. The
at least
one thrombolytic cnzymc can bc at lcast onc scicctcd from aitcplasc,
anistrcplasc,
reteplase (recombinant), streptokinase, urokinase. (See, e.g., pp. 834-66 of
Nursing
2001 Drug Handbook.)
[125] The at least one local anti-infectives can be at least one selected from
acyclovir,
amphotericin B, azelaic acid cream, bacitracin, butoconazole nitrate,
clindamycin
phosphate, clotrimazole, econazole nitrate, erythromycin, gentamicin sulfate,
kctoconazolc, mafenide acctatc, mctronidazolc (topical), miconazolc nitratc,
mupirocin,
naftifine hydrochloride, neomycin sulfate, nitrofurazone, nystatin, silver
sulfadiazine,
terbinafine hydrochloride, terconazole, tetracycline hydrochloride,
tioconazole,
tolnaftate. The at least one scabicide or pediculicide can be at least one
selected from
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43
crotamiton, lindane, permethrin, and pyrethrins. The at least one topical
corticosteroid
can be at least one selected from betamethasone dipropionate, betamethasone
valerate,
clobetasol propionate, desonide, desoximetasone, dexamethasone, dexamethasone
sodium phosphate, diflorasone diacetate, fluocinolone acetonide, fluocinonide,
flurandrenolide, fluticasone propionate, halcionide, hydrocortisone,
hydrocortisone
acetate, hydrocortisone butyrate, hydrocorisone valerate, mometasone furoate,
triamcinolone acetonide. (See, e.g., pp. 1098-1136 of Nursing 2001 Drug
Handbook.)
[126] The at least one vitamin or mineral can be at least one selected from
vitamin A,
vitamin B complex, cyanocobalamin, folic acid, hydroxocobalamin, leucovorin
calcium,
niacin, niacinamide, pyridoxine hydrochloride, riboflavin, thiamine
hydrochloride,
vitamin C, vitamin D, cholecalciferol, ergocalciferol, vitamin D analogue,
doxercalciferol, paricalcitol, vitamin E, vitamin K analogue, phytonadione,
sodium
fluoride, sodium fluoride (topical), trace elements, chromium, copper, iodine,
manganese, selenium, zinc. The at least one calorics can be at least one
selected from
amino acid infusions (crystalline), amino acid -infusions in dextrose, amino
acid
infusions with electrolytes, amino acid infusions with electrolytes in
dextrose, amino
acid infusions for hepatic failure, amino acid infusions for high metabolic
stress, amino
acid infusions for renal failure, dextrose, fat emulsions, medium-chain
triglycerides.
(See, e.g., pp. 1137-63 of Nursing 2001 Drug Handbook.)
[127] GLP-1 mimetibody or specified portion or variant compositions of the
present
invention can further comprise at least one of any suitable auxiliary, such
as, but not
limited to, diluent, binder, stabilizer, buffers, salts, lipophilic solvents,
preservative,
adjuvant or the like. Pharmaceutically acceptable auxiliaries are preferred.
Non-
limiting examples of, and methods of preparing such sterile solutions are well
known in
the art, such as, but limited to, Gennaro, Ed., Rernington.'s Phannaceutical
Sciences,
18"' Edition, Mack Publishing Co. (Easton, PA) 1990. Pharmaceutically
acceptable
carriers can be routinely selected that are suitable for the mode of
administration,
solubility and/or stability of the GLP-1 mimetibody composition as well known
in the
art or as described. herein.
[128] Pharmaceutical excipients and additives useful in the present
composition
include but are not limited to proteins, peptides, amino acids, lipids, and
carbohydrates
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(e.g., sugars, including monosaccharides, di-, tri-, tetra-, and
oligosaccharides;
derivatized sugars such as alditols, aldonic acids, esterified sugars and the
like; and
polysaccharides or sugar polymers), which can be present singly or in
combination,
comprising alone or in combination 1-99.99% by weight or volume. Exemplary
protein
excipients include serum albumin such as human serum albumin (HSA),
recombinant
human albumin (rHA), gelatin, casein, and the like. Representative amino
acid/GLP-1
mimetibody or specified portion or variant components, which can also function
in a
buffering capacity, inclu.d.e alanine, glycine, arginine, betaine, histidine,
glutamic acid,
aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine,
phenylalanine,
aspartame, and the like. One preferred amino acid is glycine.
[129] Carbohydrate excipients suitable for use in the invention include, for
example,
monosaccharides such as fructose, maltose, galactose, glucose, D-mannose,
sorbose, and
the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and
the like;
polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans,
starches, and the
like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol
sorbitol (glucitol),
myoinositol and the like. Preferred carbohydrate excipients for use in the
present
invention are mannitol, trehalose, and raffinose.
[130] GLP-1 mimetibody compositions can also include a buffer or a pH
adjusting
agent; typically, the buffer is a salt prepared from an organic acid or base.
Representative buffers include organic acid salts such as salts of citric
acid, ascorbic
acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid,
or phthalic
acid; Tris, tromethamine hydrochloride, or phosphate buffers. Preferred
buffers for use
in the present compositions are organic acid salts such as citrate.
[131] Additionally, the GLP-1 mimetibody or specified portion or variant
compositions of the invention can include polymeric excipients/additives such
as
polyvinylpyrrolidoncs, ficolls (a polymeric sugar), dcxtratcs (e.g.,
cyclodextrins, such as
2-hydroxypropyl-f3-cyclodextrin), polyethylene glycols, flavoring agents,
antimicrobial
agents, sweeteners, antioxidants, antistatic agents, surfactants (e.g.,
polysorbates such as
"TWEEN 20" and "TWEEN 90"), lipids (e.g., phospholipids, fatty acids),
steroids (e.g.,
cholesterol), and chelating agents (e.g., EDTA).
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[132] These and additional known pharmaceutical excipients and/or additives
suitable
for use in the GLP-1 mimetibody compositions according to the invention are
known in
the art, e.g., as listed in "Remington: The Science & Practice of Pharmacy",
19 th ed.,
Williams & Williams, (1995), and in the "Physician's Desk Reference", 52nd
ed.,
5 Medical Economics, Montvale, NJ (1998), the disclosures of which are
entirely
incorporated herein by reference. Preferrred carrier or excipient materials
are
carbohydrates (e.g., saccharides and alditols) and buffers (e.g., citrate) or
polymeric
agents.
[133] Formulations. As noted above, the invention provides for stable
formulations,
10 which can preferably includ.e a suitable buffer with saline or a chosen
salt, as well as
optional preserved solutions and fortnulations containing a preservative as
well as multi-
use preserved formulations suitable for pharmaceutical or veterinary use,
comprising at
least one GLP-1 mimetibody or specified portion or variant in a
pharmaceutically
acceptable formulation. Preserved formulations contain at least one known
preservative
15 or optionally selected from the group consisting of at least one phenol, m-
cresol, p-
cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuric nitrite,
phenoxyethanol,
formaldehyde, chlorobutanol, magnesium chloride (e.g., hexahydrate),
allcylparaben
(methyl, ethyl, propyl, butyl and the like), benzalkonium chloride,
benzethonium
chloride, sodium dehydroacetate and thimerosal, or mixtures thereof in an
aqueous
20 diluent. Any suitable concentration or mixture can be used as known in the
art, such as
0.001-5 fo, or any range or value therein, such as, but not limited to 0.001,
0.003, 0.005,
0.009, 0.01, 0.02, 0.03, 0.05, 0.09, 0.1, 0.2, 0.3, 0.4., 0.5, 0.6, 0.7, 0.8,
0.9, 1.0, 1.1, 1.2,
1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7,
2.8, 2.9, 3.0, 3.1, 3.2,
3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.3, 4.5, 4.6, 4.7, 4.8, 4.9, or any
range or value
25 therein. Non-limiting examples include, no preservative, 0.1-2% m-cresol
(e.g., 0.2,
0.3. 0.4, 0.5, 0.9, 1.0%), 0.1-3% benzyl alcohol (e.g., 0.5, 0.9, 1.1., 1.5,
1.9, 2.0, 2.5%),
0.001-0.5% thimerosal (e.g., 0.005, 0.01), 0.001-2.0% phenol (e.g., 0.05,
0.25, 0.28, 0.5,
0.9, 1.0%), 0.0005-1.0% alkylparaben(s) (e.g., 0.00075, 0.0009, 0.001, 0.002,
0.005,
0.0075, 0.009, 0.01, 0.02, 0.05, 0.075, 0.09, 0.1, 0.2, 0.3, 0.5, 0.75, 0.9,
1.0%), and the
30 like.
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[134] As noted above, the invention provides an article of manufacture,
comprising
packaging material and at least one vial comprising a solution of at least one
GLP-1
mimetibody or specified portion or variant with the prescribed buffers and/or
preservatives, optionally in an aqueous diluent, wherein said packaging
material
comprises a label that indicates that such solution can be held over a period
of 1, 2, 3, 4,
5, 6, 9, 12, 18, 20, 24, 30, 36, 40, 48, 54, 60, 66, 72 hours or greater. The
invention
further comprises an article of manufacture, comprising packaging material, a
first vial
comprising lyophilized at least one GLP-1 mimetibody or specified, portion or
variant,
and a second vial comprising an aqueous diluent of prescribed buffer or
preservative,
wherein said packaging material comprises a label that instructs a patient to
reconstitute
the at least one GLP-1 mimetibody or specified portion or variant in the
aqueous diluent
to form a solution that can be held over a period of twenty-four hours or
greater.
[135] The at least one GLP-1 mimetibody or specified portion or variant used
in
accordance with the present invention can be produced by recombinant means,
including from mammalian cell or transgenic preparations, or can be purified
from other
biological sources, as described herein or as known in the art.
[136] The range of amounts of at least one GLP-1 mimetibody or specified
portion or
variant in the product of the present invention includes amounts yielding upon
reconstitution, if in a wet/dry system, concentrations from about 0.01 gg/ml
to about
100 mg/ml, although lower and higher concentrations are operable and are
dependent on
the intended delivery vehicle, e.g., solution formulations will differ from
transdermal
patch, pulmonary, transmucosal, or osmotic or micro pump methods.
[137] Preferably, the aqueous diluent optionally further comprises a
pharmaceutically
acceptable preservative. Preferred preservatives include those selected from
the group
consisting of phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl
alcohol,
alkylparabcn (methyl, cthyl, propyl, butyl and the like), bcnzalkonium
chloridc,
benzethonium chloride, sodium dehydroacetate and thimerosal, or mixtures
thereof. The
concentration of preservative used in the formulation is a concentration
sufficient to
yield an anti-microbial effect. Such concentrations are dependent on the
preservative
selected and are readily determined by the slcilled artisan.
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[138] Other excipients, e.g. isotonicity agents, buffers, antioxidants,
preservative
enhancers, can be optionally and preferably added to the diluent. An
isotonicity agent,
such as glycerin, is commonly used at known concentrations. A physiologically
tolerated buffer is preferably added to provide improved pH control. The
formulations
can cover a wide range of pHs, such as from about pH 4 to about pH 10, and
preferred
ranges from about pH 5 to about pH 9, and a most preferred range of about 6.0
to about
8Ø Preferably the formulations of the present invention have pH between
about 6.8
and. about 7.8. Preferred buffers include phosphate buffers, most preferably
sodium
phosphate, particularly phosphate buffered saline (PBS).
[139] Other additives, such as a pharmaceutically acceptable solubilizers like
Tween
(polyoxyethylene (20) sorbitan monolaurate), Tween 40 (polyoxyethylene (20)
sorbitan monopalmitate), Tween 80 (polyoxyethylene (20) sorbitan monooleate),
Pluronic F68 (polyoxyethylene polyoxypropylene block copolymers), and PEG
(polyethylene glycol) or non-ionic surfactants such as polysorbate 20 or 80 or
15 poloxamer 184 or 188, Pluronic polyls, other block co-polymers, and
chelators such as
EDTA and EGTA can optionally be added to the formulations or compositions to
reduce
aggregation. These additives are particularly usefu.l if a pump or plastic
container is
used to adrninister the formulation. The presence of pharmaceutically
acceptable
surfactant mitigates the propensity for the protein to aggregate.
20 [140] The formulations of the present invention can be prepared by a
process which
comprises mixing at least one GLP-1 mimetibody or specified portion or variant
and a
preservative selected from the group consisting of phenol, m-cresol, p-cresol,
o-cresol,
chlorocresol, benzyl alcohol, alkylparaben, (methyl, ethyl, propyl, butyl and
the like),
benzalkonium chloride, benzethonium chloride, sodiu.m dehydroacetate and
thimerosal
or mixtures thereof in an aqueous diluent. Mixing the at least one GLP-1
mimetibody or
specified portion or variant and preservative in an aqueous diluent is carried
out using
conventional dissolution and mixing procedures. To prepare a suitable
formulation, for
example, a measured amount of at least one GLP-1 mimetibody or specified
portion or
variant in buffered solution is combined with the desired preservative in a
buffered
solution in quantities sufficient to provide the protein and preservative at
the desired
concentrations. Variations of this process would be recognized by one of
ordinary skill
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48
in the art. For example, the order the components are added, whether
additional
additives are used, the temperature and pH at which the formulation is
prepared, are all
factors that may be optimized for the concentration and means of
administration used.
[141] The claimed formulations can be provided to patients as clear solutions
or as
dual vials comprising a vial of lyophilizcd at least one GLP-1 mimetibody or
specified
portion or variant that is reconstituted with a second. vial containing water,
a
preservative and/or excipients, preferably a phosphate buffer and/or saline
and a chosen
salt, in an aqueous diluent. Either a single solution vial or dual vial
requiring
reconstitution can be reused multiple times and can suffice for a single or
multiple
cycles of patient treatment and thus can provide a more convenient treatment
regimen
than currently available.
[142] The present claimed articles of manufacture are useful for
administration over a
period of immediately to twenty-four hours or greater. Accordingly, the
presently
claimed articles of manufacture offer significant advantages to the patient.
Formulations
of the invention can optionally be safely stored at temperatures of from about
2 to about
40 C and retain the biologically activity of the protein for extended periods
of time,
thus, allowing a package label indicating that the solution can be held and/or
used over a
period of 6, 12, 18, 24, 36, 48, 72, or 96 hours or greater. If preserved
diluent is used,
such label can include use up to at least one of 1-12 months, one-half, one
and a half,
and/or two years.
[143] The solutions of at least one GLP-1 mimetibody or specified. portion or
variant
in the invention can be prepared by a process that comprises mixing at least
one GLP-1
mimetibody or specified portion or variant in an aqueous diluent. Mixing is
carried out
using conventional dissolution and mixing procedures. To prepare a suitable
diluent, for
example, a measured amount of at least one GLP-1 mimetibody or specified
portion or
variant in water or buffer is combined in quantities sufficient to provide the
protein and
optionally a preservative or buffer at the desired concentrations. Variations
of this
process would be recognized by one of ordinary skill in the art. For example,
the order
the components are added, whether additional additives are used, the
temperature and
pH at which the formulation is prepared, are all factors that may be optimized
for the
concentration and means of administration used.
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[144] The claimed products can be provided to patients as clear solutions or
as dual
vials comprising a vial of lyophilized at least one GLP-1 mimetibody or
specified
portion or variant that is reconstituted with a second vial containing the
aqueous diluent.
Either a single solution vial or dual vial requiring reconstitution can be
reused multiple
times and can suffice for a single or multiple cycles of patient treatment and
thus
provides a more convenient treatment regimen than currently available.
[145] The claimed products can be provided indirectly to patients by providing
to
pharmacies, clinics, or other such institutions and facilities, clear
solutions or dual vials
comprising a vial of lyophilized at least one GLP-1 mimetibody or specified
portion or
variant that is reconstitu.ted, with a second vial containing the aqueous
diluent. The clear
solution in this case can be up to one liter or even larger in size, providing
a large
reservoir from which smaller portions of the at least one GLP-1 mimetibody or
specified
portion or variant solution can be retrieved one or multiple times for
transfer into
smaller vials and provided by the pharmacy or clinic to their customers andlor
patients.
[146] Recognized devices comprising these single vial systems include those
pen-
injector devices for delivery of a solution such as HumajectQ~NovoPen , B-D
Pen,
AutoPen , and QptiPen . Recognized devices comprising a dual vial system
include
those pen-injector systems for reconstituting a lyophilized drug in a
cartridge for
delivery of the reconstituted solution such as the HumatroPen~.
[147] The products presently claimed include packaging material. The packaging
material provides, in addition to the information required by the regulatory
agencies, the
conditions under which the product can be used. The packaging material of the
present
invention provides instructions to the patient to rcconstitutc the at least
onc GLP-1
mimetibody or specified portion or variant in the aqueous diluent to form a
solution and.
to use the solution over a period of 2-24 hours or greater for the two vial,
wet/dry,
product. For the single vial, solution product, the label indicates that such
solution can
be used over a period of 2-24 hours or greater. The presently claimed products
are
useful for human pharmaceutical product use.
[148] The formulations of the present invention can be prepared by a process
that
comprises mixing at least one GLP-1 mimetibody or specified portion or variant
and a
selected buffer, preferably a phosphate buffer containing saline or a chosen
salt. Mixing
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the at least one GLP-1 mimetibody or specified portion or variant and buffer
in an
aqueous diluent is carried out using conventional dissolution and mixing
procedures.
To prepare a suitable formulation, for example, a measured amount of at least
one GLP-
1 mimetibody or specified portion or variant in water or buffer is combined
with the
5 desired buffering agent in water in quantities sufficient to provide the
protein and buffer
at the desired concentrations. Variations of this process would be recognized
by one of
ordinary skill in the art. For example, the order the components are added,
whether
additional additives are used, the temperature and. pH at which the
formulation is
prepared, are all factors that can be optirnized for the concentration and
means of
10 administration used.
[149] The claimed stable or preserved formulations can be provided to patients
as
clear solutions or as dual vials comprising a vial of lyophilized at least one
GLP-1
mimetibody or specified portion or variant that is reconstituted with a second
vial
containing a preservative or buffer and excipients in an aqueous diluent.
Either a single
15 solution vial or dual vial requiring reconstitution can be reused multiple
times and can
suffice for a single or multiple cycles of patient treatment and thus provides
a more
convenient treatment regimen than currently available.
[150] At least one GLP-1 mimetibody or specified portion or variant in either
the
stable or preserved formulations or solutions described herein, can be
administered to a
20 patient in accordance with the present invention via a variety of delivery
methods
including SC or IM injection; transdermal, pulmonary, transmucosal, implant,
osmotic
pump, cartridge, micro pump, or other means appreciated by the skilled
artisan, as well-
known in the art.
[151] Therapeutic Applications. The present invention for mimetibodies also
25 provides a method for modulating or treating obesity related disorders,
including adult
onsct or juvcnilc, insulin dcpcndcnt, non-insulin dcpcndcnt, and the likc,
including the
associated. signs and symptoms, such as but not limited to, insulin
resistance,
hyperglycemia, hypoglycemia, pancreatitis, Sushing's syndrome, acanthosis
nigricans,
lipoatrrophic diabetes, retinopathy, nephropathy, polyneuropathy,
mononeuropathy,
30 autonomic neuropathy, ulcers, foot ulcers, joint problems, infections
(e.g., fungal or
bacterial), and the like, in a cell, tissue, organ, animal, or patient.
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[152] The present invention also provides a method for modulating or treating
at least
one obesity associated immune related disease, in a cell, tissue, organ,
animal, or patient
including, but not limited to, at least one of type I or type II diabetes
mellitus, including
adult onset or juvenile, insulin dependent, non-insulin dependent, and the
like, including
the associated signs and symptoms, such as but not limited to, insulin
resistance,
hyperglycemia, hypoglycemia, pancreatitis, Sushing's syndrome, acanthosis
nigricans,
lipoatrrophic diabetes, retinopathy, nephropathy, polyneuropathy,
mononeuropathy,
autonomic neuropathy, ulcers, foot ulcers, joint problems, infections (e.g.,
fungal or
bacterial), and the like. See, e.g., the Merck Manual, 12th-17th Editions,
Merck &
Company, Rahway, NJ (1972, 1977, 1982, 1987, 1992, 1999), Pharmacotherapy
Handbook, Wells et al., eds., Second Edition, Appleton and Lange, Stamford,
Conn.
(1998, 2001), each entirely incorporated by reference.
[153] Such a method can optionally comprise administering an effective amount
of at
least one composition or pharmaceutical composition comprising at least one
GLP-1
mimetibody or specified portion or variant to a cell, tissue, organ, animal or
patient in
need of such modulation, treatment or therapy.
[154] Any method of the present invention can comprise administering an
effective
amount of a composition or pharmaceutical composition comprising at least one
GLP-1
mimetibody or specified portion or variant to a cell, tissue, organ, animal or
patient in
need of such modulation, treatment or therapy. Such a method can optionally
further
comprise co-administration or combination therapy for treating such immune
diseases,
wherein the administering of said at least one GLP-1 mimetibody, specified
portion or
variant thereof, further comprises administering, before concurrently, and/or
after, at
least one selected from at least one TNF antagonist (e.g., but not limited to
a TNF
antibody or fragment, a soluble TNF receptor or fragment, fusion proteins
thereof, or a
small molecule TNF antagonist), an antirheumatic, a muscle relaxant, a
narcotic, a non-
steroid anti-inflammatory drug (NSAID), an analgesic, an anesthetic, a
sedative, a local
anethctic, a neuromuscular blockcr, an antimicrobial (e.g., aminoglycoside, an
antifungal, an antiparasitic, an antiviral, a carbapenem, cephalosporin, a
flurorquinolone,
a macrolide, a penicillin, a sulfonamide, a tetracycline, another
antimicrobial), an
antipsoriatic, a corticosteriod, an anabolic steroid, a obesity related agent,
a mineral, a
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52
nutritional, a thyroid agent, a vitamin, a calcium related hormone, an
antidiarrheal, an
antitussive, an antiemetic, an antiulcer, a laxative, an anticoagulant, an
erythropieitin
(e.g., GLP-letin alpha), a filgrastim (e.g., G-CSF, Neupogen), a sargramostim
(GM-
CSF, Leukine), an immunization, an immunoglobulin, an immunosuppressive (e.g.,
basiliximab, cyclosporine, daclizumab), a growth hormone, a hormone
replacement
drug, an estrogen receptor modulator, a mydriatic, a cycloplegic, an
alkylating agent, an
antimetabolite, a mitotic inhibitor, a radiopharmaceutical, an antidepressant,
antimanic
agent, an antipsychotic, an anxiolytic, a hypnotic, a sympathomimetic, a
stimulant,
donepezil, tacrine, an asthma medication, a beta agonist, an inhaled steroid,
a
leukotriene inhibitor, a methylxanthine, a cromolyn, an epinephrine or analog,
dornase
alpha (Pulmozyrne), a cytolcine or a cytokine antagonist. Suitable dosages are
well
lrnown in the art. See, e.g., Wells et al., eds., Pharrnacotherapy Handbook,
2nd Edition,
Appleton and Lange, Stamford, CT (2000); PDR Pharmacopoeia, Tarascon Pocket
Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Loma Linda, CA
(2000),
each of which references are entirely incorporated herein by reference.
[155] Typically, treatment of pathologic conditions is effected by
administering an
effective amount or dosage of at least one GLP-1 rnimetibody composition that
total, on
average, a range from at least about 0.0001 to 500 milligrams of at least one
GLP-1
mimetibody or specified portion or variant /kilogram of patient per dose, and
preferably
from at least about 0.001 to 100 milligrams GLP-1 mimetibody or specified
portion or
variant /kilogram of patient per single or multiple administration, depending
upon the
specific activity of contained in the composition. Altcrnativcly, the
effective serum
concentration can comprise 0.001-5000 gg/mi serum concentration per single or
multiple adminstration. Suitable dosages are known to medical practitioners
and will, of
course, depend upon the particular disease state, specific activity of the
composition
being administered, and the particular patient undergoing treatment. In some
instances,
to achieve the desired therapeutic amount, it can be necessary to provide for
repeated
administration, i.e., repeated individual administrations of a particular
monitored or
mctcrcd dose, whcrc the individual administrations arc rcpcatcd until the
dcsircd daily
dose or effect is achieved.
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[156] Preferred doses can optionally include 0.0001, 0.0002, 0.0003, 0.0004,
0.0005.
0.0006, 0.0007, 0.0008, 00009, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006,
0.007, 0.008,
0.009, 0.01, 0.02, 0.03, 0.04, 0.05. 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3,
0.4, 0.5, 0.6, 0.7,
0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24,
25, 26, 27, 28, 29, and/or 30 mg/kg/admini5tration, or any range, value or
fraction
thereof, or to achieve a serum concentration of 0.0001, 0.0002, 0.0003,
0.0004, 0.0005.
0.0006, 0.0007, 0.0008, 00009, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006,
0.007, 0.008,
0.009, 0.01, 0.02, 0.03, 0.04, 0.05. 0.06, 0.07, 0.08, 0.09, 0.1, 0.5, 0.9,
1.0, 1.1, 1.2, 1.5,
1.9, 2.0, 2.5, 2.9, 3.0, 3.5, 3.9, 4.0, 4.5, 4.9, 5.0, 5.5, 5.9, 6.0, 6.5,
6.9, 7.0, 7.5, 7.9, 8.0,
8.5, 8.9, 9.0, 9.5, 9.9, 10, 10.5, 10.9, 11, 11.5, 11.9, 20, 12.5, 12.9, 13.0,
13.5, 13.9, 14.0,
14.5, 4.9, 5.0, 5.5., 5.9, 6.0, 6.5, 6.9, 7.0, 7.5, 7.9, 8.0, 8.5, 8.9, 9.0,
9.5, 9.9, 10, 10.5,
10.9, 11, 11.5, 11.9,12, 12.5, 12.9, 13.0, 13.5, 13.9, 14, 14.5, 15, 15.5,
15.9, 16, 16.5,
16.9, 17, 17.5, 17.9, 18, 18.5, 18.9, 19, 19.5, 19.9, 20, 20.5, 20.9, 21, 22,
23, 24, 25, 26,
27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 96, 100, 200,
300, 400,
and/or 500 g/mi serum concentration per single or multiple administration, or
any
range, value or fraction thereof.
[157] Alternatively, the dosage administered can vary depending upon known
factors,
such as the pharmacodynamic characteristics of the particular agent, and its
mode and
route of administration; age, health, and weight of the recipient; nature and
extent of
symptoms, kind of concurrent treatment, frequency of treatment, and the effect
desired.
Usually a dosage of active ingredient can be about 0.000 1 to 100 milligrams
per
kilogram of body weight. Ordinarily 0.00 1 to 10, and preferably 0.00 1 to 1-
milligrarns
per kilogram per administration or in sustained release form is effective to
obtain
desired results.
[158] As a non-limiting example, treatment of humans or animals can be
provided as
a one-time or periodic dosage of at least one GLP- 1 mimetibody or specified
portion or
variant of the present invention 0.0001 to 100 mg/kg, such as 0.0002, 0.0003,
0.0004,
0.0005. 0.0006, 0.0007, 0.0008, 00009, 0.001, 0.002, 0.003, 0.004, 0.005,
0.006, 0.007,
0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05. 0.06, 0.07, 0.08, 0.09, 0.1, 0.2,
0.3, 0.4, 0.5,
0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/kg, per day, on at least
one of day 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 26, 27, 28,
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29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively, at least
one of week 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, or any
combination
thereof, using single, infusion or repeated doses.
[159] Dosage forms (composition) suitable for internal administration
generally
contain from about 0.0001 milligram to about 500 milligrams of activc
ingredient pcr
unit or container. In these pharmaceutical compositions the active ingredient
will
ordinarily be present in an amount of about 0.5-99.999 fo by weight based on
the total
weight of the composition.
[160] For parenteral administration, the GLP-1 rnimetibody or specified
portion or
variant can be formulated as a solution, suspension, emulsion or lyophilized
powder in
association, or separately provided, with a pharmaceutically acceptable
parenteral
vehicle. Examples of such vehicles are water, saline, Ringer's solution,
dextrose
solution, and 5% human serum albumin. Liposomes and nonaqueous vehicles such
as
fixed oils may also be used. The vehicle or lyophilized powder may contain
additives
that maintain isotonicity (e.g., sodium chloride, mannitol) and chemical
stability (e.g.,
buffers and preservatives). The formulation is sterilized by known or suitable
techniques.
[161] Suitable pharmaceutical carriers are described in the most recent
edition of
Remington's Pharmaceutical Sciences, A. Osol, a standard reference text in
this field.
[162] Therapeutic Administration. Many known and developed modes of can be
used for administering pharmaceutically effective amounts of at least one GLP-
1
mimetibody or specified portion or variant according to the present invention.
A GLP-1
mimetibody of the present invention can be delivered in a carrier, as a
solution,
emulsion, colloid, or suspension, or as a powder, using any of a variety of
devices and
methods suitable for administration by inhalation or other modes described
here within
or known in the art.
[163] Parenteral Formulations and Administration. Formulations for parenteral
administration can contain as cornmon excipients sterile water or saline,
polyalkylene
glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated
naphthalenes
and the likc. Aqueous or oily suspensions for injection can be prepared by
using an
appropriate emulsifier or humidifier and a suspending agent, according to
known
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methods. Agents for injection can be a non-toxic, non-orally administrable
diluting
agent such as aquous solution or a sterile injectable solution or suspension
in a solvent.
As the usable vehicle or solvent, water, Ringer's solution, isotonic saline,
etc. are
allowed; as an ordinary solvent, or suspending solvent, sterile involatile oil
can be used.
5 For these purposes, any kind of involatile oil and fatty acid can be used,
including
natural or synthetic or semisynthetic fatty oils or fatty acids; natural or
synthetic or
semisynthtetic mono- or di- or tri-glycerides. Parental administration is
known in the art
and includes, but is not limited to, conventional means of injections, a gas
pressured
needle-less injection device as described in U.S. Pat. No. 5,851,198, and a
laser
10 perforator device as described in U.S. Pat. No. 5,839,446 entirely
incorporated herein by
reference.
[164] Alternative Delivery. The invention further relates to the
administration of at
least one GLP-1 mimetibody or specified portion or variant by parenteral,
subcutaneous,
intramuscular, intravenous, bolus, vaginal, rectal, buccal, sublingual,
intranasal, or
15 transdermal means. Protein, GLP-1 mimetibody or specified portion or
variant
compositions can be prepared for use for parenteral (subcutaneous,
intramuscular or
intravenous) administration particularly in the form of liquid solutions or
suspensions;
for use in vaginal or rectal administration particularly in semisolid forrris
such as creams
and suppositories; for buccal, or sublingual administration particularly in
the form of
20 tablets or capsules; or intranasally particularly in the form of powders,
nasal drops or
aerosols or certain agents; or transderrnally particularly in the form of a
gel, ointment,
lotion, suspension or patch dclivcry system with chemical cnhanccrs such as
dimethyl
sulfoxide to either modify the skin structure or to increase the drug
concentration in the
transdermal patch (Junginger, et al. In "Drug Permeation Enhancement"; Hsieh,
D. S.,
25 Eds., pp. 59-90 (Marcel Dekker, Inc. New York 1994, entirely incorporated
herein by
reference), or with oxidizing agents that enable the application of
formulations
containing proteins and. peptides onto the skin (WO 98/53847), or applications
of
electric fields to create transient transport pathways such as
electroporation, or to
increase the mobility of charged drugs through the skin such as iontophoresis,
or
30 application of ultrasound such as sonophoresis (U.S. Pat. Nos. 4,309,989
and 4,767,402)
(the above publications and patents being entirely incorporated herein by
reference).
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[165] Pulmonary/Nasal Administration. For pulmonary administration, preferably
at least one GLP-1 mimetibody or specified portion or variant composition is
delivered
in a particle size effective for reaching the lower airways of the lung or
sinuses.
According to the invention, at least one GLP-l mimetibody or specified portion
or
variant can be delivered by any of a variety of inhalation or nasal devices
known in the
art for administration of a therapeutic agent by inhalation. These devices
capable of
dGLP-lsiting aerosolized formulations in the sinus cavity or alveoli of a
patient include
metered dose inhalers, nebulizers, dry powder generators, sprayers, and, the
like. Other
devices suitable for directing the pulmonary or nasal administration of GLP-1
mimetibody or specified portion or variants are also known in the art. All
such devices
can use of forrn.ulations suitable for the administration for the dispensing
of GLP-1
mimetibody or specified portion or variant in an aerosol. Such aerosols can be
comprised of either solutions (both aqueous and non aqueous) or solid
particles.
Metered dose inhalers like the Ventolin metered dose inhaler, typically use a
propellent
gas and require actuation during inspiration (See, e.g., WO 94/16970, WO
98/35888).
Dry powder inhalers like TurbuhalerTM (Astra), Rotahaler (Glaxo), Diskus
(Glaxo),
SpirosTm inhaler (Dura), devices marketed by Inhale Therapeutics, and the
Spinhaler
powder inhaler (Fisons), use breath-actuation of a mixed powder (US 4668218
Astra,
EP 237507 Astra, WO 97/25086 Glaxo, WO 94/08552 Dura, US 5458135 Inhale, WO
94/06498 Fisons, entirely incorporated herein by reference). Nebulizers like
AERxTM
Aradigm, the Ultravent nebulizer (Mallinckrodt), and the Acorn II nebulizer
(Marquest Medical Products) (US 5404871 Aradigm, WO 97/22376), the above
references entirely incorporated herein by reference, produce aerosols from
solutions,
while metered dose inhalcrs, dry powdcr inhalcrs, ctc. generate small particle
aerosols.
These specific examples of commercially available inhalation d.evices are
intended to be
a representative of specific devices suitable for the practice of this
invention, and are not
intended as limiting the scope of the invention. Preferably, a composition
comprising at
least one GLP-1 mimetibody or specified portion or variant is delivered by a
dry powder
inhaler or a sprayer. There are a several desirable features of an inhalation
device for
administering at least one GLP-1 mimetibody or specified portion or variant of
the
present invention. For example, delivery by the inhalation device is
advantageously
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57
reliable, reproducible, and accurate. The inhalation device can optionally
deliver small
dry particlcs, c.g. lcss than about 10 m, prcfcrably about 1-5 m, for good
respirability.
[166] Administration of GLP-1 mimetibody or specified portion or variant
Compositions as a Spray. A spray including GLP-1 mimetibody or specified
portion
or variant composition protein can be produced by forcing a suspension or
solution of at
least one GLP-1 mimetibody or specified portion or variant through a nozzle
under
pressure. The nozzle size and configuration, the applied pressure, and the
liquid feed
rate can be chosen to achieve the desired output and particle size. An
electrospray can
be produced, for example, by an electric field in connection with a capillary
or nozzle
feed. Advantageously, particles of at least one GLP-1 mimetibody or specified
portion
or variant composition protein delivered by a sprayer have a particle size
less than about
10 m, preferably in the range of about 1 m to about 5 m, and most
preferably about
2 gm to about 3 m.
[167] Formulations of at least one GLP-1 mimetibody or specified portion or
variant
composition protein suitable for use with a sprayer typically include GLP-1
mimetibody
or specified portion or variant composition protein in an aqueous solution at
a
concentration of about 1 mg to about 20 mg of at least one GLP-1 m.imetibody
or
specified portion or variant composition protein per ml of solution. The
formulation can
include agents such as an excipient, a buffer, an isotonicity agent, a
preservative, a
surfactant, and, preferably, zinc. The formulation can also include an
excipient or agent
for stabilization of the GLP-1 mimetibody or specified portion or variant
composition
protein, such as a buffer, a reducing agent, a bulk protein, or a
carbohydrate. Bulk
proteins useful in formulating GLP-1 mimetibody or specified portion or
variant
composition proteins include albumin, protamine, or the like. Typical
carbohydrates
useful in formulating GLP-1 mimetibody or specified portion or variant
composition
proteins include sucrose, mannitol, lactose, trehalose, glucose, or the like.
The GLP-1
mimetibody or specified portion or variant composition protein formulation can
also
include a surfactant, which can reduce or prevent surface-induced aggregation
of the
GLP-1 mimetibody or specified portion or variant composition protein caused by
atomization of the solution in forming an aerosol. Various conventional
surfactants can
be employed, such as polyoxyethylene fatty acid esters and alcohols, and
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polyoxyethylene sorbitol fatty acid esters. Amounts will generally range
between 0.001
and 14% by weight of the formulation. Especially preferred surfactants for
purposes of
this invention are polyoxyethylene sorbitan monooleate, polysorbate 80,
polysorbate 20,
or the like. Additional agents known in the art for formulation of a protein
such as
mimetibodies, or specified portions or variants, can also be included in the
formulation.
[168] Administration of GLP-1 mimetibody or specified portion or variant
compositions by a Nebulizer. GLP-1 mimetibody or specified portion or variant
composition protein can be administered by a nebulizer, such as jet nebulizer
or an
ultrasonic nebulizer. Typically, in a jet nebulizer, a compressed air source
is used to
create a high-velocity air jet through an orifice. As the gas expands beyond
the nozzle,
a low-pressure region is created, which draws a solution of GLP-1 mimetibody
or
specified portion or variant composition protein through a capillary tube
connected to a
liquid reservoir. The liquid stream from the capillary tube is sheared into
unstable
filaments and droplets as it exits the tube, creating the aerosol. A range of
configurations, flow rates, and baffle types can be employed to achieve the
desired
performance characteristics from a given jet nebulizer. In an ultrasonic
nebulizer, high-
frequency electrical energy is used to create vibrational, mechanical energy,
typically
employing a piezoelectric transducer. This energy is transmitted to the
formulation of
GLP-1 mimetibody or specified portion or variant composition protein either
directly or
through a coupling fluid, creating an aerosol including the GLP-1 mimetibody
or
specified portion or variant composition protein. Advantageously, particles of
GLP-1
mimctibody or spccificd portion or variant composition protcin dclivcrcd by a
ncbulizcr
have a particle size less than about 10 m, preferably in the range of about 1
m to
about 5 m, and most preferably about 2 m to about 3 m.
[169] Formulations of at least one GLP-1 mimetibody or specified portion or
variant
suitable for use with a nebulizer, either jet or ultrasonic, typically include
GLP-1
mimetibody or specified portion or variant composition protein in an aqueous
solution at
a concentration of about 1 mg to about 20 mg of at least one GLP-1 mimetibody
or
specified portion or variant protein per ml of solution. The formulation can
include
agents such as an excipient, a buffer, an isotonicity agent, a preservative, a
surfactant,
and, preferably, zinc. The formulation can also include an excipient or agent
for
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stabilization of the at least one GLP-1 mimetibody or specified portion or
variant
composition protein, such as a buffer, a reducing agent, a bulk protein, or a
carbohydrate. Bulk proteins useful in formulating at least one GLP-1
mimetibody or
specified portion or variant composition proteins include albumin, protamine,
or the
like. Typical carbohydrates useful in formulating at least one GLP-1
mimetibody or
specified portion or variant include sucrose, mannitol, lactose, trehalose,
glucose, or the
like. The at least one GLP-1 mimetibody or specified portion or variant
forrnulation can
also include a surfactant, which can reduce or prevent surface-induced.
aggregation of
the at least one GLP-1 mimetibody or specified portion or variant caused by
atomization
of the solution in forining an aerosol. Various conventional surfactants can
be
employed, such as polyoxyethylene fatty acid esters and alcohols, and
polyoxyethylene
sorbital fatty acid esters. Amounts will generally range between 0.001 and 4%
by
weight of the formulation. Especially preferred surfactants for purposes of
this
invention are polyoxyethylene sorbitan mono-oleate, polysorbate 80,
polysorbate 20, or
the like. Additional agents known in the art for formulation of a protein such
as at least
one GLP-1 mimetibody or specified portion or variant protein can also be
included in
the formulation.
[170] Administration of GLP-1 mimetibody or specified portion or variant
compositions By A Metered Dose Inhaler. In a metered dose inhaler (MDI), a
propellant, at least one GLP-1 mimetibody or specified portion or variant, and
any
excipients or other additives are contained in a canister as a mixture
including a
liqucficd compressed gas. Actuation of the mctcring valve releases the mixture
as an
aerosol, preferably containing particles in the size range of less than about
10 pm,
preferably about 1 m to about 5 gm, and most preferably about 2 m to about 3
m.
The desired aerosol particle size can be obtained by employing a formulation
of GLP- I
mimetibody or specified portion or variant composition protein produced by
various
methods known to those of skill in the art, including jet-milling, spray
drying, critical
point condensation, or the like. Preferred metered dose inhalers include those
manufactured by 3M or Glaxo and employing a hydrofluorocarbon propellant.
[171] Formulations of at least one GLP-1 mimetibody or specified portion or
variant
for use with a metered-dose inhaler device will generally include a finely
divided
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powder containing at least one GLP-1 mimetibody or specified portion or
variant as a
suspension in a non-aqueous medium, for example, suspended in a propellant
with the
aid of a surfactant. The propellant can be any conventional material emptoyed
for this
purpose, such as chlorofluorocarbon, a hydrochlorofluorocarbon, a
hydrofluorocarbon,
5 or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane,
dichlorotetrafluoroethanol and 1, 1, 1,2-tetrafluoroethane, HFA-134a
(hydrofluroalkane-
134a), HFA-227 (hydrofluroalkane-227), or the like. Preferably the propellant
is a
hydrofluorocarbon. The surfactant can be chosen to stabilize the at least one
GLP-1
mimetibody or specified portion or variant as a suspension in the propellant,
to protect
10 the active agent against chemical degradation, and the like. Suitable
surfactants include
sorbitan trioleate, soya lecithin, oleic acid, or the like. In some cases
solution aerosols
are preferred using solvents such as ethanol. Additional agents known in the
art for
formulation of a protein such as protein can also be included in the
formulation.
[172] One of ordinary skill in the art will recognize that the methods of the
current
15 invention can be achieved by pulmonary administration of at least one GLP-1
mimetibody or specified portion or variant compositions via devices not
described
herein.
[173] Mucosal Formulations and Administration. For absorption through mucosal
surfaces, compositions and methods of administering at least one GLP-1
mimetibody or
20 specified portion or variant include an emulsion comprising a plurality of
submicron
particles, a mucoadhesive macromolecule, a bioactive peptide, and an aqueous
continuous phase, which promotes absorption through mucosal surfaces by
achieving
mucoadhesion of the emulsion particles (U.S. Pat. Nos. 5,514,670). Mucous
surfaces
suitable for application of the emulsions of the present invention can include
comeal,
25 conjunctival, buccal, sublingual, nasal, vaginal, pulmonary, stomachic,
intestinal, and
rectal routes of administration. Formulations for vaginal or rectal
administration, e.g.
suppositories, can contain as excipients, for example, polyalkyleneglycols,
vaseline,
cocoa butter, and the like. Formulations for intranasal administration can be
solid and
contain as excipients, for example, lactose or can be aqueous or oily
solutions of nasal
30 drops. For buccal administration excipients include sugars, calcium
stearate, magnesium
stearate, pregelinatined starch, and the like (U.S. Pat. Nos. 5,849,695).
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[174] Oral Formulations and Administration. Formulations for oral rely on the
co-
administration of adjuvants (e.g., i-esorcinols and nonionic surfactants such
as
polyoxyetlhylene oleyl ether and n-hexadecylpolyethylene ether) to increase
artificially
the permeability of the intestinal walls, as well as the co-administration of
enzymatic
inhibitors (e.g., pancreatic trypsin inhibitors, diisopropylfluorophosphate
(DFF) and
trasylol) to inhibit enzymatic degradation. The active constituent compound of
the
solid-type dosage form for oral administration can be mixed with at least one
additive,
includ.ing sucrose, lactose, cellulose, mannitol, trehalose, raffinose,
maltitol, dextran,
starches, agar, arginates, chitins, chitosans, pectins, gum tragacanth, gum
arabic, gelatin,
collagen, casein, albumin, synthetic or semisynthetic polymer, and glyceride.
These
dosage forms can also contain other type(s) of additives, e.g., inactive
diluting agent,
lubricant such as magnesium stearate, paraben, preserving agent such as sorbic
acid,
ascorbic acid, alpha-tocopherol, antioxidant such as cysteine, disintegrator,
binder,
thickener, buffering agent, sweetening agent, flavoring agent, perfuming
agent, etc.
[175] Tablets and pills can be further processed into enteric-coated
preparations. The
liquid preparations for oral administration include emulsion, syrup, elixir,
suspension
and solution preparations allowable for medical use. These preparations may
contain
inactive diluting agents ordinarily used in said field, e.g., water. Liposomes
have also
been described as drug delivery systems for insulin and heparin (U.S. Pat. No.
4,239,754). More recently, microspheres of artificial polymers of mixed amino
acids
(proteinoids) have been used to deliver pharmaceuticals (U.S. Pat. No.
4,925,673).
Furthcrrnorc, carrier compounds described in U.S. Pat. No. 5,879,681 and U.S.
Pat. No.
5,5,871,753 are used to deliver biologically active agents orally are known in
the art.
[176] Transdermal Formulations and Administration. For transdermal
administration, the at least one GLP-1 mimetibody or specified portion or
variant is
encapsulated in a delivery device such as a liposome or polymeric
nanoparticles,
microparticle, microcapsule, or microspheres (referred to collectively as
microparticles
unlcss otherwise stated). A number of suitable dcviccs are known, including
microparticles made of synthetic polymers such as polyhydroxy acids such as
polylactic
acid, polyglycolic acid and copolymers thereof, polyorthoesters,
polyanhydrides, and
polyphosphazenes, and natural polymers such as collagen, polyamino acids,
albumin
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62
and other proteins, alginate and other polysaccharides, and combinations
thereof (U.S.
Pat. Nos. 5,814,599).
[177] Prolonged Administration and Formulations. It can be sometimes desirable
to deliver the compounds of the present invention to the subject over
prolonged periods
of time, for cxampic, for periods of one wcck to one year from a single
administration.
Various slow release, dGLP-1t or implant dosage fortns can be utilized. For
example, a
dosage form can contain a pharmaceutically acceptable non-toxic salt of the
compounds
that has a low degree of solubility in body fluids, for example, (a) an acid
addition salt
with a polybasic acid such as phosphoric acid, sulfuric acid, citric acid,
tartaric acid,
tannic acid., pamoic acid., alginic acid, polyglutamic acid, naphthalene mono-
or di-
sulfonic acids, polygalacturonic acid, and the like; (b) a salt with a
polyvalent metal
cation such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper,
cobalt,
nickel, cadmium and the like, or with an organic cation formed from e.g., N,N'-
dibenzyl-ethylenediamine or ethylenediamine; or (c) combinations of (a) and
(b) e.g. a
zinc tannate salt. Additionally, the compounds of the present invention or,
preferably, a
relatively insoluble salt such as those just described, can be formulated in a
gel, for
example, an aluminum monostearate gel with, e.g. sesame oil, suitable for
injection.
Particularly preferred salts are zinc salts, zinc tannate salts, pamoate
Salts, and the like.
Another type of slow release dGLP-lt formulation for injection would contain
the
compound or salt dispersed for encapsulated in a slow degrading, non-toxic,
non-
antigenic polymer such as a polylactic acid/polyglycolic acid polymer for
example as
described in U.S. Pat. No. 3,773,919. The compounds or, prcfcrably, relatively
insoluble
salts such as those described above can also be formulated in cholesterol
matrix silastic
pellets, particularly for use in animals. Additional slow release, dGLP-1t or
implant
formulations, e.g. gas or liquid liposomes are known in the literature (U.S.
Pat. Nos.
5,770,222 and "Sustained and Controlled Release Drug Delivery Systems", J. R.
Robinson ed., Marcel Dekker, Inc., N.Y., 1978).
[178] Having generally described the invention, the samc will be more readily
understood by reference to the following examples, which are provided. by way
of
3 0 illustration and are not intended as limiting.
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63
[179] Example 1: Cloning and Expression of a GLP-1 mimetibody in
Mammalian Cells. A typical mammalian expression vector contains at least one
promoter element, which mediates the initiation of transcription of mRNA, the
GLP-1
mimetibody or specified portion or variant coding sequence, and signals
required for the
termination of transcription and polyadenylation of the transcript. Additional
elements
include enhancers, Kozak sequences and intervening sequences flanked by donor
and
acceptor sites for RNA splicing. Highly efficient transcription can be
achieved with the
early and late promoters from SV40, the long terminal repeats (LTRS) from
Retroviruses, e.g., RSV, HTLVI, HIVI and the early promoter of the
cytomegalovirus
(CMV). However, cellular elements can also be used (e.g., the human actin
promoter).
Suitable expression vectors for use in practicing the present invention
include, for
example, vectors such as pTRES 1 neo, pRetro-Off, pRetro-On, PLXSN, or pLNCX
(Clonetech Labs, Palo Alto, CA), pcDNA3.1 (+/-), pcDNA/Zeo (+/-) or
pcDNA3.1/Hygro (+/-) (Invitrogen), PSVL and PMSG (Pharmacia, Uppsala, Sweden),
pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC 67109).
Mammalian host cells that could be used include human Hela 293, H9 and Jurkat
cells,
mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV 1, quail QC 1-3 cells, mouse
L
cells and Chinese hamster ovary (CHO) cells.
[180] Alternatively, the gene can be expressed in stable cell lines that
contain the gene
integrated into a chromosome. The co-transfection with a selectable marker
such as
dhfr, gpt, neomycin, or hygromycin allows the identification and isolation of
the
transfcctcd cells.
[181] The transfected gene can also be amplified to express large amounts of
the
encoded GLP-1 mimetibody or specified portion or variant. The DHFR
(dihydrofolate
reductase) marker is useful to develop cell lines that carry several hundred
or even
several thousand copies of the gene of interest. Another useful selection
marker is the
enzyme glutamine synthase (GS) (Murphy, et al., Biochem. J. 227:277-279
(1991);
Bcbbington, et al., Bio/Technology 10:169-175 (1992)). Using these markcrs,
thc
mammalian cells are grown in selective medium and. the cells with the highest
resistance
are selected. These cell lines contain the amplified gene(s) integrated into a
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64
chromosome. Chinese hamster ovary (CHO) and NSO cells are often used for the
production of GLP-1 mimetibody or specified portion or variants.
[182] The expression vectors pC1 and pC4 contain the strong promoter (LTR) of
the
Rous Sarcoma Virus (Cullen, et al., Molec. Cell. Biol. 5:438-447 (1985)) pl.us
a
fragment of the CMV-cnhanccr (Boshart, ct al., Cc1141:521-530 (1985)).
Multiple
cloning sites, e.g., with the restriction enzyme cleavage sites BamHI, Xbal
and. Asp718,
facilitate the cloning of the gene of interest. The vectors contain in
addition the 3'
intron, the polyadenylation and termination signal of the rat preproinsulin
gene.
[183] Cloning and Expression in CHO Cells. The vector pC4 is used for the
expression of GLP-1 mimetibody or specified portion or variant. Plasmid pC4 is
a
derivative of the plasmid pSV2-dhfr (ATCC Accession No. 37146). The plasmid
contains the mouse DHFR gene under control of the SV40 early promoter. Chinese
hamster ovary- or other cells lacking dihydrofolate activity that are
transfected with
these plasmids can be selected by growing the cells in a selective medium
(e.g., alpha
minus MEM, Life Technologies, Gaithersburg, MD) supplemented with the
chemotherapeutic agent methotrexate. The amplification of the DHFR genes in
cells
resistant to methotrexate (MTX) has been well documented (see, e.g., F. W.
Alt, et al., J.
Biol. Chem. 253:1357-1370 (1978); J. L. Hamlin and C. Ma, Biochem. et Biophys.
Acta
1097:107-143 (1990); and M. J. Page and M. A. Sydenham, Biotechnology 9:64-68
(1991)). Cells grown in increasing concentrations of MTX develop resistance to
the
drug by overproducing the target enzyme, DHFR, as a result of amplification of
the
DHFR gene. If a second gene is linked to the DHFR gene, it is usually co-
amplified and
over-expressed. It is known in the art that this approach can be used to
develop cell
lines carrying more than 1,000 copies of the amplified gene(s). Subsequently,
when the
methotrexate is withdrawn, cell lines are obtained that contain the amplified
gene
integrated into one or more chromosome(s) of the host cell.
[184] Plasmid. pC4 contains for expressing the gene of interest the strong
promoter of
the long terminal repeat (LTR) of the Rous Sarcoma Virus (Cullen, et al.,
Molec. Cell.
Biol. 5:438-447 (1985)) plus a fragment isolated from the enhancer of the
immediate
early gene of human cytomegalovirus (CMV) (Boshart, et al., Cell 41:521-530
(1985)).
Downstream of the promoter are BamHT, XbaT, and Asp718 restriction enzyme
cleavage
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sites that allow integration of the genes. Behind these cloning sites the
plasmid contains
the 3' intron and polyadenylation site of the rat preproinsulin gene. Other
high
efficiency promoters can also be used for the expression, e.g., the human b-
actin
promoter, the SV40 early or late promoters or the long terminal repeats from
other
5 retroviruses, e.g., HIV and HTLVI. Clontech's Tet-Off and Tet-On gene
expression
systems and similar systems can be used to express the GLP- 1 in a regulated
way in
mammalian cells (M. Gossen, and H. Bujard, Proc. Natl. Acad. Sci. USA 89: 5547-
5551
(1992)). For the polyadenylation of the mRNA other signals, e.g., from the
human
growth hormone or globin genes can be used as well. Stable cell lines carrying
a gene
10 of interest integrated into the chromosomes can also be selected upon co-
transfection
with a selectable marker such as gpt, G418 or hygromycin. It is advantageous
to use
more than one selectable marker in the beginning, e.g., G418 plus
methotrexate.
[185] The plasmid pC4 is digested with restriction enzymes and then
dephosphorylated using calf intestinal phosphatase by procedures known in the
art. The
15 vector is then isolated from a 1% agarose gel.
[186] The DNA sequence encoding the complete GLP-1 mimetibody or specified
portion or variant is used, corresponding to HC and LC variable regions of a
GLP-1
mimetibody of the present invention, according to known method steps. Isolated
nucleic acid encoding a suitable human constant region (i.e., HC and LC
regions) is also
20 used in this construct.
[187] The isolated variable and constant region encoding DNA and the
dephosphorylated vector are then ligated with T4 DNA ligase. E. coli HB101 or
XL-1
Blue cells are then transformed and bacteria are identified that contain the
fragment
inserted into plasmid pC4 using, for instance, restriction enzyme analysis.
25 [188] Chinese hamster ovary (CHO) cells lacking an active DHFR gene are
used for
transfection. 5 g of the expression plasmid pC4 is cotransfected with 0.5 g
of the
plasmid pSV2-neo using lipofectin. The plasmid pSV2neo contains a dominant
selectable marker, the neo gene from Tn5 encoding an enzyme that confers
resistance to
a group of antibiotics including G418. The cells are seeded in alpha minus MEM
3 0 supplcmcntcd with 1 g /ml G418. After 2 days, the cells are trypsinized
and sccdcd in
hybridoma cloning plates (Greiner, Germany) in alpha minus MEM supplemented
with
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10, 25, or 50 ng/ml of methotrexate plus 1 g /ml G418. After about 10-14 days
single
clones are trypsinized and then seeded in 6-well petri dishes or 10 ml flasks
using
different concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800
nM).
Clones growing at the highest concentrations of methotrexate are then
transferred to
new 6-well plates containing even higher concentrations of methotrexate (1 mM,
2 mM,
5 mM, 10 mM, 20 mM). The same procedure is repeated until clones are obtained
that
grow at a concentration of 100 - 200 mM. Expression of the desired gene
product is
analyzed, for instance, by SDS-PAGE and Western blot or by reverse phase HPLC
analysis.
[189] Example 2: Non-Limiting Example of a GLP-1 mimetibody of the
Invention. GLP-1 is a 37-amino acid peptide secreted from the L-cells of the
intestine
following an oral glucosc challenge. A mimetibody construct incorporating a
biologically active GLP-1 (7-37) peptide, variant or derivative is expected to
prolong the
in vivo lifetime of the peptide and provide a novel therapy for lowering blood
glucose in
Type 2 diabetic patients. Peptides encoding the native GLP-1 (7-37) peptide or
a DPP-
IV resistant analogue can be incorporated into the mimetibody scaffold.
Several of
these molecules have been made, and the resulting mimetibod.ies have
d.emonstrated.
activity in functional in vitro cell-based assays. It should be noted that
different in vitro
assays and in vivo models can be used in these studies and the potencies may
not be
comparable to each other or to results presented herein.
[190] To generate GLP-1 mimetibody variants, the GLP-1 peptide, the linker,
the
hinge, or the CH2 and CH3 sequences in the mirnetibody could be deleted,
added,
substituted, mutated or modified to improve expression, potency, stability, or
effector
functions.
[191] The wild-type GLP-1 sequence as well as DPP-IV resistant GLP-1 variants,
such as GLP-1 (A2S) or GLP-1 (A2G) can be incorporated into a mimetibody
scaffold.
Mutations of the peptide could be made to improve the properties of a GLP-1
mimetibody. For example mutations in the amino terminal residues may improve
signaling while mutations in the helical domain may stabilize the helix and
thereby
improve binding to the receptor and/or stability of the mimetibody.
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[192] The length and composition of the linker could be mutated to vary the
flexibility
or stability of the attachment between the GLP-1 peptide and the Fe region.
Different
isotypes could be incorporated into the hinge region of the molecute. In
addition,
mutations could be made within the hinge region of the mimetibody to stabilize
the
molecule. For example, the human IgG4 hinge could be mutated to make the
Serz2$-
>Pro variant, to stabilize the interchain disulfide bonds in the mimetibody.
Variations
within the Fc portion of the mimetibody could be made to improve the stability
of the
molecule and, to change effector functions such as FcR binding. For example,
one could
use human or murine isotypes (or variations of these molecules) such as IgG4
with
Ala/Ala mutations.
[193] GLP-1 mimetibody of the Present Invention. A specific, non-limiting,
example of this invention is the GLP-1 mimetibody construct (SEQ ID NO:2)
according
to Formula (I):
[194] ((P(n)-L(o)-V(p)-H(q)-CH2(r)-CH3(s))(t),
[195] where P is a single copy of the bioactive GLP-1 peptide (7-36), L is a
tandem
repeat of either Gly-Ser or Gly-Gly-Gly-Ser flexible linker, V is the C-
terminal of VH
sequence, i.e., the J region of a naturally occurring IgG, H is the complete
IgGl hinge
region and CH2 & CH3 are of the 1gG 1 isotype subclass. It is expected that
the half-life
of this construct will be many times that of the GLP-1 peptide alone or its
variant or
derivative and similar to that of an TgG.
[196] In addition to the basic structure described above, variants with
potentially
favorable biological characteristics are described. These include constructs
that may
have a decreased tendency to self-associate, reduced immune effector functions
or
decreased immunogenicity. Other modifications that confer desired
characteristics such
as improved conformation of the biologically active peptide, and transfer
across the
blood-brain barrier are envisioned. The proposed variants and modifications
may be
combined in any fashion to yield constructs with desired activities.
[197] Using recombinant DNA methods, the GLP-1 peptide was inserted into an
intermediate vector between an immunoglobulin signal peptide and a human J
sequence.
This was done using complementary synthetic oligonucletides with ends
compatible
with the restriction sites present in the vector. These oligonucleotides
comprised, coding
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sequences for the GLP-1 peptide, and a flexible linker composed of two GGGS
repeats.
A restriction fragment containing the above-mentioned functional elements was
then
transferred into an expression vector. This vector contained the anti-CD4
immunoglobulin promoter and enhancer, and the coding sequence for the human
IgGl
hinge sequence, HC constant region 2 (CH2) and constant region 3 (CH3) as well
as the
necessary elements for plasrnid replication and selection in bacteria and
selection for
stable expressers in mammalian cells.
[198] This plasmid was introduced into the HEK293E cells and expression of the
wt
GLP-1 MMB was achieved in transiently transfected cells. Purification of GLP-1
MMB
was accomplished. by standard protein A and Superose 12 affin.ity
chromatography,
yielding approximately 1.5 mg/L of transfected cells. This protein was the
starting
material for the experiments described below.
[199] The amino acid sequence of GLP-1 rnimetibody is shown in Table 2.
Functional domains are annotated above the peptide coding sequence. It is
thought that
the J sequence will provide even more flexibility to allow the GLP- 1 dimer to
assume
the proper conformation, and allow the dimer to protrude from the globular
structure of
the immunoglobulin and penetrate into the cleft between two GLP-1 receptors.
There
are three cysteines in the IgG1 hinge region. The first would normally pair to
the
immunoglobulin light chain (LC) and the other two participate in interchain
bonds
between two HCs. CH2 and CH3 regions constitute the bulk of the protein. One
of the
reasons that imrnunoglobulins are believed to have a long serum half-life is
their ability
to bind the FcRn that extends the serum half-life by returning pinocytosed
immunoglobulin back to the extracellular space. The binding site of the FcRn
overlaps
the junction of the CH2 and CH3 regions (Sheilds et al, 2001, J. Biol. Chem.,
vol. 276
(9), 6591-6604).
[200] It is well known that two IgG heavy chains are assembled during cellular
processing via disulfide bonds between cysteines located in the hinge region
to form a
homodimer. It is expected that this will also occur between the modified
peptides to
form the assembled GLP-1 mimetibody construct. In addition, it is expected
that the
intrachain disulfide bond between the two cysteines in the GLP-1 peptide will
also fonm.
The expected structure of GLP-1 mimetibody contains two GLP-1 peptides. The
spatial
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69
arrangement of the peptides at the N-terminus along with the flexibility of
adjoining
sequences should allow the peptides to form the bioacive dimer.
[201] Example 3: FACS Binding Assay. The activity of GLP-1 mimetibody was
tested in an in vitro FACS binding assay. To determine whether the GLP-1 MMB
binds
thc GLP-1R, HEK293 cc11s (1x106 cclls) over-expressing the GLP-1R were
incubated
with GLP-1 MMB (20 nM) for 2 hours at 4 C. The cells were washed, and a
fluorescently labeled secondary detection antibody (1 g/mL goat anti-human
1gG, Fc
gamma specific) was added for 30 minutes at 4 C. The fluorescence intensity of
the
cells was monitored via flow cytometry. GLP-1 MMB binds to HEK293 cells ovcr-
expressing the GLP-1R. GLP-1 MMB does not bind. to the control HEK293 cells.
GLP-1 peptide analogue (A2S) is able to compete with GLP-1 MMB for binding to
HEK293 cells over-expressing the GLP-1R.
[202] Example 4: cAMP Assay. GLP-1 binds to its receptor, a G-protein coupled
receptor, resulting in a dose-dependent increase in the signaling molecule,
3',5'-cyclic
AMP (cAMP). cAMP can be measured with an in vitro assay in cells expressing
the
GLP-1R (Applied Biosysterns). Briefly, Rinm cells (100,000 cells) were
incubated with
increasing concentrations of GLP-1 peptide (0-30 nM) or GLP-1 MMB (0-100 nM).
The cells were lysed, and the amount of cAMP was determined using a
competitive
assay that employs an alkaline-phosphatase labeled cAMP conjugate and a
chemiluminescent substrate (Tropix0 CDPDO). The concentration dependent cAMP
activity for the wt GLP-1 MMB is comparable to the GLP-1 peptide (EC50=11 nM
vs.
0.4 nM, respectively). In a similar experiment, GLP-1 (A2G) MMB in IgG4
scaffold
and GLP-1 (A2S) MMB in TgG4 scaffold both increased cAMP levels in Rinm cells
to
a significantly higher level than wt GLP-l MMB in IgG4 scaffold
[203] Example 5: DPP-1V cleavage assay. Since GLP-1 is rapidly inactivated by
DPP-IV, an in vitro assay was established to quantitate intact (i.e.
uncleaved) GLP-1
MMB. Briefly, GLP-1 MMB or peptide (1.2 nM) was incubated at room temperature
with DPP-IV (1 g/mL, R&D Systems). After various times (0, 5, 10, 15, 20, 30,
40
minutes), a DPP-IV inhibitor (100 M, Linco) was added to quench the reaction.
The
amount of intact GLP-1 MMB or peptide was mcasurcd using the GLP-1 Active
ELISA
(Linco) and. the GLP-1 MMB or peptid.es for the respective standard curves.
The GLP-1
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MMB was significantly more resistant to cleavage by DPP-IV, relative to the
GLP-1
peptide.
[204] Example 6: Human Serum stability assay. The stability of the GLP-1 MMB
in serum was also measured to ensure that other serum proteases were not able
to cleave
5 and inactivate the GLP-1 MMB. Bricfly, GLP1 pcptidc or the GLP1 MMB (30 nM)
was incu.bated. in human serum at 37 C. After variou.s times, the reactions
were
quenched with a DPP-IV inhibitor (100 M, Linco), and the samples were
analyzed
using the GLP-1 Active ELISA from Linco. The GLP-1 MMB is stable in human
serum for 24 hours while the peptide is decayed rapidly.
10 [205] Example 7: GLP-1 MMB causes insulin secretion in RINm cells. To test
the
effect of GLP-1 MMB in insulin secretion, RINm cells were treated with
increasing
concentrations of GLP-1 (7-36) peptide (0-5 nM), exendin-4 peptide (0-5 nM),
or
various GLP-1 rnimetibodies (5 or 50 nM) and the amount of insulin secreted
was
measured via ELISA. All GLP-1 MMBs tcstcd had activities in stimulating
insulin
15 secretion in RINm cells. At 50 nM, the MMBs had activities comparable to
that of the
wide-type GLP-1 (7-36) peptide.
[206] Example 8: GLP-1 MMB lowers glucose level in db/db mice. Six week old
db/db mice were fasted for two hours and then dosed intravenously with
vehicle, GLP-1
peptide, or GLP-1 (A2S) MMB. Blood glucose was monitored 0.5, 1, 2, 3, and 4
hours
20 post-dosing. The GLP-1 peptide lowered blood glucose at 30 minutes, but by
60
minutes, the blood glucose began to increase again likely due to the short
half-life of the
GLP-1 peptide. In comparison, GLP-1 (A2S) MMB at a dose 100-fold lower than
the
GLP-1 peptide dose induced a decrease in blood glucose throughout the entire 4
hour
period. In addition, the decrease in blood glucose was dose dependent.
25 [207] Example 9: Pharmacokinetics of GLP-1 MMBs in mice and in cynomolgus
monkeys. To measure the pharinacokinetics of four GLP-1 mimetibodies (A2G,
A2S,
exedin-cap, and wt), C57/B16 mice were intravenously dosed with lmg/kg of the
MMBs. Plasma was obtained via cardiac puncture after sacrificing mice at
different
time point. Various ELISAs were used to measure Fc, total MMB, active MMB, and
30 acive peptide as they were metabolized in the animal. Active MMB reflects
the intact
N-terminus of the peptide still attached to the Fe region of the mimetibody.
Substitution
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71
of the second amino acid in the peptide (alanine) with either a serine or a
glycine
prolonged the lifetime of the active MMB in circulation.
[208] Cynomolgus monkey were injected intravenously with 1.0 mg/kg of four
different
GLP-1 MMB constructs and serum samples were taken at different time points
from 10
minutes to 5 days following dosing. Scruxn samplcs wcrc evaluated by ELISA to
quantify
intact MMB. All four MMBs exhibit a rapid distribution phase, followed by a
slower
clearance phase. Pharmacokinetic constants were calculated for each of the
constructs to
indicate a T1/z of approximately 3 days with similar exposure determined by
AUC from T
= 0 to T = 120 hours.
[209] Example 10: Intracerebroventricular (icv) Dosing of CNTO 736 to Normal
Rats Demonstrating Evidence Supporting Treatment for Obesity.
[210] Sprague-Dawley rats weighing 250-350g were cannulized into the lateral
ventricle at the following coordinates: -0.80mm anterior, +1.2mm lateral to
bregma, -
3.2mm ventral to bregma. Animals were submitted for Angiotensin ll testing to
confirm
that the cannulas were placed appropriately. Rats were acclimated to a
reversed
light/dark cycle and to handling. A 24-hour fasted/refed measurement was taken
after a
single PBS injection in order to establish a feeding baseline. Animals were
evenly
distributcd according to 24-hour food intakc from PBS injcctions (3 groups of
10
animals). Seven-ten days following PBS injection, rats were fasted for 24
hours and
were dosed icv prior to entering the dark cycle. The rats were dosed with
either CNTO
736 (3 nmols), excndin-4 (3 nmols), or PBS in a volume of 5 L. Food intake
and water
intake were measured from 0-12 and 12-24 hours after dosing and body weight
was
measured after 24 and 48 hours. In the first 12 hours following dosing, there
was a
significant reduction in food and water intake with exendin-4 and CNTO 736
treatments. In the second 12 hours following dosing (12-24 hours), thcrc was a
significant reduction in food intake with both exendin-4 and CNTO 736
treatments, but
a reduction in water intake was observed only with exendin-4 treatment. There
was a
significant reduction in body weight with exendin-4 treatment at both 24 and
48 hours,
but not with CNTO 736.
[211] Example 11 Intravenous (iv) Dosing of CNTO 736 to Normal Rats
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[212] Sprague-Dawley rats weighing 250-350g were acclimated to a reversed
light/dark cycle and to handling. A 24-hour fasted/refed measurement was taken
after a
single PBS injection in order to establish a feeding baseline. Animals were
evenly
distributed according to 24-hour food intake from PBS injections (3 groups of
10
animals). Seven-ten days following PBS injection, rats were fasted for 24
hours and
were dosed iv prior to entering the dark cycle. The rats were dosed with
either CNTO
736 (12 nmols), exendin-4 (12 nmols), or PBS. Food intake and water intake
were
measured from 0-12 and 12-24 hours after dosing and body weight was measured
after
24 hours. In the first 12 hours following dosing, there was a significant
reduction in
food and water intake with exendin-4 and CNTO 736 treatments. In the second 12
hours following dosing (12-24 hours), there was a significant reduction in
food intake
only in the CNTO 736 treated animals, and a significant increase in water
intake was
observed in both exendin-4 and CNTO 736 treated animals. There was a
significant
reduction in body weight with exendin-4 and CNTO 736 treatments at 24 hours
after
dosing.
[213] ExamAle 12 Subcutaneous (sc) Dosing of CNTO 736 to Normal Rats
[214] Sprague-Dawley rats weighing 250-350g were acclimated to a reversed
light/dark cycle and to handling. A 24-hour fasted/refed measurement was taken
after a
single PBS inj ection in order to establish a feeding baseline. Animals were
evenly
distributed according to 24-hour food intake from PBS injections (3 groups of
10
animals). Seven-ten days following PBS injection, rats were fasted for 24
hours and
were dosed subcutaneoulsy prior to entering the dark cycle. The rats were
dosed with
either CNTO 736 (12 mnols), exendin-4 (12 nm.ols), or PBS. Food intake and
water
intake \were measured from 0-6, 6-12 and 12-24 hours after dosing and body
weight
was measured after 24 hours and 48 hours. In the first 6 hours following
dosing, there
was a significant reduction in food and water intake with exendin-4 treatment
only. In
the second 6 hours following dosing (6-12 hours), there was a significant
reduction in
food intake in both the cxendin-4 and CNTO 736 treated animals, and a
significant
decrease in water intake in the exendin-4 treated animals (not significant for
the CNTO
736 treated animals). This data is consistent with previous data showing that
the Cmax
for CNTO 736 after sc dosing is approximately six hours. There was a
significant
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73
reduction in body weight with exendin-4 and CNTO 736 treatments at 24 hours
after
dosing but not at 48 hours.
[215] Example 13: Chronic Dosing of CNTO 736 in DIO mice.
[216] Prior to the start of the study (seven days), an IPGTT was done in DIO
mice.
Briefly, mice were fasted overnight (21 h), and a basal fasting blood glucose
measurement was taken at t=-5 min (relative to glucose challenge). At t = 0
min, mice
wcrc dosed i.p. with D-glucosc (1.0 mg/g). Blood glucose lcvcls were mcasurcd
at 15,
30, 60, 90, 120, 150, and 180 minutes. The data for the IPGTT was plotted as
blood
glucose versus time, and the AUC was used to randomize the mice into groups of
4
(n=7). Blood for insulin and HbAlc measurements was taken three days before
the start
of the study.
[217] Beginning on day 1 of the study, mice were dosed with CNTO 736 or
vehicle
three times per week (Monday, Wednesday, and Friday) or daily for a total of 6
weeks
(Table 3). Body weight was measured daily, and fasting blood glucose was
measured
twice per week. An IPGTT was repeated after two weeks of treatment and again
after
five weeks of treatment. Blood for insulin and HbAlc was taken after six weeks
of
dosing. Also after six weeks of dosing, animals in groups 1 and 2 were
sacrificed and
immediately analyzed for body composition via P1X.Lmus.
Table 3
Group n Test article Dose Dosing
(mg/kg) Schedule
1 7 Vehicle N/A 1X per day
2 7 CNTO 736 1 1X per day
3 7 CNTO 736 0.1 1X per day
4 7 CNTO736 1 M,W,F
[218] The fasting blood glucose in the animals treated with CNTO 736 daily or
three
times per week was reduced throughout the six-weeks in a dose dependent
manner.
Body weight in the treated animals was also reduced relative to the vehicle
treated
2 5 animals. The PBS treated animals gained 6.5% of their starting body weight
during the
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course of the study while the CNTO 736 treated animals dosed daily lost 13.2%
or 1.2%
of their body weight after six weeks (1.0 and 0.1 mg/kg, respectively). The
animals
dosed with CNTO 736 (1.0 mg/kg) three times per week lost 5.8% of their body
weight.
PIXImus analysis revealed that the reduction in body weight was due to a loss
of fat
mass rather than lean mass.
[219] Summary: The data d.escribed here suggest that display of the GLP-1
peptide
on the mimetibody scaffold results in a novel molecule with properties that
enable its
utility as a therapeutic for obesity. It has been shown that GLP-1 peptide
analogues
cause a reduction in food intake and body weight. However, the mechanisms
underlying
this effect are not well understood.. It is possible that the effect is
mediated via
activation of GLP-1 receptors in the brain or alternatively, peripherally
expressed GLP-
1 could activate the afferent vagal pathways that are involved in body weight
regulation.
The data presented here suggest that CNTO 736 was able to reduce food intake
and
body weight. The effect of CNTO 736 on food and water intake as well as body
weight
was observed after either peripheral or central administration, suggesting
that central
and peripheral mechanisms could play a role in the GLP-1 mediated effect on
energy
balance. CNTO 736 is a large molecule (6010) that is unlilcely to cross the
blood brain
barrier. Thus, it is likely that peripherally administered CNTO 736 exerts its
effect on
feeding via activation of the vagal system. Alternatively, CNTO 736 could
activate
GLP-1 receptors located in brain areas outside of the blood brain barrier to
affect
feeding and water intake. Both hypotheses are being tested.
[220] Advantaaes: The use of this novel molecule as a therapeutic to treat
obesity
provides several advantages over other GLP-1 analogues.
[221] CNTO 736 has a sustained pharmacokinetic profile (T1/2 - 5 days in
cynomolgus monkeys) relative to the other GLP-1 peptide analogues. This may
allow
for the drug to remain in circulation for a longcr period of timc, thcreby
reducing food
intake in a more sustained manner relative to other analogues.
[222] CNTO 736 is a significantly larger molecule than other GLP-1 peptide
analogues, and it may not be able to cross the blood brain barrier. If the
side effects
observed with other GLP-1 analogues (nausea and vomiting) are mediated
centrally, it is
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possible that patients treated with CNTO 736 would not experience these
adverse
events.
[223] The MimetibodyTM platform results in expression of two peptides on each
MimetibodyTM molecule. This may allow the GLP-1 peptides to interact with each
5 other, forming a dimeric ligand with increased affinity to the cell surface
GLP-1
receptor.
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9. Daniel J. Drucker, J.P., Svetlana Mojsov, William L. Chick, and Joel F.
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increases cyclic AMP levels in a rat islet cell line. Proc. Natl. Acad. Sci.
USA,
35 1987. 84: p. 3431-3438.
10. S. Suzuki, K.K., S. Ohashi, H. Mukai and K. Yamashita, Comparison of the
effects of various C-terminal and N-ternainal fragrnent peptides ofglucagon-
like peptide-1 on insulin and glucagon release froin the isolated perfused rat
pancreas. Endocrinology, 1989. 125: p. 3109-3114.
40 11. A Wettergren, B.S., PE Mortensen, J Myhre, J Christiansen, JJ Holst,
Truncated
GLP-1 (proglucagon 78-107-amide) inhibits gastric and pancreatic functions in
rnan. Dig Dis Sci, 1993. 38(4): p. 665-673.
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12. M Tang-Christensen, P.L., R Goke, A Fink-Jensen, DS Jessop, M Moller, SP
Sheikh, Central administration of GLP-1 (7-36) amide inhibits food and watef=
intake in rats. American Journal of Physiology, 1996. 271: p. R848-R856.
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M.A. Ghatei, J. Hcrbcrt and S.R. Bloom, A role for glucagon-like peptide-1 in
the central regulation qffeeding. Nature, 1996. 379: p. 69-72.
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and satiety. Nature, 1997. 385: p. 214.
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[224] Additional Advantages: The use of this novel molecule as a therapeutic
to
treat obesity related disorders several advantages over othcr GLP-1 agonists.
For
example, it is likely to prolong the half-life of the GLP-1 peptide. Also, the
wild-type
GLP-1 peptide in the mimetibody scaffold is resistant to protease degradation,
specifically DPP-IV. This may allow for treatment with the wild-type GLP-1
peptide
rather than a mutant peptide. Since GLP-1 is a native peptide, there may be
less
immune response in patients treated with a GLP-1 mimetibody than in patients
treated
with a mutated GLP-1 analogue. In addition, the large size of the GLP-1 MMB
may
preclude it from crossing the blood brain barrier. This may offer an advantage
since
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nausea and anxiety have been associated with GLP-1 engaging the GLP-1R in the
brain.
Furthermore, the mimetibody platform results in expression of two peptides on
each
mimetibody molecule. This may allow the GLP-1 peptides to interact with each
other,
forming a dimeric ligand that could increase affinity to the cell surface GLP-
1 receptor.
[225] It will bc clear that the invention can be practiced otherwisc than as
particularly
described in the foregoing description and examples.
[226] Numerous modifications and variations of the prescnt invention arc
possible in
light of the above teachings and, therefore, are within the scope of the
present invention.
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Table 1
SEQ TOTAL REGIONS
ID NO AA NO FRI FR4
47 Vh1 125 1-31 81-125
48 Vh2 97 1-30 80-97
49 Vh3a 102 1-30 80-102
50 Heavy Vh3b 102 1-30 80-102
chain
51 Vh3c 94 1-30 80-94
variable
52 region Vh4 106 1-30 80-106
53 Vh5 97 1-30 80-97
54 Vh6 91 1-30 80-91
55 Vh7 91 1-30 80-91
SEQ AA NO REGIONS
ID NO hingel hinge2 hinge3 hinge4 CH2 CH3
56 IgAl 354 103-122 123-222 223-354
57 IgA2 340 103-108 109-209 210-340
58 IgD 384 102-135 319-497 160-267 268-384
59 Heavy IgE 497 104-210 211-318
chain
60 IgG1 339 99-121 122-223 224-339
constant
61 region IgG2 326 99-117 118-219 220-326
62 IgG3 377 99-115 131-145 146-168 169-270 271-377
63 IgG4 327 99-110 324-476 111-220 221-327
64 IgM 476 105-217 218-323
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Table 2: MM13 CNTO 736 GLP-1 amino acid sequence
SIGNAL SEQUENCE
...............................................................................
...................................................
Met Ala Trp Val Trp Thr Leu Leu Phe Leu Met Ala Ala Ala Gln
ATG GCT TGG GTG TGG ACC TTG CTA TTC CTG ATG GCG GCC GCC CAA
.......................................... . GLP-
1..............................................................................
................................. .
Ser Ile Gln Ala His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser
AGT ATA CAG GCC CAT GCT GAA GGG ACC TTT ACT AGT GAT GTA AGT
...............................................................................
...............................................................................
...................
Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe I1e Ala Trp Leu
TCT TAT TTG GAA.GGC CAA GCT GCC AAG GAA TTC ATT GCT TGG CTG
LINKER
...........................................................................
VH........................ .
Val Lys Gly Arg Gly Gly Gly Ser Gly Gly Gly Ser Gly Thr Leu
GTG AAA GGC CGA GGA GGT GGA TCC GGT GGA GGC TCC GGT ACC TTA
........................................................
HINGE..........................................................................
..........................
Val Thr Val Ser Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr
GTC ACC GTC TCC TCA GAG CCC AAA TCT TGT GAC AAA ACT CAC ACG
......................................................... C H
2..............................................................................
........................... .
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
TGC CCA CCG TGC CCA GCA CCT GAA CTC CTG GGG GGA CCG TCA GTC
...............................................................................
...............................................................................
................ .
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
TTC CTC TTC CCC CCA AAA CCC AAG GAC ACC CTC ATG ATC TCC CGG
...............................................................................
...............................................................................
................ .
Thr Pro glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
ACC CCT GAG GTC ACA TGC GTG GTG GTG GAC GTG AGC CAC GAA GAC
...............................................................................
...............................................................................
................ .
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
CCT GAG GTC AAG TTC AAC TGG TAC GTG GAC GGC GTG GAG GTG CAT
...............................................................................
...............................................................................
................ .
Asn Ala Lys Thr Lys Pro Arg GLu Glu Gln Tyr Asn Ser Thr tyr
AAT GCC AAG ACA AAG CCG CGG GAG GAG CAG TAC AAC AGC ACG TAC
...............................................................................
...............................................................................
................ .
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
CGG GTG GTC AGC GTC CTC ACC GTC CTG CAC CAG GAC TGG CTG AAT
...............................................................................
...............................................................................
................ .
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
GGC AAG GAG TAC AAG TGC AAG GTC TCC AAC AAA GCC CTC CCA GCC
.... C H 3 ............................................. .
Pro Ile Glu Lys Thr I1e Ser Lys Ala Lys Gly Gln Pro Arg Glu
CCC ATC GAG AAA ACC ATC TCC AAA GCC A.AA GGG CAG CCC CGA GAA.
...............................................................................
...............................................................................
................ .
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
CCA CAG GTG TAC ACC CTG CCC CCA TCC CGG GAT GAG CTG ACC AAG
...............................................................................
...............................................................................
................ .
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Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
AAC CAG GTC AGC CTG ACC TGC CTG GTC AAA GGC TTC TAT CCC AGC
...............................................................................
...............................................................................
................ .
5 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
GAC ATC GCC GTG GAG TGG GAG AGC AAT GGG CAG CCG GAG AAC AAC
...............................................................................
...............................................................................
................ .
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
10 TAC AAG ACC ACG CCT CCC GTG CTG GAC TCC GAC GGC TCC TTC TTC
...............................................................................
...............................................................................
................ .
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
CTC TAC AGC AAG CTC ACC GTG GAC AAG AGC AGG TGG CAG CAG GGG
...............................................................................
...............................................................................
............... .
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
AAC GTC TTC TCA TGC TCC GTG ATG CAT GAG GCT CTG CAC AAC CAC
...............................................................................
............................................................ STOP
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys (SEQ ID NO:69)
TAC ACG CAG AAG AGC CTC TCC CTG TCT CCG GGT AAA TGA (SEQ ID NO:70)
