U.S. patent application number 15/139657 was filed with the patent office on 2017-11-30 for antibody therapy for modulating function of intestinal receptors and methods of treating diabetes and obesity.
The applicant listed for this patent is Circle 33 LLC. Invention is credited to Barbara S. Fox.
Application Number | 20170342149 15/139657 |
Document ID | / |
Family ID | 40341967 |
Filed Date | 2017-11-30 |
United States Patent
Application |
20170342149 |
Kind Code |
A1 |
Fox; Barbara S. |
November 30, 2017 |
ANTIBODY THERAPY FOR MODULATING FUNCTION OF INTESTINAL RECEPTORS
AND METHODS OF TREATING DIABETES AND OBESITY
Abstract
The present invention provides pharmaceutical compositions
formulated for direct delivery to the GI tract of a patient
comprising an antibody specific for a target apical intestinal
receptor. The present invention further provides methods of
treating diseases and conditions in a patient comprising
administering directly to the GI tract of the patient, compositions
of the present invention wherein modulation of the target apical
intestinal receptor by the antibody treats the condition.
Inventors: |
Fox; Barbara S.; (Wayland,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Circle 33 LLC |
New York |
NY |
US |
|
|
Family ID: |
40341967 |
Appl. No.: |
15/139657 |
Filed: |
April 27, 2016 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
14658569 |
Mar 16, 2015 |
|
|
|
15139657 |
|
|
|
|
14061301 |
Oct 23, 2013 |
|
|
|
14658569 |
|
|
|
|
13590755 |
Aug 21, 2012 |
8592560 |
|
|
14061301 |
|
|
|
|
12687660 |
Jan 14, 2010 |
8268971 |
|
|
13590755 |
|
|
|
|
PCT/US08/70235 |
Jul 16, 2008 |
|
|
|
12687660 |
|
|
|
|
60950029 |
Jul 16, 2007 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 29/00 20180101;
C07K 2317/76 20130101; A61P 9/12 20180101; A61P 5/48 20180101; A61P
25/00 20180101; A61P 9/10 20180101; A61P 3/06 20180101; A61P 13/12
20180101; A61P 1/04 20180101; A61P 27/02 20180101; A61P 19/06
20180101; C07K 16/28 20130101; A61P 3/04 20180101; A61P 9/04
20180101; A61P 7/10 20180101; C07K 2317/12 20130101; A61K 2039/505
20130101; C07K 2317/34 20130101; A61P 35/00 20180101; A61P 3/00
20180101; C07K 16/04 20130101; C07K 16/2896 20130101; A61P 3/10
20180101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; C07K 16/04 20060101 C07K016/04 |
Claims
1. (canceled)
2. A composition comprising an antibody specific for a target
apical intestinal receptor present in the gastrointestinal (GI)
tract of a patient formulated for oral or rectal delivery to the GI
tract of the patient, wherein the antibody modulates at least one
biological function of the target apical intestinal receptor.
3. The composition of claim 2, wherein the target apical intestinal
receptor is selected from the group consisting of a nutrient
receptor, a nutrient transporter, a pattern recognition receptor, a
chemokine receptor, a cytokine receptor, a bile salt transporter,
an inorganic ion transporter, a mineral transporter, peptidases,
saccharases, and growth factor receptors.
4. The composition of claim 2 wherein the target apical intestinal
receptor modulates a condition selected from a metabolic condition,
inflammation, cancer, drug toxicity, conditions modulated by
receptors for neurotransmitters, and conditions modulated by
receptors for inorganic ions.
5. A method of modulating at least one biological function of the
target apical intestinal receptor in a patient by administering the
composition of claim 2.
6. The method of claim 5, wherein the biological function of the
target intestinal receptor is associated with a condition selected
from: a. a metabolic condition, inflammation, cancer, drug
toxicity, conditions modulated by apical receptors for
neurotransmitters and conditions modulated by apical intestinal
receptors for inorganic ions; or b. cancerous tumor of the GI
tract, and the antibody is specific for a target on the surface of
the tumor; or elevated levels of low density lipoprotein (LDL), and
the antibody is specific for a bile acid receptor; or c.
inflammation, and the antibody is specific for atoll-like receptor
(TLR), a proteinase-activated receptor (PAR), an inflammatory
cytokine receptor or a chemokine receptor; or d. abnormal calcium
homeostasis, and the antibody is specific for a calcium receptor;
or e. hyperlipidemia, diabetes and adiposity, and the antibody is
specific for a cell surface peptidase or saccharase.
Description
RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
application Ser. No. 14/658,569, filed Mar. 16, 2015, which is a
continuation application of U.S. application Ser. No. 14/061,301,
filed on Oct. 23, 2013, now abandoned, which is a divisional of
U.S. application Ser. No. 13/590,755, filed on Aug. 21, 2012, now
U.S. Pat. No. 8,592,560, issued on Nov. 26, 2013, which is a
continuation of U.S. application Ser. No. 12/687,660, filed on Jan.
14, 2010, now U.S. Pat. No. 8,268,971, issued on Sep. 18, 2012,
which is a Continuation of International Application No.
PCT/US08/70235, which designated the United States and was filed on
Jul. 16, 2008, published in English, which claims the benefit of
U.S. Provisional Application No. 60/950,029, filed on Jul. 16,
2007. The entire teachings of the above applications are
incorporated herein by reference.
FIELD OF THE INVENTION
[0002] This invention relates to the modulation of the function of
membrane-bound proteins expressed on the luminal surface of the
gastrointestinal tract by administration of antibodies specific for
such membrane-bound proteins in the form of compositions formulated
for delivery directly to the gastrointestinal (GI) tract.
BACKGROUND OF THE INVENTION
[0003] The gut responds to a large variety of stimuli, including
nutrients, chemicals, mechanical factors, hormones and
micro-organisms {Dockray, 2003, J Physiol Pharmacol, 54 Suppl 4,
9-17}. Many of these stimuli are detected through specific
receptors that are expressed on the luminal surface of the
gastrointestinal tract. In particular, multiple receptors are
expressed in the small intestine that recognize sugars.
[0004] Sugar transport across the intestinal membranes is tightly
regulated and is mediated by a specific set of receptors (reviewed
in {Drozdowski and Thomson, 2006, World J Gastroenterol, 12,
1657-70}). Dietary glucose crosses the apical membrane of the
enterocyte in the small intestine by the Na+/glucose cotransporter
(SGLT1). Dietary fructose is transported across the apical membrane
by the facilitative transporter GLUTS. The transporter GLUT2 is
important in transporting glucose, particularly at high
concentrations {Drozdowski and Thomson, 2006, World J
Gastroenterol, 12, 1657-70}. The transporter GLUT7 is also
expressed in the small intestine {Li et al., 2004, Am J Physiol
Gastrointest Liver Physiol, 287, G236-42}.
[0005] GLUT2 expression on the apical surface of enterocytes is
regulated by both SGLT1 {Kellett and Brot-Laroche, 2005, Diabetes,
54, 3056-62} and by sweet taste receptors {Mace et al., 2007, J
Physiol}. At high glucose concentrations, GLUT2 is inserted into
the apical membrane, thereby providing a cooperative mechanism by
which glucose absorptive capacity is rapidly and precisely matched
to dietary intake immediately after a meal {Mace et al., 2007, J
Physiol}. GLUT2 has been identified as a potential therapeutic
target for small molecule inhibitors, and quercitin and similar
flavonoids have been shown to be GLUT2 inhibitors {Kwon et al.,
2007, FASEB J, 21, 366-77}. GLUT2 inhibition could be therapeutic
for diabetes and / or obesity.
[0006] The intestine expresses taste receptors on the epithelial
cells of the stomach and duodenum known as brush cells {Hofer et
al., 1996, Proc Natl Acad Sci U S A, 93, 6631-4} {Bezencon et al.,
2007, Chem Senses, 32, 41-9}. Taste receptors are also expressed on
the enteroendocrine cells of the intestinal tract {Masuho et al.,
2005, Chem Senses, 30, 281-90}. The sweet taste receptors (T1Rs),
including T1R1, T1R2 and T1R3, belong to the guanine nucleotide
regulatory protein (G protein)-coupled receptor (GPCR) superfamily.
The receptors have a long extracellular NH2-terminal segment, seven
transmembrane a-helices, three extracellular loops, three
cytoplasmic loops and a COOH-terminal segment. The T1Rs function as
molecular complexes, with the heterodimeric T1R2/T1R3 receptor
binding to sweet stimuli while the T1R1/T1R3 complex recognizes
amino acids {Rozengurt, 2006, Am J Physiol Gastrointest Liver
Physiol, 291, G171-7}.
[0007] Polyclonal antibodies have been described that are specific
for the receptors present in the GI tract as research agents useful
in the detection of the receptor of interest by immunostaining. For
example, antibodies have been described to alpha-gustducin, the
GTP-binding subunit of taste receptors {Hofer et al., 1996, Proc
Natl Acad Sci USA, 93, 6631-4}. Several antibodies specific for
SGLT1, GLUTS, GLUT2, TAS1R1, TAS1R2, TAS1R3 and T2R1 are
commercially available as research reagents for the detection of
the receptor of interest. However, oral delivery of protein
therapeutics to modulate cellular receptors located in the lumen of
the GI tract to treat various conditions is an unexplored area.
Compositions and methods for administration of therapeutic
antibodies directly to the GI tract to target apical intestinal
receptors to treat conditions modulated by such target receptors,
are therefore needed.
SUMMARY OF THE INVENTION
[0008] The present invention provides pharmaceutical compositions
formulated for direct delivery to the GI tract of a patient
comprising an antibody specific for a target apical intestinal
receptor. The present invention further provides methods of
treating diseases and conditions in a patient comprising
administering directly to the GI tract of the patient compositions
of the present invention wherein modulation of the target apical
intestinal receptor by the antibody treats the condition.
DETAILED DESCRIPTION OF THE INVENTION
[0009] The intestinal tract expresses many different receptors and
proteins whose function is to sense and respond to nutrients,
microorganisms and other matter contained within the
gastrointestinal tract. These receptors and proteins are
collectively referred to herein as "apical intestinal receptors".
"Apical intestinal receptors" are endogenous transmembrane
proteins, expressed in the cell membrane of cells facing the
luminal side of the intestinal tract. Classes of apical intestinal
receptors described in this invention include but are not limited
to: nutrient receptors and transporters (including sugar receptors
and transporters, taste receptors, amino acid transporters, and
free fatty acid receptors); pattern recognition receptors
(including the Toll-like receptors); chemokine and cytokine
receptors; bile salt transporters; transporters for calcium iron,
and other ions and minerals; peptidases; disaccharidases; growth
factor receptors (including epidermal growth factor receptor) and
proteins expressed on the surface of cancerous cells in the GI
tract. Apical intestinal receptors may be expressed in the stomach,
the small intestine or the colon. Preferably, this invention
utilizes antibodies directed against apical intestinal receptors
expressed in the small intestine or the colon, most preferably
those apical intestinal receptors expressed in the small intestine.
Apical intestinal receptors may also be expressed in tumors of the
gastrointestinal tract.
[0010] "Molecular sensing by GI cells plays a critical role in the
control of multiple fundamental functions in digestion, including
secretory activity of GI glands, absorptive activity, motility, and
blood supply of the intestinal tract. Furthermore, molecular
sensing of luminal contents also initiates hormonal and/or neural
pathways leading to the regulation of caloric intake, pancreatic
insulin secretion, and metabolism. Molecular sensing in the GI
tract is also responsible for the detection of ingested harmful
drugs and toxins, thereby initiating responses critical for
survival" (Rozengurt, 2006, Am J Physiol Gastrointest Liver
Physiol, 291, G171-7). Many of these responses are mediated by
apical intestinal receptors. Antibodies specific for such apical
intestinal receptors can be used to inhibit or modulate the
function of these receptors including partially blocking at least
one biological function of the target receptors.
[0011] Accordingly, the present invention provides pharmaceutical
compositions formulated for direct delivery to the GI tract of a
patient comprising an antibody specific for a target apical
intestinal receptor. In one embodiment the target receptor
modulates a condition that is treatable by administering an
antibody capable of modulating at least one biological function of
the target receptor.
[0012] As used herein the term "direct delivery to the GI tract of
a patient" is oral or rectal delivery to the patient. As used
herein the terms "target apical intestinal receptor" and "target
receptor" are used interchangeably to refer to an apical intestinal
receptor to which an antibody of the invention will selectively
bind. The terms "specific for", "selective for" or "selectively
binds" when describing the ability of an antibody of the invention
to bind to a target apical intestinal receptor means that the
antibody can be demonstrated to bind to the target receptor
generating a signal greater than that seen from control antibody
using any assay known to those experienced in the field, including
but not limited to ELISA, RIA, flow cytometry, inhibition or
augmentation of biological function, or equilibrium dialysis. The
term "receptor" also includes receptors that transmit a signal upon
binding the appropriate ligand and receptors that function as
transporters.
[0013] The GI tract contains receptors specific for sugars. In one
embodiment of the invention, antibodies specific to certain target
apical intestinal receptors can be used to modulate the uptake of
glucose, to treat obesity or diabetes. Such antibodies are
selective for apical intestinal receptors that recognize sugars.
Such receptors include but are not limited to SGLT1, GLUTS, GLUT2,
GLUT7, TAS1R1, TAS1R2, TAS1R3, and T2R1. In one embodiment of the
invention, antibodies specific for a sugar receptor can be used to
modulate the uptake of sugars such as glucose and fructose, to
treat metabolic diseases including but not limited to disease
associated with hyperglycemia, diabetes (especially postprandial
hyperglycemia), impaired glucose tolerance, impaired fasting
glycemia, diabetic complications (e.g., retinopathy, neuropath,
nephropathy, ulcer, macroangiopathy), obesity, hyperinsulinemia,
hyperlipidemia, hyper-cholesterolemia, hypertriglyceridemia, lipid
metabolism disorder, atherosclerosis, hypertension, congestive
heart failure, edema, hyperuricemia, gout or the like. In one
embodiment, the antibodies specific for a sugar receptor are
delivered orally to the patient. Specific sugar receptors are also
present in the lower intestine and in some embodiments it may be
preferable to deliver antibodies rectally such as by suppository or
similar formulation for direct delivery to the colon. Although not
intended to imply a mechanism of action or to limit this invention
to antibodies that function with this mechanism, in one embodiment,
antibodies specific for sugar receptors in the lumen will block or
partially block the binding of sugar to the target receptors and
thereby reduce the amount of sugar absorbed from the lumen. Upon
ingestion of a meal containing a sugar such as glucose, the amount
of glucose absorbed from the lumen of the intestine will be
reduced, thus minimizing the caloric intake of the individual and
minimizing the post-prandial increase in glucose which is
detrimental to, for example, diabetic patients. In another
embodiment, antibodies specific for sugar receptors may be capable
of preventing the development of diabetes or obesity in a patient
at high risk of developing diabetes or obesity.
[0014] The GI tract expresses receptors that recognize fatty acids
and amino acids as well as sugars. Fats are the most effective food
group in stimulating endocrine cells of the distal duodenum and
jejunum, increasing the secretion of cholesystokinin (CCK),
glucose-dependent insulinotropic polypeptide (GIP) and secretin.
Glucose stimulates the release of GIP and CCK, but not secretin, in
the upper small intestine. The amino acids histidine and arginine
stimulate the secretion of GIP, and tryptophan and phenylalanine
stimulate CCK. These hormones drive the correct processing of
ingested food, by inducing the pancreas to secrete proteolytic
enzymes, inducing the secretion of bile to promote the formation of
chylomicrons for absorption of triglyceride and long-chain fatty
acids, and driving secretion of insulin from the pancreas to
facilitate uptake of the absorbed glucose, amino acids, and fat. In
one embodiment of this invention, antibodies specific for receptors
for fatty acids and amino acids can be used to modulate the release
of enteroendocrine hormones in response to ingested food. The fatty
acid transporters SMCT1, SMCT2, and MCT1, as well as the G protein
coupled receptor (GPCR) GPR40 are involved in fatty acid
recognition. Therefore, without being limited to any theory, an
antibody specific for SMCT1, SMCT2, MCT1, or the GPCR GPR40
receptor may be capable of inhibiting or partially inhibiting the
response to ingested fat. In one embodiment of this invention,
antibodies specific for the amino acid carriers EAAC1, EAAT3, PAT1,
LYAAT-1, tramdorin 3, B'', B.degree. , or the di- and tripeptide
transporter, PepT1, would be useful in the treatment or prevention
of metabolic syndrome, obesity or diabetes or inflammatory
diseases, including Crohn's disease, ulcerative colitis,
necrotizing enterocolitis, celiac disease, inflammation due to
infection with invasive organisms such as Salmonella and
Escherichia coli, or inflammation secondary to injury caused by
surgery, trauma, ionizing radiation, or toxic chemicals, including
NSAIDs. Other transporters in the GI tract which may serve as
targets for the antibodies of the invention include but are not
limited to: the multivitamin transporter SMVT which transport,
biotin, lipoate and panthothenate among others; the serotonin
transporter SERT; the taurine transporter TAUT; the IMINO
transporter system; the dicarboxylate transporter NaDC-1; and the
nucleoside transporter CNT1.
[0015] The GI tract contains receptors that are involved in
regulating the response to inflammation. Cells in the GI tract
express toll-like receptors (TLRs). TLR1, TLR2, TLR3, TLR4, TLRS,
TLR7 and TLR9 are expressed in the small intestine and TLR1, TLR2,
TLR3, TLR4, TLRS, TLR6, TLR7, TLR8 and TLR9 are expressed in the
epithelium of the large intestine. Stimulation of TLR receptors in
the intestine induces the production of chemokines, defensins and
CCK {Palazzo et al., 2007, J Immunol, 178, 4296-303}. Antibodies to
TLRs may be used to treat inflammatory diseases and disorders of
the GI tract, including Crohn's disease, ulcerative colitis,
necrotizing enterocolitis, celiac disease, inflammation due to
infection with invasive organisms such as Salmonella and
Escherichia coli, or inflammation secondary to injury caused by
surgery, trauma, ionizing radiation, or toxic chemicals, including
NSAIDs. In one specific embodiment of the invention, antibodies
that inhibit signaling through TLR4 are used to treat necrotizing
enterocolitis. In one specific embodiment of the invention,
antibodies that enhance signaling through TLR2, TLRS or TLR9 are
use to treat intestinal inflammation. It is understood by those
skilled in the art that the precise method of treatment of
intestinal inflammation by antibodies specific for TLRs will depend
on the nature of the inflammation and the characteristics of the
patient requiring treatment.
[0016] The GI tract contains receptors for calcium and other
inorganic ions. These receptors and transporters include the
inorganic phosphate transporter NaPi-IIb, and the transporter DMT1
(also known as NRAMP2, DCT1) for Fe2+, Mn2+, Ni2+, Co2+. The
calcium sensing receptor (CaSR) is expressed in epithelial cells
throughout the small and large intestine. Antibodies specific for
CaSR and for other receptors for inorganic ions can be used to
treat diseases or disorders of one or more of the following types:
those characterized by abnormal inorganic ion homeostasis; those
characterized by an abnormal amount of an extracellular or
intracellular messenger whose production can be affected by
inorganic ion receptor activity; those characterized by an abnormal
effect of stimulation through the inorganic ion receptor (e.g., a
different effect in kind or magnitude) levels, for example, as
assessed by bone mineral density measurements; those characterized
by an abnormal absorption of dietary calcium or other inorganic
ions. The abnormal increase or decrease in these different aspects
of inorganic ion homeostasis is relative to that occurring in the
general population and is generally associated with a disease or
disorder. Diseases and disorders characterized by abnormal
inorganic ion homeostasis can be due to different cellular defects
such as a defective inorganic ion receptor activity or a defective
intracellular protein acted on by a receptor for an inorganic ion.
Patients in need of treatment involving modulation of inorganic ion
receptors can be identified using standard techniques known to
those in the medical profession. Preferably, a patient is a human
having a disease or disorder characterized by one or more of the
following: (1) abnormal inorganic ion homeostasis, more preferably
abnormal calcium homeostasis; (2) an abnormal level of a messenger
whose production or secretion is affected by inorganic ion receptor
activity, more preferably affected by calcium receptor activity;
and (3) an abnormal level or activity of a messenger whose function
is affected by inorganic ion receptor activity, more preferably
affected by calcium receptor activity. In one embodiment of this
invention, antibodies that enhance the activity of CaSR are used to
modulate secretion and absorption of electrolytes, this being used
in the treatment of diarrheal disease.
[0017] The GI tract contains proteinase-activated receptors (PARs),
receptors that are activated by proteinases involved in digestion
and host defense. Both PAR-1 and PAR-2 are expressed on intestinal
epithelial cells. Intestinal PARs are involved in regulation of
cell proliferation, inflammation and chloride secretion. Among the
conditions to be treated using antibodies to PARs are characterized
by inappropriate expression or activity of PARs such as when the
PAR expression or activity level is too high or too low. Specific
medical conditions that are treatable or preventable using
antibodies to PARs include the treatment or prevention of cancers
of the GI tract, treatment of secretory disorders or disorders
associated with abnormal calcium secretion or absorption, or
inflammatory diseases of the GI tract including, but not limited
to: celiac disease, Crohn's disease; ulcerative colitis; idiopathic
gastroparesis; inflammatory bowel disease and ulcers, including
gastric and duodenal ulcers.
[0018] The GI tract contains receptors for bile acids. Bile acids
undergo passive absorption in the proximal small intestine and
active transport in the terminal ileum. Active transport is
mediated by the apical sodium co-dependent bile acid transporter
(ASBT) localized to the distal one-third of the ileum. An
equilibrium generally exists between hepatic cholesterol and the
bile acid pool. Inhibition of ileal ASBT by oral administration of
a specific antibody interrupts enterohepatic recirculation of bile
acids, resulting in a decrease in the liver bile acid pool. This
stimulates increased hepatic synthesis of bile acids from
cholesterol, eventually depleting the liver's pool of esterified
cholesterol, increasing the de novo synthesis of cholesterol in
hepatocytes and increasing the uptake of serum cholesterol by
upregulating the number of cell surface low density lipo-protein
cholesterol receptors ("LDL receptors"). The number of hepatic LDL
receptors directly impacts serum low density lipoprotein ("LDL")
cholesterol levels, with an increase in the number of LDL receptors
resulting in a decrease in serum cholesterol. The net result,
therefore, is that serum LDL cholesterol levels decrease when
intestinal bile acid reabsorption is reduced.
[0019] The GI tract contains cell surface peptidases and
saccharases. Oral administration of antibodies specific for
saccharases may be used in the treatment of diabetes, hyperlipaemia
and adiposity, and in animal nutrition, for the better utilization
of feed and for influencing the lean meat/fat ratio in favor of the
proportion of lean meat.
[0020] The GI tract also contains receptors for cytokines,
chemokines and other related immune mediators. Such receptors
include receptors specific for inflammatory cytokines such as
TNF-alpha, TNF-Kappa, IL-6, IFN-gamma, IL-1 beta, IL-12, IL-13,
11-23, and IL-2. Targeting any one or more of these receptors
expressed in the lumen with an orally administered therapeutic
antibody is useful for the treatment of a number of conditions
modulated by these receptors including but not limited to irritable
bowel syndrome, including Crohn's disease, ulcerative colitis,
necrotizing enterocolitis, celiac disease, inflammation due to
infection with invasive organisms such as Salmonella and
Escherichia coli, or inflammation secondary to injury caused by
surgery, trauma, ionizing radiation, or toxic chemicals, including
NSAIDs.
[0021] Cancers of the GI tract are composed of cells that express
receptors in the lumen of the GI tract that may be modulated by
antibodies to inhibit the growth of the tumor and/or to kill the
tumor. This invention encompasses antibodies that are directed
against GI tumors, preferably colon cancers. Targets include MS412,
PMEPA1, EGFR, CXCR2, VEGFR, PAR-1, CCK2R, TMPRS54, NMB-R,
Neuropilin-1 (NRP-1), GLUT1, STIM1, and the voltage-gated L-type
calcium channel alpha(1C), most preferably EGFR.
[0022] The terms "antibody" or "antibodies" as used herein refer to
a polypeptide comprising a framework region from an immunoglobulin
gene or fragments thereof that specifically binds and recognizes an
antigen. The recognized immunoglobulin genes include the kappa,
lambda, alpha, gamma, delta, epsilon, and mu constant region genes,
as well as the myriad immunoglobulin variable region genes. Light
chains are classified as either kappa or lambda. Heavy chains are
classified as gamma, mu, alpha, delta, or epsilon, which in turn
define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE,
respectively. Typically, the antigen-binding region of an antibody
will be most critical in specificity and affinity of binding to a
target receptor. An exemplary immunoglobulin (antibody) structural
unit comprises a tetramer. Each tetramer is composed of two
identical pairs of polypeptide chains, each pair having one "light"
(about 25 kD) and one "heavy" chain (about 50-70 kD). The
N-terminus of each chain defines a variable region of about 100 to
110 or more amino acids primarily responsible for antigen
recognition. The terms variable light chain (VL) and variable heavy
chain (VH) refer to these light and heavy chains respectively.
[0023] Antibodies exist, e.g., as intact immunoglobulins or as a
number of well-characterized fragments produced by digestion with
various peptidases that are able to compete with the intact
antibody for specific binding, unless otherwise specified herein.
Thus, for example, pepsin digests an antibody below the disulfide
linkages in the hinge region to produce F(ab)'2, a dimer of Fab
which itself is a light chain joined to V.sub.H-CH1 by a disulfide
bond. The F(ab)'2 may be reduced under mild conditions to break the
disulfide linkage in the hinge region, thereby converting the
F(ab)'2 dimer into an Fab' monomer. The Fab' monomer is essentially
Fab with part of the hinge region (see Fundamental Immunology (Paul
ed., 3d ed. 1993)). While various antibody fragments are defined in
terms of the digestion of an intact antibody, one of skill will
appreciate that such fragments may be synthesized de novo either
chemically or by using recombinant DNA methodology. Thus, the term
"antibody", as used herein, also includes antibody fragments either
produced by the modification of whole antibodies, or those
synthesized de novo using chemical or recombinant DNA methodologies
(e.g., single chain Fv, complementarity determining region (CDR)
fragments, or polypeptides that contain at least a portion of an
immunoglobulin that is sufficient to confer specific receptor
binding to the polypeptide) or those identified using phage display
libraries (see, e.g., McCafferty et al., Nature 348: 552-554
(1990)).
[0024] The terms "monoclonal antibody" or "monoclonal antibodies"
as used herein refer to a preparation of antibodies of single
molecular composition. A monoclonal antibody composition displays a
single binding specificity and affinity for a particular epitope of
a target receptor.
[0025] An "epitope" is the portion of a molecule that is bound by
an antibody. An epitope can comprise non-contiguous portions of the
molecule (e.g., in a polypeptide, amino acid residues that are not
contiguous in the polypeptide's primary sequence but that, in the
context of the polypeptide's tertiary and quaternary structure, are
near enough to each other to be bound by an antibody).
[0026] The term "polyclonal antibody" as used herein refers to a
composition of different antibody molecules which is capable of
binding to or reacting with several different specific antigenic
determinants on the same or on different antigens. The variability
in antigen specificity of a polyclonal antibody is located in the
variable regions of the individual antibodies constituting the
polyclonal antibody, in particular in the complementarity
determining regions (CDR)1, CDR2 and CDR3 regions. Preferably, the
polyclonal antibody is prepared by immunization of an animal with
the target receptor or portions thereof as specified below.
Alternatively, the polyclonal antibody may be prepared by mixing
multiple monoclonal antibodies (e.g. Nowakowski, A. et al. 2002.
Proc Natl Acad Sci USA 99, 11346-11350 and U.S. Pat. No. 5,126,130)
having desired specificity to a target receptor.
[0027] Polyclonal antibody preparations isolated from the blood,
milk, colostrum or eggs of immunized animals typically include
antibodies that are not specific for the immunogen in addition to
antibodies specific for the target receptor. Antibodies specific
for the target receptor may be purified from the polyclonal
antibody preparation or the polyclonal antibody preparation may be
used without further purification. Thus, the term "polyclonal
antibody" as used herein refers both to antibody preparations in
which the antibody specific for the target receptor has been
enriched and to preparations that are not purified. Numerous
techniques are known to those in the art for enriching polyclonal
antibodies for antibodies to specific targets. Recently a
technology for recombinant production of highly specific polyclonal
antibodies suitable for prophylactic and therapeutic administration
has been developed (WO 2004/061104). The recombinant polyclonal
antibody (rpAb) can be purified from a production bioreactor as a
single preparation without separate handling, manufacturing,
purification, or characterization of the individual members
constituting the recombinant polyclonal protein.
[0028] A "chimeric antibody" is an antibody molecule in which (a)
the constant region, or a portion thereof, is altered, replaced or
exchanged so that the antigen binding site (variable region) is
linked to a constant region of a different or altered class,
effector function and/or species, or an entirely different molecule
which confers new properties to the chimeric antibody, e.g., an
enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the
variable region, or a portion thereof, is altered, replaced or
exchanged with a variable region having a different or altered
antigen specificity. See, e.g., U.S. Pat. No. 4,816,567 and
Morrison, 1985, Science 229:1202-07.
[0029] The invention further contemplates the use of molecules
intended to mimic antibodies, such as aptamers. The invention also
contemplates the use of "fusion proteins" in which a portion of an
antibody molecule is fused to the ligand for the target receptor
and thereby made specific for the target receptor. In another
aspect, the present invention provides a derivative of an antibody
specific for a target apical intestinal receptor. The derivatized
antibody can comprise any molecule or substance that imparts a
desired property to the antibody, such as increased half-life in a
particular use. The derivatized antibody can comprise, for example,
a detectable (or labeling) moiety (e.g., a radioactive,
colorimetric, antigenic or enzymatic molecule, a detectable bead
(such as a magnetic or electrodense (e.g., gold bead), or a
molecule that binds to another molecule (e.g., biotin or
streptavidin)), a therapeutic or diagnostic moiety (e.g., a
radioactive, cytotoxic, or pharmaceutically active moiety), or a
molecule that increases the suitability of the antibody for a
particular use (e.g., administration to a subject, such as a human
subject, or other in vivo or in vitro uses). Examples of molecules
that can be used to derivatize an antibody include albumin (e.g.,
human serum albumin) and polyethylene glycol (PEG). Albumin-linked
and PEGylated derivatives of antibodies can be prepared using
techniques well known in the art. In one embodiment, the antibody
is conjugated or otherwise linked to transthyretin (TTR) or a TTR
variant. The TTR or TTR variant can be chemically modified with,
for example, a chemical selected from the group consisting of
dextran, poly(n-vinyl pyurrolidone), polyethylene glycols,
propropylene glycol homopolymers, polypropylene oxide/ethylene
oxide co-polymers, polyoxyethylated polyols and polyvinyl
alcohols.
[0030] Derivitized antibodies are also suitable for in-vivo or
in-vitro detection of expression of a target receptor. In one
preferred embodiment, an antibody derivitized with a
physiologically acceptable label detectable by standard imaging
equipment such as ultrasound, is used for in-vivo diagnostic
imaging to detect aberrant expression of a target receptor. Such
diagnostic techniques are useful in identifying patients who have
elevated expression, activation or activity of a target receptor
associated with one or more diseases thereby identifying patients
who may benefit most from treatment with an antibody of the
invention.
[0031] The present invention further comprises nucleic acid
molecules encoding all or a part of an antibody of the invention,
for example, one or both chains of the antibody of the invention or
a fragment, derivative, or variation thereof. The nucleic acids can
be single-stranded or double stranded and can comprise RNA and/or
DNA nucleotides or variants there of such as peptide nucleic acids.
The present invention further comprises host cells into which a
recombinant expression vector or transfectoma is introduced and is
capable of expressing an antibody of the invention or fragment
thereof. A host cell can be any prokaryotic cell or eukaryotic
cell. Vector DNA can be introduced into a host cell via
conventional transformation or transfection techniques.
[0032] In one embodiment, the antibody of the invention is capable
of at least partially blocking at least one biological activity of
a target apical intestinal receptor. In another embodiment, the
antibody of the invention has a binding affinity (K.sub.a) for the
target receptor of at least 10.sup.6. In other embodiments, the
antibody exhibits a K.sub.a of at least 10.sup.7, at least
10.sup.8, at least 10.sup.9, or at least 10.sup.10. In another
embodiment, the present invention provides an antibody that has a
low dissociation rate from a target receptor. In one embodiment,
the antibody has a K.sub.off of 1.times.10.sup.-4 s.sup.-1 or
lower. In another embodiment, the K.sub.off is 5.times.10.sup.-5
s.sup.-1 or lower. It is understood by those skilled in the art
that these affinities and dissociation rates refer to average
affinities and dissociation rates when used to describe polyclonal
antibodies. It is further understood by those skilled in the art
that affinity is defined broadly and includes avidity as well as
affinity. In another aspect, the present invention provides an
antibody that inhibits at least one biological activity of a target
receptor, for example, an antibody to the PAR-2 receptor may
inhibit Ca2+mobilization. In one embodiment, the antibody has an
IC.sub.50 of 1000 nM or lower. In another embodiment, the IC.sub.50
is 100 nM or lower; in another embodiment, the IC.sub.50 is 10 nM
or lower.
[0033] In one embodiment, monoclonal antibodies are preferred. In
another embodiment polyclonal antibodies are preferred. Monoclonal
antibodies are more controllable, but their specificity is limited.
Polyclonal antibodies are more difficult to characterize, but their
broad specificity means that they can interfere with target
receptors in several different ways. In addition, the manufacture
of polyclonal antibodies can be very inexpensive.
[0034] Methods of producing polyclonal and monoclonal antibodies
that react specifically with the target receptors of the invention
are known to those of skill in the art (see, e.g., Coligan, Current
Protocols in Immunology (1991); Harlow & Lane, Antibodies, A
Laboratory Manual (1988); Goding, Monoclonal Antibodies: Principles
and Practice (2d ed. 1986); and Kohler & Milstein, Nature 256:
495-497 (1975). Such techniques include antibody preparation by
selection of antibodies from libraries of recombinant antibodies in
phage or similar vectors, as well as preparation of polyclonal and
monoclonal antibodies by immunizing suitable animals (see, e.g.,
Huse et al., Science 246: 1275-1281 (1989); Ward et al., Nature
341: 544-546 (1989)).
[0035] A number of immunogens comprising target receptors or
portions of target receptors may be used to produce antibodies
specifically reactive with the target receptor. For example, an
antigenic fragment or protein portion of a target receptor can be
isolated using known procedures. Recombinant protein can be
expressed in eukaryotic or prokaryotic cells as described above,
and purified as generally described above. Alternatively, a
synthetic peptide derived from a target receptor can be used as an
immunogen. Preferably, the peptide is derived from a portion of the
target receptor that is expressed extracellularly. The synthetic
peptide may be conjugated to a carrier protein prior to
immunization. Naturally occurring protein may also be used either
in pure or impure form. The product is then injected into an animal
capable of producing antibodies. Animals may also be immunized with
cells that have been transfected with the target receptor or may be
immunized with DNA encoding the target receptor. Either monoclonal
or polyclonal antibodies may be generated accordingly.
[0036] Monoclonal antibodies may be obtained by various techniques
familiar to those skilled in the art. Briefly, spleen cells from an
animal immunized with a desired antigen are immortalized, commonly
by fusion with a myeloma cell (see, Kohler & Milstein, Eur. J.
Immunol., 6: 511-519 (1976)). Alternative methods of
immortalization include transformation with Epstein Barr Virus,
oncogenes, or retroviruses, or other methods well known in the art.
Colonies arising from single immortalized cells are screened for
production of antibodies of the desired specificity and affinity
for the antigen, and yield of the monoclonal antibodies produced by
such cells may be enhanced by various techniques, including
injection into the peritoneal cavity of a vertebrate host.
Alternatively, one may isolate DNA sequences which encode a
monoclonal antibody or a binding fragment thereof by screening a
DNA library from human B cells according to the general protocol
outlined by Huse, et al., Science 246: 1275-1281 (1989).
[0037] Methods of production of polyclonal antibodies are known to
those of skill in the art. An appropriate animal is immunized with
the protein using a standard adjuvant, such as Freund's adjuvant,
and a standard immunization protocol. The animal's immune response
to the immunogen preparation may be monitored by taking test bleeds
and determining the titer of reactivity to target receptor. When
appropriately high titers of antibody to the immunogen are
obtained, blood is collected from the animal and antisera are
prepared. Further fractionation of the antisera to enrich for
antibodies reactive to the protein can be done if desired (see,
Harlow & Lane, supra).
[0038] Alternatively, eggs can be collected from immunized birds
and antibody is isolated from the yolks of the eggs. Alternatively,
milk or colostrum can be collected from immunized female animals
and antibody is isolated from the milk or colostrum.
[0039] In one embodiment the antibody is isolated from the yolk of
eggs from a bird such as a chicken, duck, or goose that has been
immunized with a target receptor and/or peptide or antigenic
portion derived from a target receptor and a suitable adjuvant. In
another embodiment, the antibody is isolated from the serum of an
animal such as a cow, horse, rabbit, or goat that has been
immunized with a sweet taste receptor and/or peptide derived from a
sweet taste receptor and a suitable adjuvant.
[0040] In one embodiment, the antibody is a polyclonal antibody
derived from milk or colostrum. In one embodiment, the polyclonal
antibody is derived from the milk or colostrum of a ruminant such
as a cow, goat, sheep, camel or water buffalo. In another
embodiment, the antibody is isolated from the milk or colostrum of
a human. In a preferred embodiment, the polyclonal antibody is
isolated from the milk or colostrum of a bovine, preferably an
immunized cow. Bovine colostrum (early milk) is a preferred source
of antibodies for this invention. In cows, antibody does not cross
the placenta, and thus all passive immunity is transferred to the
newborn calf through the milk. As a result, cows secrete a large
bolus of antibody into the colostrum immediately after parturition
and approximately 50% of the protein in colostrum is
immunoglobulin. In the first 4 hours after birth, immunoglobulin
concentrations of 50 mg/ml are typically found in the colostrum
{Butler and Kehrli, 2005, Mucosal Immunology, 1763-1793}, dropping
to 25 - 30 mg/ml 24 hours later {Ontsouka et al., 2003, J Dairy
Sci, 86, 2005-11}.
[0041] Colostrum and milk are a uniquely safe source of polyclonal
antibody for oral delivery. There is already extensive human
exposure to bovine immunoglobulin, as regular milk contains 1.5 g/L
IgG.
[0042] In one aspect, the invention provides methods of treating a
patient using the therapeutic compositions of the invention. The
term "patient" as used herein refers to an animal. Preferably the
animal is a mammal. More preferably the mammal is a human. A
"patient" also refers to, for example, dogs, cats, horses, cows,
pigs, guinea pigs, fish, birds and the like. Thus, the compositions
and methods of the invention are equally suitable for veterinary
treatments.
[0043] The terms "treatment" "treat" and "treating" encompasses
alleviation, cure or prevention of at least one symptom or other
aspect of a disorder, disease, illness or other condition
(collectively referred to herein as a "condition"), or reduction of
severity of the condition, and the like. A composition of the
invention need not effect a complete cure, or eradicate every
symptom or manifestation of a disease, to constitute a viable
therapeutic agent. As is recognized in the pertinent field, drugs
employed as therapeutic agents may reduce the severity of a given
disease state, but need not abolish every manifestation of the
disease to be regarded as useful therapeutic agents. Similarly, a
prophylactically administered treatment need not be completely
effective in preventing the onset of a condition in order to
constitute a viable prophylactic agent. Simply reducing the impact
of a disease (for example, by reducing the number or severity of
its symptoms, or by increasing the effectiveness of another
treatment, or by producing another beneficial effect), or reducing
the likelihood that the disease will occur or worsen in a subject,
is sufficient. In one embodiment, an indication that a
therapeutically effective amount of a composition has been
administered to the patient is a sustained improvement over
baseline of an indicator that reflects the severity of the
particular disorder.
[0044] In one embodiment, the invention provides a method of
treating a condition comprising administering directly to the G.I.
tract of a patient, a composition comprising an antibody specific
for a target apical intestinal receptor as described above wherein
the target receptor modulates a condition including, but not
limited to: a metabolic condition, inflammation, cancer, drug
overdose or toxicity, conditions modulated by receptors for
neurotransmitters located on the luminal surface of the G.I. tract,
and conditions modulated by receptors for inorganic ions.
[0045] The pharmaceutical compositions of the present invention
comprise a therapeutically effective amount of an antibody of the
present invention formulated together with one or more
pharmaceutically acceptable carriers or excipients. By a
"therapeutically effective amount" of an antibody of the invention
is meant an amount of the composition which confers a therapeutic
effect on the treated subject, at a reasonable benefit/risk ratio
applicable to any medical treatment. The therapeutic effect is
sufficient to "treat" the patient is that term is used herein. As
used herein, the term "pharmaceutically acceptable carrier or
excipient" means a non-toxic, inert solid, semi-solid or liquid
filler, diluent, encapsulating material or formulation auxiliary of
any type. Some examples of materials which can serve as
pharmaceutically acceptable carriers are sugars such as lactose,
glucose and sucrose; starches such as corn starch and potato
starch; cellulose and its derivatives such as sodium carboxymethyl
cellulose, ethyl cellulose and cellulose acetate; powdered
tragacanth; malt; gelatin; talc; excipients such as cocoa butter
and suppository waxes; oils such as peanut oil, cottonseed oil,
safflower oil, sesame oil, olive oil, corn oil and soybean oil;
glycols such as propylene glycol; esters such as ethyl oleate and
ethyl laurate; agar; buffering agents such as magnesium hydroxide
and aluminun hydroxide; alginic acid; pyrogen-free water; isotonic
saline; Ringer's solution; ethyl alcohol, and phosphate buffer
solutions, as well as other non-toxic compatible lubricants such as
sodium lauryl sulfate and magnesium stearate, as well as coloring
agents, releasing agents, coating agents, sweetening, flavoring and
perfuming agents, preservatives and antioxidants can also be
present in the composition, according to the judgment of the
formulator.
[0046] The pharmaceutical compositions of this invention are
administered directly to the G.I. tract of a patient. As used
herein the phrase "administered directly to the G.I. tract of a
patient" means oral or rectal administration. Thus the
pharmaceutical compositions of the invention are appropriately
formulated for administration directly to the G.I. tract of the
patient such that they are suitable for oral or rectal
administration to the patient.
[0047] Liquid dosage forms for oral administration include
pharmaceutically acceptable emulsions, microemulsions, solutions,
suspensions, syrups and elixirs. In addition to the active
compounds, the liquid dosage forms may contain inert diluents
commonly used in the art such as, for example, water or other
solvents, solubilizing agents and emulsifiers such as ethyl
alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl
alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,
dimethylformamide, oils (in particular, cottonseed, groundnut,
corn, germ, olive, castor, and sesame oils), glycerol,
tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid
esters of sorbitan, and mixtures thereof. Besides inert diluents,
the oral compositions can also include adjuvants such as wetting
agents, emulsifying and suspending agents, sweetening, flavoring,
and perfuming agents.
[0048] Compositions for rectal administration are preferably
suppositories which can be prepared by mixing the compounds of this
invention with suitable non-irritating excipients or carriers such
as cocoa butter, polyethylene glycol or a suppository wax which are
solid at ambient temperature but liquid at body temperature and
therefore melt in the rectum or vaginal cavity and release the
active compound. In one embodiment, compositions for rectal
administration are in the form of an enema.
[0049] Solid dosage forms for oral administration include capsules,
tablets, pills, powders, and granules. In such solid dosage forms,
the active compound is mixed with at least one inert,
pharmaceutically acceptable excipient or carrier such as sodium
citrate or dicalcium phosphate and/or: a) fillers or extenders such
as starches, lactose, sucrose, glucose, mannitol, and silicic acid,
b) binders such as, for example, carboxymethylcellulose, alginates,
gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants
such as glycerol, d) disintegrating agents such as agar-agar,
calcium carbonate, potato or tapioca starch, alginic acid, certain
silicates, and sodium carbonate, e) solution retarding agents such
as paraffin, f) absorption accelerators such as quaternary ammonium
compounds, g) wetting agents such as, for example, cetyl alcohol
and glycerol monostearate, h) absorbents such as kaolin and
bentonite clay, and i) lubricants such as talc, calcium stearate,
magnesium stearate, solid polyethylene glycols, sodium lauryl
sulfate, and mixtures thereof. In the case of capsules, tablets and
pills, the dosage form may also comprise buffering agents.
[0050] Solid compositions of a similar type may also be employed as
fillers in soft and hard-filled gelatin capsules using such
excipients as lactose or milk sugar as well as high molecular
weight polyethylene glycols and the like.
[0051] Oral delivery of protein therapeutics is challenging because
the GI tract is designed to degrade and digest ingested material.
However, bovine immunoglobulin is partially stable to gastric
digestion. Several studies have directly examined the stability of
bovine immunoglobulin in the human GI tract. Human subjects have
been administered oral preparations of bovine colostral
immunoglobulin and material has been recovered in ileal fluid
effluent {Roos et al., 1995, J Nutr, 125, 1238-44; Warny et al.,
1999, Gut, 44, 212-7}. Both intact IgG and functional activity were
recovered in the ileum, with quantities ranging from 19% to 49%.
Immunoglobulin has also been recovered in the stool of patients
dosed with bovine IgG {McClead et al., 1988, Am J Med, 85, 811-6;
Kelly et al., 1997, Antimicrob Agents Chemother, 41, 236-41}.
Recovery rates ranged from 0.6% to 8.8% of the administered dose.
The ability of bovine IgG to survive digestion by gastric and
pancreatic proteases as well as the microbial proteases found in
the colon highlights the unusual stability of these
immunoglobulins.
[0052] Should it be desirable to avoid gastric degradation, there
are many options for enteric coating (see for example U.S. patents
4,330,338 and 4,518,433). In one embodiment, enteric coatings take
advantage of the post-gastric change in pH to dissolve a film
coating and release the active ingredient. Coatings and
formulations have been developed to deliver protein therapeutics to
the small intestine and these approaches could be adapted for the
delivery of an antibody of the invention. For example, an
enteric-coated form of insulin has been developed for oral delivery
{Toorisaka et al., 2005, J Control Release, 107, 91-6}.
[0053] In addition, the solid dosage forms of tablets, dragees,
capsules, pills, and granules can be prepared with other coatings
and shells well known in the pharmaceutical formulating art. They
may optionally contain opacifying agents and can also be of a
composition that they release the active ingredient(s) only, or
preferentially, in a certain part of the intestinal tract,
optionally, in a delayed manner. Examples of embedding compositions
that can be used include polymeric substances and waxes.
[0054] Antibodies that are directly engineered or modified to
improve gastric stability would be preferred for use in this
invention. Such engineering and modification could be accomplished
by the addition or removal of glycosylation sites, by the addition
of agents such as polyethylene glyclol, by the removal or
modification of sites that confer acid instability, or by the
removal or modification of sites that confer sensitivity to
proteases present in the stomach and small intestine, including
pepsin, trypsin and chymotrypsin.
[0055] Effective doses will vary depending on route of
administration, as well as the possibility of co-usage with other
agents. It will be understood, however, that the total daily usage
of the compounds and compositions of the present invention will be
decided by the attending physician within the scope of sound
medical judgment. The specific therapeutically effective dose level
for any particular patient will depend upon a variety of factors
including the disorder being treated and the severity of the
disorder; the activity of the specific compound employed; the
specific composition employed; the age, body weight, general
health, sex and diet of the patient; the time of administration,
route of administration, and rate of excretion of the specific
compound employed; the timing of delivery of the compound relative
to food intake; the duration of the treatment; drugs used in
combination or contemporaneously with the specific compound
employed; and like factors well known in the medical arts.
[0056] Particular embodiments of the present invention involve
administering a pharmaceutical composition comprising an antibody
specific for a target receptor at a dosage of from about 1 mg per
day to about 1 g/day, more preferably from about 10 mg/day to about
500 mg/day, and most preferably from about 20 mg/day to about 100
mg/day, to a subject. In one embodiment, a polyclonal antibody
preparation is administered at a dosage of antibody from about 100
mg to about 50 g/day, more preferably from about 500 mg/day to
about 10 g/day, and most preferably from about 1 g/day to about 5
g/day, to a subject, wherein the polyclonal antibody preparation
has not been enriched for antibodies specific for the target
receptor.
[0057] Treatment regimens include administering an antibody
composition of the invention one time per day, two times per day,
or three or more times per day, to treat a medical disorder
disclosed herein. In one embodiment, an antibody composition of the
invention is administered one time per week, two times per week, or
three or more times per week, to treat a medical disorder disclosed
herein.
[0058] The methods and compositions of the invention include the
use of an antibody of the invention in combination with one or more
additional therapeutic agents useful in treating the condition with
which the patient is afflicted. Examples of such agents include
both proteinaceous and non-proteinaceous drugs. When multiple
therapeutics are co-administered, dosages may be adjusted
accordingly, as is recognized in the pertinent art.
"Co-administration" and combination therapy are not limited to
simultaneous administration, but also include treatment regimens in
which an antibody of the invention is administered at least once
during a course of treatment that involves administering at least
one other therapeutic agent to the patient.
[0059] The pharmaceutical compositions of this invention can be
administered orally to animals, for example, by blending said
pharmaceutical compositions into animal feed or said pharmaceutical
compositions may be dissolved in water that the animals drink. The
dosage for the treatment of an animal differs depending upon the
purpose of administration (prevention or cure of disease) and type
of administration and of the animal to be treated. Generally, a
dosage of 1-1000 mg, preferably 20-100 mg, per kg of body weight of
the animal may be administered per day, either at one time or
divided into several times. It will be recognized that the
above-specified dosage is only a general range which may be reduced
or increased depending upon the age, body weight, condition of
disease, etc. of the animal.
[0060] The following prophetic examples are provided for the
purpose of illustrating specific embodiments or features of the
invention and are not intended to limit its scope.
EXAMPLES
[0061] Example 1. Generation of bovine immunoglobulin specific for
GLUT2
[0062] The GLUT2 peptide derived from the extracellular loop
between transmembrane regions 1 and 2 (aa 40-55) is synthesized
with an additional cysteine residue at the C-terminus
(SHYRHVLGVPLDDRRAC) (SEQ ID NO: 1) and coupled to maleimide
activated mcKLH (Pierce Protein Research Products) using procedures
supplied by the manufacturer.
[0063] The GLUT2-KLH conjugate is dissolved in PBS at 0.1 mg/mL and
emulsified 1:1 (vol/vol) with EMULSIGEN.RTM.-D (purchased from MVP
Laboratories, Omaha, Neb.), an oil-in-water adjuvant containing an
immunostimulant. Pregnant, healthy, mastitis-free Holstein dairy
cows are immunized subcutaneously in the rear thigh with 100 .mu.g
of gliadin in a total volume of 2 mL. All vaccinations are
performed under the direction of a licensed veterinarian and health
records are maintained. Vaccinations are given on days 0, 21 and
35. The immunizations are timed such that the final boost is given
approximately three weeks before parturition.
[0064] Colostrums are collected on days 1-4 after parturition.
Colostrum is collected from each vaccinated cow separately and
immediately frozen. Small (15 mL) samples of each milking are taken
from cows prior to freezing bulk colostrum. These samples are used
to measure immunogenicity of the vaccine regimen on an individual
cow basis. Colostrums are pooled and frozen at -20.degree. C. until
further use.
[0065] Colostral whey is prepared using standard methods {Su and
Chiang, 2003, J Dairy Sci, 86, 1639-45}. Samples from individual
animals are processed independently. Colostrum collected on days
1-4 post-parturition is thawed and pooled. Colostrum is centrifuged
at 4000.times.g to remove fat. The pH is slowly adjusted to 4.6
using 1 N HCl, incubated for 30 min at 37.degree. C. to precipitate
casein, and centrifuged. Whey is stored at -20.degree. C.
[0066] The titer of anti-GLUT2 antibody in each whey sample is
assessed by ELISA. Microtiter plates are coated with GLUT2-BSA
conjugates, prepared as above, at 1 .mu.g/ml and blocked with 1%
ovalbumin. Serial dilutions of colostral whey are added to the
plates in triplicate wells and incubated for 1 hr at room
temperature. Plates are washed and developed with horseradish
peroxidase (HRP)-labeled sheep anti-bovine IgG (h+l) (Bethyl
Laboratories, Montgomery, Tex.) and substrate OPD using standard
techniques. The second antibody will recognize all bovine
immunoglobulin isotypes through detection of the light chain.
Colostral whey from cows immunized with influenza is used as a
negative control. Antibody levels are expressed as titer, the
reciprocal of the dilution yielding a half-maximal absorbance in
the ELISA. Antibodies specific for GLUT2 are generated by
immunization with the GLUT2 peptide conjugate, as demonstrated by a
positive response in the ELISA using colostrum from immunized cows
but not from cows immunized with influenza.
[0067] The concentration of immunoglobulin in each whey sample is
also determined by ELISA. ELISA plates are coated with sheep anti
bovine IgG (h+1) (Bethyl Laboratories) and blocked with 1%
ovalbumin. Serial dilutions of whey are added to the plates and
developed as in the anti-TNF ELISA above. Purified bovine
immunoglobulin is used as a control. The specific activity of each
colostral sample is calculated (titer per mg immunoglobulin). A
pool is created from all colostral samples that contain levels of
anti-GLUT2 antibody more than 2.times. above background and used
for all subsequent work.
[0068] Example 2. Generation of bovine immunoglobulin specific for
GLUTS
[0069] The GLUTS peptide derived from the extracellular loop
between transmembrane regions 1 and 2 (aa 62-77) is synthesized
with an additional cysteine residue at the C-terminus
(LLMQQFYNETYYGRTC) (SEQ ID NO: 2) and coupled to maleimide
activated mcKLH (Pierce Protein Research Products) using procedures
supplied by the manufacturer.
[0070] The GLUTS-KLH conjugate is dissolved in PBS at 0.1 mg/mL and
emulsified 1:1 (vol/vol) with CARBIGEN.RTM. (purchased from MVP
Laboratories, Omaha, NE), a carbomer-based adjuvant. Pregnant,
healthy, mastitis-free Holstein dairy cows are immunized
subcutaneously in the rear thigh with 100 .mu.g of the GLUTS-KLH
conjugate in a total volume of 2 mL. All vaccinations are performed
under the direction of a licensed veterinarian and health records
are maintained. Vaccinations are given on days 0, 21 and 35. The
immunizations are timed such that the final boost is given
approximately three weeks before parturition.
[0071] Colostrums are collected on days 1-4 after parturition.
Colostrum is collected from each vaccinated cow separately and
immediately frozen. Small (15 mL) samples of each milking are taken
from cows prior to freezing bulk colostrum. These samples are used
to measure immunogenicity of the vaccine regimen on an individual
cow basis. Colostrums are pooled and frozen at -20.degree. C. until
further use.
[0072] Colostral whey is prepared using standard methods {Su and
Chiang, 2003, J Dairy Sci, 86, 1639-45}. Samples from individual
animals are processed independently. Colostrum collected on days
1-4 post-parturition is thawed and pooled. Colostrum is centrifuged
at 4000.times. g to remove fat. The pH is slowly adjusted to 4.6
using 1 N HCl, incubated for 30 min at 37.degree. C. to precipitate
casein, and centrifuged. Whey is stored at -20.degree. C.
[0073] The titer of anti-GLUTS antibody in each whey sample is
assessed by ELISA. Microtiter plates are coated with GLUTS-BSA
conjugates, prepared as above, at 1 .mu.g/ml and blocked with 1%
ovalbumin. Serial dilutions of colostral whey are added to the
plates in triplicate wells and incubated for 1 hr at room
temperature. Plates are washed and developed with horseradish
peroxidase (HRP)-labeled sheep anti-bovine IgG (h+1) (Bethyl
Laboratories, Montgomery, Tex.) and substrate OPD using standard
techniques. The second antibody will recognize all bovine
immunoglobulin isotypes through detection of the light chain.
Colostral whey from cows immunized with influenza is used as a
negative control. Antibody levels are expressed as titer, the
reciprocal of the dilution yielding a half-maximal absorbance in
the ELISA. Antibodies specific for GLUTS are generated by
immunization with the GLUTS peptide conjugate, as demonstrated by a
positive response in the ELISA using colostrum from immunized cows
but not from cows immunized with influenza.
[0074] Example 3. Generation of bovine immunoglobulin specific for
GLUT7
[0075] The GLUT7 peptide derived from the extracellular loop
between transmembrane regions 1 and 2 (aa 50-77) is synthesized
with an additional cysteine residue at the C-terminus
(KVGTSCGWGNVFQVFKSFYNETYFERHC) (SEQ ID NO: 3) and coupled to
maleimide activated ovalbumin (Pierce Protein Research Products)
using procedures supplied by the manufacturer.
[0076] The GLUT?-OVA conjugate is dissolved in PBS at 0.2 mg/mL and
emulsified 1:1 (vol/vol) with the adjuvant EMULSIGEN-D.RTM.
(purchased from MVP Laboratories, Omaha, Neb.). Pregnant, healthy,
mastitis-free Holstein dairy cows are immunized subcutaneously in
the rear thigh with 200 .mu.g of the GLUT?-OVA conjugate in a total
volume of 2 mL. All vaccinations are performed under the direction
of a licensed veterinarian and health records are maintained.
Vaccinations are given on days 0, 21 and 35. The immunizations are
timed such that the final boost is given approximately three weeks
before parturition.
[0077] Colostrums are collected on days 1-4 after parturition.
Colostrum is collected from each vaccinated cow separately and
immediately frozen. Small (15 mL) samples of each milking are taken
from cows prior to freezing bulk colostrum. These samples are used
to measure immunogenicity of the vaccine regimen on an individual
cow basis. Colostrums are pooled and frozen at -20.degree. C. until
further use.
[0078] Colostral whey is prepared using standard methods. Samples
from individual animals are processed independently. Colostrum
collected on days 1-4 post-parturition is thawed and pooled.
Colostrum is centrifuged at 4000.times. g to remove fat. The pH is
slowly adjusted to 4.6 using 1 N HCl, incubated for 30 min at
37.degree. C. to precipitate casein, and centrifuged. Whey is
stored at -20.degree. C.
[0079] The titer of anti-GLUT7 antibody in each whey sample is
assessed by ELISA. Microtiter plates are coated with GLUT7-KLH
conjugates, prepared as above, at 1 .mu.g/ml and blocked with 1%
immunoglobulin-free BSA. Serial dilutions of colostral whey are
added to the plates in triplicate wells and incubated for 1 hr at
room temperature. Plates are washed and developed with horseradish
peroxidase (HRP)-labeled sheep anti-bovine IgG (h+1) (Bethyl
Laboratories, Montgomery, Tex.) and substrate OPD using standard
techniques. The second antibody will recognize all bovine
immunoglobulin isotypes through detection of the light chain.
Colostral whey from cows immunized with influenza is used as a
negative control. Antibody levels are expressed as titer, the
reciprocal of the dilution yielding a half-maximal absorbance in
the ELISA. Antibodies specific for GLUT7 are generated by
immunization with the GLUT7 peptide conjugate, as demonstrated by a
positive response in the ELISA using colostrum from immunized cows
but not from cows immunized with influenza.
[0080] Example 4. Antibody inhibition of GLUT7-mediated uptake of
glucose Anti-GLUT7 antibody is generated as described in Example 3
and used to inhibit glucose uptake by Xenopus oocytes expressing
GLUT7. Plasmid containing the hGLUT gene is produced and
transcribed as described {Li et al., 2004, Am J Physiol
Gastrointest Liver Physiol, 287, G236-42}. Stage V/VI oocytes are
harvested from anesthetized Xenopus laevis and placed in Modified
Barth's Medium (MBM). The follicular layer is removed by treatment
for 2 h with 0.02 g/ml type I collagenase (Sigma Aldrich), followed
by hypertonic phosphate treatment. Oocytes are incubated at
16-18.degree. C. for 24 hr in MBM and injected with 20 ng GLUT7
synthetic mRNA transcript and incubated for 3-5 days at
16-18.degree. C. before use in functional uptake assays. Control
oocytes are injected with water alone.
[0081] Uptake experiments are performed at 20.degree. C. with 5-10
oocytes for each condition. GLUT7-transfected or control oocytes
are preincubated fo 30 min with varying doses of GLUT7-specific
antibody or control influenza-specific bovine antibody. Three doses
are examined: the dose that generates a half-maximal response in a
GLUT7-specific ELISA, and doses 10-fold higher and 10-fold lower.
[3H]glucose (100 uM, 1 uCi/ml) is added and incubated for an
additional 30 min. Oocytes are washed with cold MBM to stope the
incubation and individual oocytes are placed in vials and dissolved
in 0.5 ml 5% SDS for 30 min. Scintillation fluid is added to each
vial and radioactivity measured. Data are expressed as pmoles
glucose taken up over 30 min. Reduced levels of glucose uptake are
seen in the presence of GLUT7-specific antibody than in the
controls.
[0082] Example 5. Antibody inhibition of glucose uptake by
GLUT2-specific antibody
[0083] Male Wistar rats (240-270 g) are anesthetized using an ip
injection of Hypnorm and Hypnovel. A mid to distal loop of jejumum
is cannulated at 10 and 35 cm from the Ligament of Treitz and
perfused in vivo in a single-pass mode with perfusate comprising
nutrient at the stated concentration in modified Krebs-Henseleit
buffer (KHB) containing 201 mM NaCl, 4.5 mM KCl, 1.0 mM MgSO.sub.4,
1.8 mM Ha.sub.2HPO.sub.4, 0.2 mM NaH.sub.2PO.sub.4, 1.25 mM
CaCl.sub.2 and 25 mM NaHCO.sub.3, gassed to pH 7.4 with 19:1
O.sub.2-CO.sub.2 before use. The flow rate of perfusate is 0.37
ml/min and that of gas 0.19 ml/min. The jejunal loop is perfused
with 20 mM glucose for 30 min and then switched to glucose mixed
with GLUT2-specific antibody for an additional 30 min. Three
concentrations of antibody are tested: the antibody concentration
that generates a half-maximal response in a GLUT2-specific ELISA,
and concentrations 10-fold higher and 10-fold lower. Glucose
absorption rates are expressed as the rate of loss from the luminal
perfusate expressed in umol/min (g dry weight).sup.-1. Reduced
glucose absorption is seen in the presence of GLUT2-specific
antibody when compared to controls.
[0084] Example 6. Generation of bovine immunoglobulin specific for
T1R3 Peptides are synthesized that are based on the extracellular
domain of the sweet taste receptor T1R3 from either mouse
(HEGLVPQHDTSCQQLGK) (SEQ ID NO: 4) or human (EEAGLRSRTRPSSP) (SEQ
ID NO: 5). The peptides are dissolved in PBS at 0.1 mg/mL and
emulsified 1:1 (vol/vol) with the adjuvant EMULSIGEN-D.RTM.
(purchased from MVP Laboratories, Omaha, Neb.). Pregnant, healthy,
mastitis-free Holstein dairy cows are immunized subcutaneously in
the rear thigh with 100 .mu.g of the T1R3 peptides in a total
volume of 2 mL. All vaccinations are performed under the direction
of a licensed veterinarian and health records are maintained.
Vaccinations are given on days 0, 21 and 35. The immunizations are
timed such that the final boost is given approximately three weeks
before parturition.
[0085] Colostrums are collected on days 1-4 after parturition.
Colostrum is collected from each vaccinated cow separately and
immediately frozen. Small (15 mL) samples of each milking are taken
from cows prior to freezing bulk colostrum. These samples are used
to measure immunogenicity of the vaccine regimen on an individual
cow basis. Colostrums are pooled and frozen at -20.degree. C. until
further use.
[0086] Colostral whey is prepared using standard methods. Samples
from individual animals are processed independently. Colostrum
collected on days 1-4 post-parturition is thawed and pooled.
Colostrum is centrifuged at 4000.times. g to remove fat. The pH is
slowly adjusted to 4.6 using 1 N HCl, incubated for 30 min at
37.degree. C. to precipitate casein, and centrifuged. Whey is
stored at -20.degree. C.
[0087] The titer of anti-T1R3 antibody in each whey sample is
assessed by ELISA. Microtiter plates are coated with the murine or
human T1R3 peptide at 1 .mu.g/ml and blocked with 1% ovalbumin.
Serial dilutions of colostral whey are added to the plates in
triplicate wells and incubated for 1 hr at room temperature. Plates
are washed and developed with horseradish peroxidase (HRP)-labeled
sheep anti-bovine IgG (h+1) (Bethyl Laboratories, Montgomery, Tex.)
and substrate OPD using standard techniques. Colostral whey from
cows immunized with influenza is used as a negative control.
Antibody levels are expressed as titer, the reciprocal of the
dilution yielding a half-maximal absorbance in the ELISA.
Antibodies specific for T1R3 are generated by immunization with
T1R3, as demonstrated by a positive response in the ELISA using
colostrum from immunized cows but not from cows immunized with
influenza.
[0088] Example 7. Inhibition of GLP-1 release by antibodies
specific for T1R3
[0089] C57BL/6 mice (8 mice per group) are fasted overnight and
administered glucose by gastric gavage (2 g/kg body weight) in the
presence or absence of polyclonal bovine antibody specific for the
murine T1R3 sweet taste receptor. The antibody dose is selected
that is 10-fold higher than that calculated to bind 90% of the
intestinal T1R3 receptors. An additional control group is
administered bovine antibody isolated from non-immunized cows.
Blood samples (0.1 ml) are collected immediately before and 10, 20
and 40 minutes after glucose administration. Dipeptidyl peptidase
IV inhibitor is added to the blood samples upon collection. Blood
samples are analyzed for the levels of glucose using a glucometer,
for plasma insulin by ELISA (ALPCO Diagnostics, Salem, N.H.), and
for plasma GLP-1 by ELISA (ALPCO Diagnostics). In the presence of
antibody specific for T1R3, glucose dosing results in reduced
levels of blood glucose, of plasma insulin and of plasma GLP-1 when
compared to glucose dosing in the absence of antibody.
[0090] Example 8. Inhibition of LPS-induced cytokine production by
anti-TLR4 antibody
[0091] An anti-TLR4 monoclonal antibody is generated by
immunization of rats with the Ba/F3 cell line expressing mouse TLR4
and MD-2. Hybridomas are generated using standard techniques and
cells secreting specific antibody are identified by the presence in
the supernatant of antibody capable of binding to TLR4.
Alternatively, anti-TLR4 monoclonal antibody is obtained from a
commercial source, such as Imgenix. Murine splenocytes are cultured
at 5.times.10.sup.5 cells/ml with lipopolysaccharide (LPS) from E.
coli at 100 ng/ml in the presence of varying concentrations of the
anti-TLR4 monoclonal antibody. After 72 hr, supernatants are
removed and assayed for the presence of TNFalpha using an ELISA
kit. The anti-TLR4 antibody inhibits the production of LPS-induced
cytokine production.
[0092] Example 9. Inhibition of necrotizing enterocolitis with oral
anti-TLR4 antibody
[0093] Necrotizing enterocolitis is induced in neonatal mice as
described {Jilling et al., 2006, J Immunol, 177, 3273-82}. Briefly,
C3HeB/FeJ or C3H/HeJ mouse pups are delivered by Cesarean section
between E20-21. Pups are stabilized, dried and maintained in an
incubator at 37degC, and bowel/bladder function are stimulated by a
soft cotton-tip applicator every 3 h. Two hours after delivery,
animals are fed Esbilac puppy formula by non-sanitized feeding
orogastric catheter every 2 h starting with 0.03 ml, increasing to
0.04 ml in the subsequent 24 h to deliver approximately 200
kcal/kg/day. Asphyxia stress is accomplished by exposure to 100%
nitrogen for 60s, followed by exposure to cold (4degC) for 10 min
twice daily. Animals are euthanized at 72 h and intestines are
collected in 10% formalin and processed for H&E staining.
Sections are analyzed and tissue injury scored from 1-4 by a
blinded investigator using an established scoring system {Jilling
et al., 2006, J Immunol, 177, 3273-82}.
[0094] To test the effect of an anti-TLR4 antibody on necrotizing
enterocolitis, a monoclonal antibody specific for murine TLR4/MD2
is purchased from InvivoGen (San Diego, Calif.). Antibody is mixed
with the Esbilac immediately prior to feeding at a dose of 1 ug/ml.
Four groups of mice are compared: C3HeB/FeJ mice with and without
antibody and C3H/HeJ mice with and without antibody. The incidence
of necrotizing enterocolitis in the 4 groups is assessed. Antibody
treatment reduces the incidence of necrotizing enterocolities in
the C3HeB/FeJ mice, but has no effect on the C3H/HeJ mice (C3H/HeJ
do not express TLR4).
[0095] Example 10. Generation of a monoclonal antibody that
inhibits the apical sodium-dependent bile acid transporter
(ASBT)
[0096] Mice are immunized with 10 ug of a peptide derived from the
extracellular loop EL1 of human ASBT (VVLIIGCCPGGTASNILAYWVDGDMDLS)
(SEQ ID NO: 6) in complete Freund's adjuvant and boosted at day 14
with the same peptide in incomplete Freund's adjuvant (IFA). An
additional boost of antigen in IFA is given at day 28 and at day 35
the mice are sacrificed. Splenic B cells are fused with a myeloma
line to produce B cell hybridomas using standard techniques.
Supernatants from antibody-secreting hybridomas are screened for
their ability to bind the immunizing peptide by ELISA. Positive
antibodies are further screened for their ability to inhibit bile
acid uptake in an in vitro system using the human intestinal Caco-2
cell line as described {Alrefai et al., 2005, Am J Physiol
Gastrointest Liver Physiol, 288, G978-85}. Briefly, confluent
Caco-2 cells are equilibrated at room temperature and then washed
and incubated for 15 min at 25degC with 110 mM NaCl, 4 mM KCl, 1 mM
MgSO.sub.4, 1 mM CaCl, 50 mM mannitol and 10 mM HEPES, pH 7.4 in
the presence or absence of varying concentrations of the anti-ASBT
antibody. Cells are washed and incubated with the same buffer and
same antibody concentration with 10 uM 1(uCi/ml) of
.sup.3H-taurocholic acid. After 5 minutes, the transport process is
stopped by washing the cells with ice cold PBS and solubilizing the
cells with 0.5 N NaOH. Protein concentration is measured and uptake
of [.sup.3H]-taurocholic acid is expressed as picomole per
milligram protein per 5 minutes. Inhibitory antibody reduces the
uptake of taurocholic acid by the apical sodium-dependent bile acid
transporter (ASBT).
[0097] The patent and scientific literature referred to herein
establishes the knowledge that is available to those with skill in
the art. All United States patents and published or unpublished
United States patent applications cited herein are incorporated by
reference. All published foreign patents and patent applications
cited herein are hereby incorporated by reference. All other
published references, documents, manuscripts and scientific
literature cited herein are hereby incorporated by reference.
[0098] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims. It
should also be understood that the embodiments described herein are
not mutually exclusive and that features from the various
embodiments may be combined in whole or in part in accordance with
the invention.
Sequence CWU 1
1
6117PRTArtificial SequenceSynthetic 1Ser His Tyr Arg His Val Leu
Gly Val Pro Leu Asp Asp Arg Arg Ala 1 5 10 15 Cys 216PRTArtificial
SequenceSynthetic 2Leu Leu Met Gln Gln Phe Tyr Asn Glu Thr Tyr Tyr
Gly Arg Thr Cys 1 5 10 15 328PRTArtificial SequenceSynthetic 3Lys
Val Gly Thr Ser Cys Gly Trp Gly Asn Val Phe Gln Val Phe Lys 1 5 10
15 Ser Phe Tyr Asn Glu Thr Tyr Phe Glu Arg His Cys 20 25
417PRTArtificial SequenceSynthetic 4His Glu Gly Leu Val Pro Gln His
Asp Thr Ser Cys Gln Gln Leu Gly 1 5 10 15 Lys 514PRTArtificial
SequenceSynthetic 5Glu Glu Ala Gly Leu Arg Ser Arg Thr Arg Pro Ser
Ser Pro 1 5 10 628PRTArtificial SequenceSynthetic 6Val Val Leu Ile
Ile Gly Cys Cys Pro Gly Gly Thr Ala Ser Asn Ile 1 5 10 15 Leu Ala
Tyr Trp Val Asp Gly Asp Met Asp Leu Ser 20 25
* * * * *