U.S. patent application number 12/319883 was filed with the patent office on 2009-08-13 for compositions and methods for diagnosis and treatment of type 2 diabetes.
Invention is credited to Catherine R. Auge, Cohava Gelber, Pranvera Ikonomi, Liping Liu, John R. Simms, Zhidong Xie.
Application Number | 20090203602 12/319883 |
Document ID | / |
Family ID | 42316869 |
Filed Date | 2009-08-13 |
United States Patent
Application |
20090203602 |
Kind Code |
A1 |
Gelber; Cohava ; et
al. |
August 13, 2009 |
Compositions and methods for diagnosis and treatment of type 2
diabetes
Abstract
The present invention relates generally to the identification of
biological markers associated with an increased risk of developing
Diabetes, as well as methods of using such biological markers in
diagnosis and prognosis of Diabetes. The biological markers of the
invention may indicate new targets for therapy or constitute new
therapeutics for the treatment or prevention of Diabetes.
Inventors: |
Gelber; Cohava; (Nokesville,
VA) ; Liu; Liping; (Manassas, VA) ; Xie;
Zhidong; (Manassas, VA) ; Ikonomi; Pranvera;
(Manassas, VA) ; Simms; John R.; (Haymarket,
VA) ; Auge; Catherine R.; (Haymarket, VA) |
Correspondence
Address: |
MINTZ LEVIN COHN FERRIS GLOVSKY & POPEO
ONE FINANCIAL CENTER
BOSTON
MA
02111
US
|
Family ID: |
42316869 |
Appl. No.: |
12/319883 |
Filed: |
January 12, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11901925 |
Sep 18, 2007 |
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12319883 |
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PCT/US2007/007875 |
Mar 28, 2007 |
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11901925 |
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60841717 |
Sep 1, 2006 |
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Current U.S.
Class: |
514/5.9 ;
435/184; 514/20.1; 514/8.6; 530/324; 530/350; 536/23.5 |
Current CPC
Class: |
C12Q 2600/118 20130101;
A61P 3/10 20180101; C12Q 1/6883 20130101; C12Q 2600/158
20130101 |
Class at
Publication: |
514/12 ; 530/324;
536/23.5; 435/184; 530/350 |
International
Class: |
A61K 38/17 20060101
A61K038/17; C07K 14/47 20060101 C07K014/47; C12N 15/12 20060101
C12N015/12; C12N 9/99 20060101 C12N009/99; A61P 3/10 20060101
A61P003/10 |
Claims
1. An isolated peptide comprising an amino acid sequence selected
from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID
NO: 3.
2. An isolated nucleic acid sequence encoding an amino acid
sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID
NO: 2, and SEQ ID NO: 3.
3. A pharmaceutical composition for inhibiting one or more kinases,
comprising as an active ingredient the isolated peptide of claim 1,
and a pharmaceutically acceptable carrier or diluent.
4. A protein kinase inhibitor comprising as an active ingredient
the isolated peptide of claim 1, and optionally, a pharmaceutically
acceptable carrier or diluent.
4. A method of inhibiting one or more kinases in a cell, comprising
contacting the cell with the isolated peptide of claim 1.
5. A method of inhibiting one or more kinases in a subject,
comprising administering to the subject the pharmaceutical
composition of claim 3 and measuring the inhibition of the one or
more kinases.
6. A method of treating type 2 Diabetes or a pre-diabetic condition
in a subject, comprising administering to the subject the
pharmaceutical composition of claim 3.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 11/901,925, filed on Sep. 18, 2007, which is a
continuation-in-part of International Application No.
PCT/US2007/007875, filed on Mar. 28, 2007, which claims priority
from U.S. Provisional Application Ser. No. 60/841,717, filed on
Sep. 1, 2006.
[0002] Each of the applications and patents cited in this text, as
well as each document or reference cited in each of the
applications and patents (including during the prosecution of each
issued patent; "application cited documents"), and each of the U.S.
and foreign applications or patents corresponding to and/or
claiming priority from any of these applications and patents, and
each of the documents cited or referenced in each of the
application cited documents, are hereby expressly incorporated
herein by reference. More generally, documents or references are
cited in this text, either in a Reference List before the claims,
or in the text itself; and, each of these documents or references
("herein-cited references"), as well as each document or reference
cited in each of the herein-cited references (including any
manufacturer's specifications, instructions, etc.), is hereby
expressly incorporated herein by reference. Documents incorporated
by reference into this text may be employed in the practice of the
invention.
FIELD OF THE INVENTION
[0003] The present invention relates generally to the
identification of biological markers associated with an increased
risk of developing Diabetes, as well as methods of using such
biological markers in diagnosis and prognosis of Diabetes.
Furthermore, selected biological markers of the present invention
present new targets for therapy and constitute new therapeutics for
treatment or prevention of Diabetes.
BACKGROUND OF THE INVENTION
[0004] Diabetes mellitus comprises a cluster of diseases
distinguished by chronic hyperglycemia that result from the body's
failure to produce and/or use insulin, a hormone produced by
.beta.-cells in the pancreas that plays a vital role in metabolism.
Symptoms include increased thirst and urination, hunger, weight
loss, chronic infections, slow wound healing, fatigue, and blurred
vision. Often, however, symptoms are not severe, not recognized, or
are absent. Diabetes can lead to debilitating and life-threatening
complications including retinopathy leading to blindness, memory
loss, nephropathy that may lead to renal failure, cardiovascular
disease, neuropathy, autonomic dysfunction, and limb amputation.
Several pathogenic processes are involved in the development of
Diabetes, including but not limited to, processes which destroy the
insulin-secreting .beta.-cells with consequent insulin deficiency,
and changes in liver and smooth muscle cells that result in
resistance to insulin uptake. Diabetes can also comprise
abnormalities of carbohydrate, fat, and protein metabolism
attributed to the deficient action of insulin on target tissues
resulting from insulin insensitivity or lack of insulin.
[0005] Type 2 Diabetes is the most common form of Diabetes, which
typically develops as a result of a relative, rather than absolute,
insulin deficiency, in combination with the body's failure to use
insulin properly (also known in the art as "insulin resistance").
Type 2 Diabetes often manifests in persons, including children, who
are overweight; other risk factors include high cholesterol, high
blood pressure, ethnicity, and genetic factors, such as a family
history of Diabetes. The majority of patients with Type 2 Diabetes
are obese, and obesity itself may cause or aggravate insulin
resistance. Apart from adults, an increasing number of children are
also being diagnosed with Type 2 Diabetes. Due to the progressive
nature of the disease, Diabetes complications often develop by the
time these children become adults. A study by the American Diabetes
Association (ADA) involved 51 children that were diagnosed with
Diabetes before the age of 17. By the time these children reached
their early 30s, three had kidney failure, one was blind, and two
died of heart attacks while on dialysis. This study reinforces the
severity of the disease, the serious damage inflicted by Diabetes
complications, and the need for early diagnosis of the disease.
[0006] The incidence of Diabetes has been rapidly escalating to
alarming numbers. Diabetes currently affects approximately 170
million people worldwide with the World Health Organization (WHO)
predicting 300 million diabetics by 2025. The United States alone
has 20.8 million people suffering from Diabetes (approximately 6%
of population and the 6.sup.th most common cause of death). The
annual direct healthcare costs of Diabetes worldwide for people in
the 20-79 age bracket are estimated at $153-286 billion and is
expected to rise to $213-396 billion in 2025.
[0007] Along with the expansion of the diagnosed diabetic
population, the undiagnosed diabetic population has also continued
to increase, primarily because Type 2 Diabetes is often
asymptomatic in its early stages, or the hyperglycemia is often not
severe enough to provoke noticeable symptoms of Diabetes. It is
believed that approximately 33% of the 20.8 million diabetics in
the United States remain undiagnosed. Due to the delay in
diagnosis, Diabetes complications have already advanced and thus,
the future risk of further complication and derailment is severely
increased. To obviate complications and irreversible damage to
multiple organs, Diabetes management guidelines advocate initiation
of therapeutic intervention early in the prognosis of the
disease.
[0008] This modern epidemic requires new tools for early detection
of Type 2 Diabetes, before the disease instigates significant and
irreparable damage. In addition, new treatment paradigms are needed
to halt, delay, or ameliorate the massive deterioration in patient
health, ideally reversing the course of the disease to partial or
complete cure as an alternative or a substitute for current
treatments, which merely address chronic management of disease
symptoms. Diabetic hyperglycemia can be decreased by weight
reduction, increased physical activity, and/or therapeutic
treatment modalities. Several biological mechanisms are associated
with hyperglycemia, such as insulin resistance, insulin secretion,
and gluconeogenesis, and there are several agents available that
act on one or more of these mechanisms, such as but not limited to
metformin, acarbose, and rosiglitazone.
[0009] It is well documented that the pre-diabetic state can be
present for ten or more years before the detection of glycemic
disorders like Diabetes. Treatment of pre-diabetics with
therapeutic agents can postpone or prevent Diabetes; yet few
pre-diabetics are identified and treated. Thus, there remains a
need in the art for methods of identifying and diagnosing these
individuals who are not yet diabetics, but who are at significant
risk of developing Diabetes.
SUMMARY OF THE INVENTION
[0010] The present invention is premised on the discovery that
disease-associated biomarkers can be identified in serum or other
bodily fluids long before overt disease is apparent. The presence
or absence of these biomarkers from the serum footprints of
patients suffering from Type 2 Diabetes precede disruptions in
blood glucose control and can be used as early diagnostic tools,
for which treatment strategies can be devised and administered to
prevent, delay, ameliorate, or reverse irreversible organ damage.
One or several of the disease-associated biomarkers of the present
invention can be used to diagnose subjects suffering from Type 2
Diabetes or related diseases, or advantageously, to diagnose those
subjects who are asymptomatic for Type 2 Diabetes and related
diseases. The biomarkers of the present invention can also be used
for the design of new therapeutics. For instance, a biomarker
absent in a diabetic patient and found in a healthy individual can
constitute a new protective or therapeutic agent which, upon
administration to the patient, may alleviate symptoms or even
reverse the disease.
[0011] The present inventors have found a peptide fragment from the
Cohen diabetic (CD) rat model that is homologous to human SERPINA1
and SERPINA3. In the CD rat model, the sensitive strain (CDs)
develops Diabetes within 30 days when maintained on a high
sucrose/copper-poor diet (HSD), whereas the resistant strain (CDr)
retains normal blood glucose levels. When maintained indefinitely
on regular rodent diet (RD), neither strain develop symptoms of
T2D. The peptide fragment was found in the serum of CDr-RD and
CDr-HSD, but not in the serum of CDs-RD or CDs-HSD (Example 1),
suggesting that the peptide is only found in rats that have not
progressed to a diabetic phenotype. The present inventors have
further studied the human homolog of this peptide and found that
the human homolog exhibits strong kinase inhibitory activity.
Accordingly, in one aspect of the present invention, an isolated
peptide comprising an amino acid sequence selected from the group
consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 is
provided. The present invention also concerns an isolated nucleic
acid sequence encoding an amino acid sequence selected from the
group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO:
3.
[0012] Another aspect of the invention provides a pharmaceutical
composition for inhibiting one or more kinases, comprising as an
active ingredient the isolated peptide of the invention, and a
pharmaceutically acceptable carrier or diluent.
[0013] The present invention also concerns a protein kinase
inhibitor, comprising as an active ingredient the isolated peptide
of the invention and optionally, a pharmaceutically acceptable
carrier or diluent.
[0014] In another aspect, the present invention provides a method
of inhibiting one or more kinases in a cell, comprising contacting
the cell with the isolated peptide of invention. The invention
further concerns a method of inhibiting one or more kinases in a
subject, comprising administering to the subject the pharmaceutical
composition of the invention and measuring the inhibition of one or
more kinases.
[0015] In another aspect, the present invention provides a method
of treating type 2 Diabetes or a pre-diabetic condition in a
subject, comprising administering to the subject the pharmaceutical
composition of the invention.
[0016] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention pertains.
Although methods and materials similar or equivalent to those
described herein can be used in the practice of the present
invention, suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are expressly incorporated by reference in their
entirety. In cases of conflict, the present specification,
including definitions, will control. In addition, materials,
methods, and examples described herein are illustrative only and
are not intended to be limiting.
[0017] Other features and advantages of the invention will be
apparent from and are encompassed by the following detailed
description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] The following Detailed Description, given by way of example,
but not intended to limit the invention to specific embodiments
described, may be understood in conjunction with the accompanying
Figures, incorporated herein by reference, in which:
[0019] FIG. 1 is a graphical comparison of serum samples from
CDr-RD, CDs-RD, CDr-HSD, and CDs-HSD on a SELDI Q10 anion exchange
surface chip. A median peak is present in CDr-RD and CDr-HSD
(marked by an arrow), but not in CDs-RD and CDs-HSD. A protein
fragment from this differentially expressed peak was identified as
the C-terminal fragment of Serpina 3M.
[0020] FIG. 2 is an MS/MS spectrum of the 4.2 kilodalton fragment
identified by SELDI.
[0021] FIG. 3A depicts a BLAST alignment of the 38-amino acid
Serpina 3M (also referred to as "D3") peptide and proteins
identified as having similar sequence identity.
[0022] FIG. 3B shows a BLAST alignment of nucleic acid sequences
encoding the 38-amino acid Serpina 3M peptide and proteins
identified in 3A.
[0023] FIG. 4A is a summary of bioinformatic analysis of the D3
peptide.
[0024] FIG. 4B shows the critical amino acid positions that may
define the mechanism of action of the D3 peptide and its inhibitory
activity.
[0025] FIG. 5 is a photograph of Western blots depicting the
reactivity of the D3-hyperimmune rabbit serum with the 4 kD protein
fragment present in CDr-RD and CDr-HSD rat serum. In the left
photograph, a higher molecular weight doublet (in the range of 49
and 62 kD) also reacted with the hyperimmune sera, indicating that
a parent protein (and a protein complex) is expressed by all
strains under both RD and HSD treatment modalities, while the
derivative of smaller size is differentially expressed only in the
CDr strain. As a negative control, the right photograph shows a
Western blot membrane incubated in the absence of the D3
hyperimmune rabbit serum.
[0026] FIG. 6 depicts a Western blot of proteins identified using
polyclonal anti-D3 antibodies and the relative abundance of the
protein by quantification of band intensity.
[0027] FIG. 7 depicts a Western blot of PNGase-treated (RTF) CDR-RD
pancreatic protein and (RTF) CDR-CD pancreatic amylase with the
monoclonal hybridoma clone MAb-P2.10B8.KA8.
[0028] FIG. 8 shows a Western blot depicting the reactivity of
MAb-P2-4-H5-K-B4 and an SDS-PAGE of CDR-HSD pancreatic proteins for
MALDI/TOF/TOF analysis.
[0029] FIG. 9 is a graph depicting the fold changes in expression
of 48 markers common to models of progression of Diabetes and
models of resistance to Diabetes.
[0030] FIG. 10 is a summary graph of expression of selected markers
measured in pancreatic tissue.
[0031] FIG. 11A depicts a network derived from biomarkers
identified in epididymal fat from a rat model of Diabetes
resistance.
[0032] FIG. 11B depicts a network derived from biomarkers
identified in epididymal fat from a rat model of Diabetes
progression.
[0033] FIG. 11C shows a network that combined the networks depicted
in FIGS. 11A and 11B.
[0034] FIG. 12A depicts a network combining most of the biomarkers
common to models of progression of Diabetes and models of
resistance to Diabetes.
[0035] FIG. 12B shows a simplified version of the network depicted
in FIG. 12A.
[0036] FIG. 12C is a bar graph depicting the top canonical pathways
implicated in the bioinformatics assays of Example 3.
[0037] FIG. 12D is a bar graph depicting the top biological
functions implicated in the bioinformatics assays described in
Example 3.
[0038] FIG. 13 is a chart showing the expression of selected
transcripts from pancreatic and epididymal fat tissue over time
[0039] FIG. 14 is a graph showing onset of diabetes in STZ treated
animals. No significant differences were noted in the time to onset
or rate of disease in D3 treated animals, when compared to
untreated diabetic controls.
[0040] FIG. 15 are graphs showing that treatment with the D3
peptide increases survival in diabetic mice induced with
streptozocin.
[0041] FIG. 16 is a graph depicting that animals administered STZ
alone demonstrate a significant increase in mean blood glucose
levels compared to normal controls over a 36 day period. In
contrast, blood glucose levels in STZ-induced animals also
receiving D3 peptide show a marked reduction.
DETAILED DESCRIPTION OF THE INVENTION
[0042] The present invention relates to, inter alia, the
identification of biomarkers associated with subjects having
Diabetes or a pre-diabetic condition, or who are pre-disposed to
developing Diabetes or a pre-diabetic condition. Accordingly, the
biomarkers and methods of the present invention allow one of skill
in the art to identify, diagnose, or otherwise assess those
subjects who do not exhibit any symptoms of Diabetes or a
pre-diabetic condition, but who nonetheless may be at risk for
developing Diabetes or experiencing symptoms characteristic of a
pre-diabetic condition. The biomarkers can also be used
advantageously to identify subjects having or at risk for
developing complications relating to Type 2 Diabetes. These
biomarkers are also useful for monitoring subjects undergoing
treatments and therapies for Diabetes or pre-diabetic conditions,
and for selecting therapies and treatments that would be effective
in subjects having Diabetes or a pre-diabetic condition, wherein
selection and use of such treatments and therapies slow the
progression of Diabetes or pre-diabetic conditions, or
substantially delay or prevent its onset. The biomarkers of the
present invention can be in the form of a pharmaceutical
composition used to treat subjects having type 2 Diabetes or
related conditions.
[0043] The present inventors have used the Cohen diabetic (CD) rat
as a model comprised of 2 strains that manifest many of the common
features of type 2 diabetes (T2D) in humans. The sensitive strain
(CDs) develops diabetes within 30 days of high sucrose/copper poor
diet (HSD), whereas the resistant strain (CDr) retains normal blood
glucose levels. Neither strain shows any signs of diabetes when
provided regular diet (RD). Thus, incidence of T2D in the CD rat
model results from synergistic effects of genetic susceptibility
and dietary influence.
[0044] Microarray transcriptome profiling revealed a number of
biomarkers related to resistance, predisposition or progression of
the disease. Particularly, upregulation of Gyk, Scd2 and Nr1h3 and
downregulation of Lypla3, Acaa2 and Anxa1 were associated with the
resistance to Diabetes. Additionally, forty-eight transcripts
showing statistically significant opposite expression trends in
resistance or progression of the disease. A decrease in the levels
of transcripts involved in angiogenesis and endothelial regulation,
such as those encoding, for example, Angiomotin, Folate Receptor 1
and Occludin, was associated with progression of type 2 Diabetes.
Similarly, a decrease of expression of Cyp4f4 that mediates
leukotriene B(4) metabolism was seen in Diabetes. On the contrary,
increased levels of the same markers were associated with
resistance to disease. Another interesting finding is a Diabetes
associated increase of adipocytic expression of Sox17, a pancreatic
progenitor marker. Changes in level of transcripts were observed as
early as 10 days after exposure to HSD and became more pronounced
after 30 days of the diet. The present invention thus seeks to
define predisposition to the development of the type 2 Diabetes as
well as be explored as potential drug targets.
[0045] In particular, the present inventors have determined that
one biomarker in particular, a peptide fragment from the Cohen
diabetic (CD) rat model, is homologous to human SERPINA1 and
SERPINA3. In the CD rat model, the sensitive strain (CDs) develops
Diabetes within 30 days when maintained on a high
sucrose/copper-poor diet (HSD), whereas the resistant strain (CDr)
retains normal blood glucose levels. When maintained indefinitely
on regular rodent diet (RD), neither strain develop symptoms of
T2D. The peptide fragment was found in the serum of CDr-RD and
CDr-HSD, but not in the serum of CDs-RD or CDs-HSD (Example 1),
suggesting that the peptide is only found in rats that have not
progressed to a diabetic phenotype. The present inventors have
further studied this peptide and found that the peptide exhibits
strong kinase inhibitory activity. Thus, the present invention also
concerns biomarkers that can act as peptide inhibitors of kinases
involved in type 2 Diabetes or pre-diabetic conditions that can be
used, for example, in pharmaceutical compositions to treat type 2
Diabetes, pre-diabetic conditions, or related conditions, such as
complications related to type 2 Diabetes.
[0046] As used herein, "a," an" and "the" include singular and
plural referents unless the context clearly dictates otherwise.
Thus, for example, reference to "an active agent" or "a
pharmacologically active agent" includes a single active agent as
well as two or more different active agents in combination,
reference to "a carrier" includes mixtures of two or more carriers
as well as a single carrier, and the like.
[0047] The term "antibody" is meant to include polyclonal
antibodies, monoclonal antibodies (mAbs), chimeric antibodies,
anti-idiotypic (anti-Id) antibodies to antibodies that can be
labeled in soluble or bound form, as well as fragments, regions,
conjugates, or derivatives thereof, provided by any known
technique, such as, but not limited to, enzymatic cleavage, peptide
synthesis or recombinant techniques.
[0048] As used herein, the term "antigen binding region" refers to
that portion of an antibody molecule which contains the amino acid
residues that bind and interact with an antigen and confer on the
antibody its specificity and affinity for the antigen. The antibody
region includes the "framework" amino acid residues necessary to
maintain the proper conformation of the antigen-binding
residues.
[0049] An "antigen" is a molecule or a portion of a molecule
capable of being bound by an antibody which is additionally capable
of inducing an animal to produce antibody capable of binding to an
epitope of that antigen. An antigen can have one or more than one
epitope. The specific reaction referred to above is meant to
indicate that the antigen will react, in a highly selective manner,
with its corresponding antibody and not with the multitude of other
antibodies which can be evoked by other antigens. Preferred
antigens that bind antibodies, fragments and regions of antibodies
of the present invention include at least one, preferably two,
three, four, five, six, seven, eight, nine, ten or more amino acid
residues of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, but can
also bind to any one or more biomarkers of the invention, or
metabolites thereof, such as those set forth in Table 1 herein.
[0050] The term "biomarker" in the context of the present invention
encompasses, without limitation, proteins, peptides (including the
peptide inhibitors disclosed herein), nucleic acids, polymorphisms
of proteins and nucleic acids, splice variants, fragments of
proteins or nucleic acids, elements, metabolites, and other
analytes. Biomarkers can also include mutated proteins or mutated
nucleic acids. The biomarkers disclosed herein are used
interchangeably with the term "T2DBMARKER".
[0051] "Complications related to type 2 Diabetes" or "complications
related to a pre-diabetic condition" can include, without
limitation, diabetic retinopathy, diabetic nephropathy, blindness,
memory loss, renal failure, cardiovascular disease (including
coronary artery disease, peripheral artery disease, cerebrovascular
disease, atherosclerosis, and hypertension), neuropathy, autonomic
dysfunction, hyperglycemic hyperosmolar coma, or combinations
thereof.
[0052] "Diabetes Mellitus" in the context of the present invention
encompasses Type 1 Diabetes, both autoimmune and idiopathic and
Type 2 Diabetes (together, "Diabetes"). The World Health
Organization defines the diagnostic value of fasting plasma glucose
concentration to 7.0 mmol/l (126 mg/dl) and above for Diabetes
Mellitus (whole blood 6.1 mmol/l or 110 mg/dl), or 2-hour glucose
level.gtoreq.11.1 mmol/L (.gtoreq.2200 mg/dL). Other values
suggestive of or indicating high risk for Diabetes Mellitus include
elevated arterial pressure.gtoreq.140/90 mm Hg; elevated plasma
triglycerides (.gtoreq.1.7 mmol/L; 150 mg/dL) and/or low
HDL-cholesterol (<0.9 mmol/L, 35 mg/dl for men; <1.0 mmol/L,
39 mg/dL women); central obesity (males:waist to hip ratio>0.90;
females:waist to hip ratio>0.85) and/or body mass index
exceeding 30 kg/m.sup.2; microalbuminuria, where the urinary
albumin excretion rate.gtoreq.20 .mu.g/min or albumin:creatinine
ratio.gtoreq.30 mg/g).
[0053] The term "epitope" is meant to refer to that portion of any
molecule capable of being recognized by and bound by an antibody at
one or more of the Ab's antigen binding regions. Epitopes usually
consist of chemically active surface groupings of molecules such as
amino acids or sugar side chains and have specific three
dimensional structural characteristics as well as specific charge
characteristics. An epitope can comprise the antibody binding
region of any one or more of T2DBMARKERS disclosed herein, or a
metabolite thereof. An epitope can also comprise at least one,
preferably two, three, four, five, six, seven, eight, nine, ten or
more amino acid residues of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID
NO: 3. The amino acid residues of the epitope that are recognized
by the isolated antibodies of the invention need not be
contiguous.
[0054] "Impaired glucose tolerance" (IGT) is defined as having a
blood glucose level that is higher than normal, but not high enough
to be classified as Diabetes Mellitus. A subject with IGT will have
two-hour glucose levels of 140 to 199 mg/dL (7.8 to 11.0 mmol) on
the 75 g oral glucose tolerance test. These glucose levels are
above normal but below the level that is diagnostic for Diabetes.
Subjects with impaired glucose tolerance or impaired fasting
glucose have a significant risk of developing Diabetes and thus are
an important target group for primary prevention.
[0055] "Insulin resistance" refers to a condition in which the
cells of the body become resistant to the effects of insulin, that
is, the normal response to a given amount of insulin is reduced. As
a result, higher levels of insulin are needed in order for insulin
to exert its effects.
[0056] "Normal glucose levels" is used interchangeably with the
term "normoglycemic" and refers to a fasting venous plasma glucose
concentration of less than 6.1 mmol/L (110 mg/dL). Although this
amount is arbitrary, such values have been observed in subjects
with proven normal glucose tolerance, although some may have IGT as
measured by oral glucose tolerance test (OGTT). A baseline value,
index value, or reference value in the context of the present
invention and defined herein can comprise, for example, "normal
glucose levels."
[0057] A "pre-diabetic condition" refers to a metabolic state that
is intermediate between normal glucose homeostasis, metabolism, and
states seen in frank Diabetes Mellitus. Pre-diabetic conditions
include, without limitation, Metabolic Syndrome ("Syndrome X"),
Impaired Glucose Tolerance (IGT), and Impaired Fasting Glycemia
(IFG). IGT refers to post-prandial abnormalities of glucose
regulation, while IFG refers to abnormalities that are measured in
a fasting state. The World Health Organization defines values for
IFG as a fasting plasma glucose concentration of 6.1 mmol/L (100
mg/dL) or greater (whole blood 5.6 mmol/L; 100 mg/dL), but less
than 7.0 mmol/L (126 mg/dL)(whole blood 6.1 mmol/L; 110 mg/dL).
Metabolic Syndrome according to National Cholesterol Education
Program (NCEP) criteria are defined as having at least three of the
following: blood pressure.gtoreq.130/85 mm Hg; fasting plasma
glucose.gtoreq.6.1 mmol/L; waist circumference>102 cm (men) or
>88 cm (women); triglycerides.gtoreq.1.7 mmol/L; and HDL
cholesterol<1.0 mmol/L (men) or 1.3 mmol/L (women).
[0058] A "sample" in the context of the present invention is a
biological sample isolated from a subject and can include, for
example, serum, blood plasma, blood cells, endothelial cells,
tissue biopsies, lymphatic fluid, pancreatic juice, ascites fluid,
interstitital fluid (also known as "extracellular fluid" and
encompasses the fluid found in spaces between cells, including,
inter alia, gingival crevicular fluid), bone marrow, sputum,
saliva, tears, or urine.
[0059] A "subject" in the context of the present invention is
preferably a mammal. The mammal can be a human, non-human primate,
mouse, rat, dog, cat, horse, or cow, but are not limited to these
examples. Mammals other than humans can be advantageously used as
subjects that represent animal models of type 2 Diabetes Mellitus
or pre-diabetic conditions. A subject can be male or female. A
subject can be one who has been previously diagnosed with or
identified as suffering from or having type 2 Diabetes, one or more
complications related to type 2 Diabetes, or a pre-diabetic
condition, and optionally, but need not have already undergone
treatment for the type 2 Diabetes, the one or more complications
related to type 2 Diabetes, or the pre-diabetic condition. A
subject can also be one who is not suffering from type 2 Diabetes
or a pre-diabetic condition. A subject can also be one who has been
diagnosed with or identified as suffering from type 2 Diabetes, one
or more complications related to type 2 Diabetes, or a pre-diabetic
condition, but who show improvements in known Diabetes risk factors
as a result of receiving one or more treatments for type 2
Diabetes, one or more complications related to type 2 Diabetes, or
the pre-diabetic condition. Alternatively, a subject can also be
one who has not been previously diagnosed as having Diabetes, one
or more complications related to type 2 Diabetes, or a pre-diabetic
condition. For example, a subject can be one who exhibits one or
more risk factors for Diabetes, complications related to Diabetes,
or a pre-diabetic condition, or a subject who does not exhibit
Diabetes risk factors, or a subject who is asymptomatic for
Diabetes, one or more Diabetes-related complications, or a
pre-diabetic condition. A subject can also be one who is suffering
from or at risk of developing Diabetes or a pre-diabetic condition.
A subject can also be one who has been diagnosed with or identified
as having one or more complications related to type 2 Diabetes or a
pre-diabetic condition as defined herein, or alternatively, a
subject can be one who has not been previously diagnosed with or
identified as having one or more complications related to type 2
Diabetes or a pre-diabetic condition.
Biomarkers of the Invention
[0060] Proteins, peptides, nucleic acids, polymorphisms, and
metabolites whose levels are changed in subjects who have Diabetes
or a pre-diabetic condition, or are predisposed to developing
Diabetes or a pre-diabetic condition are summarized in Table 1 and
are collectively referred to herein as, inter alia,
"Diabetes-associated proteins", "T2DBMARKER polypeptides", or
"T2DBMARKER proteins". The corresponding nucleic acids encoding the
polypeptides are referred to as "Diabetes-associated nucleic
acids", "Diabetes-associated genes", "T2DBMARKER nucleic acids", or
"T2DBMARKER genes". Unless indicated otherwise, "T2DBMARKER",
"Diabetes-associated proteins", "Diabetes-associated nucleic acids"
are meant to refer to any of the sequences disclosed herein. The
corresponding metabolites of the T2DBMARKER proteins or nucleic
acids can also be measured, herein referred to as "T2DBMARKER
metabolites". Calculated indices created from mathematically
combining measurements of one or more, preferably two or more of
the aforementioned classes of T2DBMARKERS are referred to as
"T2DBMARKER indices". Proteins, nucleic acids, polymorphisms,
mutated proteins and mutated nucleic acids, metabolites, and other
analytes are, as well as common physiological measurements and
indices constructed from any of the preceding entities, are
included in the broad category of "T2DBMARKERS".
[0061] Five hundred and forty-eight (548) biomarkers have been
identified as having altered or modified presence or concentration
levels in subjects who have Diabetes, or who exhibit symptoms
characteristic of a pre-diabetic condition, such as those subjects
who are insulin resistant, have altered beta cell function or are
at risk of developing Diabetes based upon known clinical parameters
or risk factors, such as family history of Diabetes, low activity
level, poor diet, excess body weight (especially around the waist),
age greater than 45 years, high blood pressure, high levels of
triglycerides, HDL cholesterol of less than 35, previously
identified impaired glucose tolerance, previous Diabetes during
pregnancy ("gestational Diabetes Mellitus") or giving birth to a
baby weighing more than nine pounds, and ethnicity.
[0062] Table 1 comprises the five hundred and forty-eight (548)
T2DBMARKERS of the present invention. One skilled in the art will
recognize that the T2DBMARKERS presented herein encompasses all
forms and variants, including but not limited to, polymorphisms,
isoforms, mutants, derivatives, precursors including nucleic acids,
receptors (including soluble and transmembrane receptors), ligands,
and post-translationally modified variants, as well as any
multi-unit nucleic acid, protein, and glycoprotein structures
comprised of any of the T2DBMARKERS as constituent subunits of the
fully assembled structure.
TABLE-US-00001 TABLE 1 T2DBMARKERS T2DBMARKER Common Name
Alternative Name 1 Serpina 3M C-terminal fragment of a predicted
protein, similar to serine protease inhibitor 2.4 2 Spin 2a 3
Fetuin beta Fetub; Fetuin .beta.; Fetuin B 4 Apolipoprotein C-III
Apoc3 precursor 5 Predicted protein, similar to Apoc2, predicted
Apolipoprotein C2 6 Alpha-2-HS-glycoprotein
.alpha.-2-HS-glycoprotein; Ahsg; Fetuin .alpha.; Fetuin A; Aa2-066
7 T-kininogen II precursor 8 Alpha-1-macroglobulin
.alpha.-1-macroglobulin; A2MG; Pzp; pregnancy-zone protein 9 Serpin
C1 Serine/cysteine proteinase inhibitor, clade C, member 1
(predicted) 10 Coagulation factor 2 F2 11 Inter-alpha-inhibitor H4
ITIH4 heavy chain 12 Vitamin D binding protein Gc; VTDB prepeptide
13 Low-molecular weight T- Kininogen; LMW T-kininogen I precursor;
major kininogen I precursor acute phase alpha-1 protein precursor
14 Apolipoprotein A-1 Preapolipoprotein A-1; ApoA1 15 Predicted
protein, similar to Apoc2, precursor apolipoprotein C-II precursor
16 Thrombin Prothrombin precursor; THRB 17 Apolipoprotein E ApoE 18
Liver regeneration-related Tf protein LRRG03 19 Apolipoprotein A-IV
ApoA4 20 Alpha-1-inhibitor 3 LOC297568 precursor 21 XP_579384 22
Histidine-rich glycoprotein Hrg 23 XP_579477 24 Complement
component C9 C9 precursor 25 Apolipoprotein H ApoH 26 B-factor,
properdin Cfb 27 Hemopexin Hpx 28 Calnexin Ca(2+)-binding
phosphoprotein p90 29 Reg3a Rn.11222; regenerating islet-derived 3
alpha 30 LOC680945 Rn.1414; similar to stromal cell-derived factor
2-like 1 31 Pap Rn.9727; pancreatitis-associated protein 32 Ptf1a
Rn.10536; Pancreas specific transcription factor, 1a 33 Mat1a
Rn.10418; methionine adenosyltransferase I, alpha 34 Nupr1
Rn.11182; nuclear protein 1 35 Rn.128013 36 Chac1 (predicted)
Rn.23367; ChaC; cation transport regulator-like 1 37 Slc7a3
Rn.9804; solute carrier family 7 (cationic amino acid transporter,
y+ system), member 3 38 LOC312273 Rn.13006; trypsin V-A 39 Rn.47821
40 Ptger3 Rn.10361; prostaglandin E receptor 3 (subtype EP3 41
RGD1562451 Rn.199400; similar to Pabpc4 predicted protein 42
RGD1566242 Rn.24858; similar to RIKEN cDNA 1500009M05 43 Cyp2d26
Rn.91355; Cytochrome P450, family 2, subfamily d, polypeptide 26 44
Rn.17900 Similar to aldehyde dehydrogenase 1 family, member L2 45
LOC286960 Rn.10387; preprotrypsinogen IV 46 Gls2 Rn.10202;
glutaminase 2 (liver, mitochondrial) 47 Nme2 Rn.927; expressed in
non-metastatic cells 2 48 Rn.165714 49 P2rx1 Rn.91176; purinergic
receptor PX2, ligand-gated ion channel, 1 50 Pdk4 Rn.30070;
pyruvate dehydrogenase kinase, isoenzyme 4 51 Amy1 Rn.116361;
amylase 1, salivary 52 Cbs Rn.87853; cystathionine beta synthase 53
Mte1 Rn.37524; mitochondrial acyl-CoA thioesterase 1 54 Spink1
Rn.9767; serine protease inhibitor, Kazal type 1 55 Gatm Rn.17661;
glycine amidinetransferase (L- arginine:glycine amidinotransferase)
56 Tmed6_predicted Rn.19837; transmembrane emp24 protein transport
domain containing 6 57 Tff2 Rn.34367; trefoil factor 2 (spasmolytic
protein 1) 58 Hsd17b13 Rn.25104; hydroxysteroid (17-beta)
dehydrogenase 13 59 Rn.11766 Similar to LRRGT00012 60 Gnmt
Rn.11142; glycine N-methyltransferase 61 Pah Rn.1652; phenylalanine
hydroxylase 62 Serpini2 Rn.54500; serine/cysteine proteinase
inhibitor, clade I, member 2 63 RGD1309615 Rn.167687 64 LOC691307
Rn.79735; similar to leucine rich repeat containing 39 isoform 2 65
Eprs Rn.21240; glutamyl-prolyl-tRNA synthetase 66 Pck2_predicted
Rn.35508; phosphoenolpyruvate carboxykinase 2 (mitochondrial) 67
Chd2_predicted Rn.162437; chromodomain helicase DNA binding protein
2 68 Rn.53085 69 Rn.12530 70 NIPK Rn.22325; tribbles homolog; cDNA
clone RPCAG66 3' end, mRNA sequence 71 Slc30a2 Rn.11135; solute
carrier family 30 (zinc transporter), member 2 72 Serpina10
Rn.10502; serine/cysteine peptidase inhibitor, clade A, member 10
73 Cfi Rn.7424; complement factor I 74 Cckar Rn.10184;
cholecystokinin A receptor 75 LOC689755 Rn.151728; LOC689755 76
Bhlhb8 Rn.9897; basic helix-loop-helix domain containing class B, 8
77 Anpep Rn.11132; alanyl (membrane) aminopeptidase) 78 Asns
Rn.11172; asparagine synthetase 79 Slc7a5 Rn.32261; solute carrier
family 7 (cationic amino acid transporter, y+ system), member 5 80
Usp43_predicted Rn.12678; ubiquitin specific protease 43 81 Csnk1a1
Rn.23810; casein kinase 1, alpha 1 82 Cml2 Rn.160578; camello-like
2 83 Pabpc4 Rn.199602 84 Gjb2 Rn.198991; gap junction membrane
channel protein beta 2 85 Ngfg Rn.11331; nerve growth factor, gamma
86 Clca2_predicted Rn.48629 87 RGD1565381 Rn.16083; similar to
RIKEN cDNA 181003M07 88 Qscn6 Rn.44920; quiescin Q6 89
Cldn10_predicted Rn.99994; claudin 10 90 Spink3 Rn.144683; serine
protease inhibitor, Kazal type 3 91 LOC498174 Rn.163210; similar to
NipSnap2 protein (glioblastoma amplified sequence) 92 Rn.140163
Similar to methionine-tRNA synthetase 93 Cyr61 Rn.22129; cysteine
rich protein 61 94 RGD1307736 Rn.162140; Similar to KIAA0152 95
Ddit3 Rn.11183; DNA damage inducible transcript 3 96 Reg1 Rn.11332;
regenerating islet derived 1 97 Eif4b Rn.95954; eukaryotic
translation initiation factor 4B 98 Rnase4 Rn.1742; ribonuclease,
RNase A family 4 99 Cebpg Rn.10332; CCAAT/enhancer binding protein
(C/EBP), gamma 100 siat7D Rn.195322; alpha-2,6-sialyltransferase
ST6GalNAc IV 101 Herpud1 Rn.4028; homocysteine-inducible,
ubiquitin-like domain member 1 102 Unknown rat cDNA 103 Gcat
Rn.43940; glycine C-acetyltransferase (2-amino-3-
ketobutyrate-coenzyme A ligase) 104 RGD1562860 Rn.75246; similar to
RIKEN cDNA 2310045A20 105 pre-mtHSP70 Rn.7535; 70 kD heat shock
protein precursor; Hspa9a_predicted; heat shock 70 kD protein 9A
106 Dbt Rn.198610; dihydrolipoamide branched chain transacylase E2
107 Bspry Rn.53996; B-box and SPRY domain containing 108 Fut1
Rn.11382; fucosyltransferase 1 109 Rpl3 Rn.107726; ribosomal
protein L3 110 Rn.22481 Similar to NP_083520.1 acylphosphatase 2,
muscle type 111 Vldlr Rn.9975; very low density lipoprotein
receptor 112 RGD1311937 Rn.33652; similar to MGC17299 113
RGD1563144 Rn.14702; Similar to EMeg32 protein 114 Rn.43268 115
Ddah1 Rn.7398; dimethylarginine dimethylaminohydrolase 1 116 RAMP4
Rn.2119; ribosome associated membrane protein 4 117 Rn.169405 118
Ccbe1_predicted Rn.199045; collagen and calcium binding EGF domains
1 119 Dnajc3 Rn.162234; DnaJ (Hsp40) homolog, subfamily C, member 3
120 Mtac2d1 Rn.43919; membrane targeting (tandem)C2 domain
containing 1 121 RGD1563461 Rn.199308 122 Gimap4 Rn.198155; GTPase,
IMAP family member 4 123 Klf2_predicted Rn.92653; Kruppel-like
factor 2 (lung) 124 RGD1309561 Rn.102005; similar to FLH31951 125
NAP22 Rn.163581 126 Sfrs3_predicted Rn.9002; splicing factor,
arginine/serine-rich 3 (SRp30) 127 Rn.6731 128 Cd53 Rn.31988; CD53
antigen 129 RGD1561419 Rn.131539; similar to RIKEN cDNA 6030405P05
gene; ARHGAP30; Hs.389374; Rho GTPase activating protein 130 Il2rg
Rn.14508; interleukin 2 receptor, gamma 131 LOC361346 Rn.31250;
similar to chromosome 18 open reading frame 54 132 Plac8_predicted
Rn.2649; placenta-specific 8 133 LOC498335 Rn.6917; similar to
small inducible cytokine B13 precursor (CXCL13) 134 Igfbp3
Rn.26369; insulin-like growth factor binding protein 3 135 Ptprc
Rn.90166; Hs.192039; protein tyrosine phosphatase, receptor type C;
CD45 136 RT1-Aw2 Rn.40130; RT1 class Ib, locus Aw2 137 Rac2
Rn.2863; RAS-related C3 botulinum substrate 2 138 Rn.9461 139 Fos
Rn.103750; FBJ murine osteosarcoma viral oncogene homolog 140 Sgne1
Rn.6173; secretory granule neuroendocrine protein 1 141 Fcgr2b
Rn.33323; Fc receptor, IgG, low affinity IIb 142 Slfn8 Rn.137139;
Schlafen 8 143 Rab8b Rn.10995; RAB8B, member RAS oncogene family
144 Rn.4287 145 RGD1306939 Rn.95357; similar to mKIAA0386 protein
146 Tnfrsf26_predicted Rn.162508; tumor necrosis factor receptor
superfamily, member 26 147 Ythdf2_predicted Rn.21737; YTH domain
family 2 148 RGD1359202 Rn.10956; similar to immunoglobulin heavy
chain 6 (Igh-6); IGHG1 in humans; immunoglobulin heavy constant
gamma 1 149 RGD1562855 Rn.117926; similar to Ig kappa chain 150
Igha_mapped Rn.109625; immunoglobulin heavy chain (alpha
polypeptide) (mapped) 151 Ccl21b Rn.39658; chemokine (C-C motif)
ligand 21b (serine) 152 IGHM Rn.201760; Hs.510635; IGHM;
immunoglobulin heavy constant mu 153 LCK Rn.22791; Hs.470627;
lymphocyte protein tyrosine kinase 154 ARHGD1B Rn.15842; Hs.
507877; Rho GDP dissociation inhibitor (CDI) beta 155 CD38
Rn.11414; Hs.479214; CD38 antigen 156 S100B Rn.8937; Hs.422181;
S100 calcium binding protein B, beta polypeptide 157 RGD1306952
Rn.64439; Similar to Ab2-225 158 Dmrt2 Rn.11448; Doublesex and
mab-3 related transcription factor 2 (predicted) 159 AA819893
Rn.148042; unknown cDNA 160 Gpr176 Rn.44656; G-protein coupled
receptor 176 161 Tmem45b Rn.42073; transmembrane protein 45b 162
Nfkbil1 Rn.38632; nuclear factor of kappa light polypeptide gene
enhancer in B-cells inhibitor-like 1 163 Dctn2 Rn.101923; Dynactin
2 164 Itpkc Rn.85907; Inositol 1,4,5-trisphosphate 3-kinase C 165
BM389613 Rn.171826; unknown cDNA 166 Prodh2 Rn.4247; proline
dehydrogenase (oxidase) 2 167 BF288777 Rn.28947; unknown cDNA 168
Abi3 Rn.95169; ABI gene family, member 3 169 AW531966 Rn.8606;
unknown cDNA 170 RGD1560732 Rn.100399; Similar to LIM and senescent
cell antigen-like domains 1 (predicted) 171 Oxsr1 Rn.21097;
oxidative-stress responsive 1 (predicted) 172 MGC114531 Rn.39247;
unknown cDNA 173 BF418465 Rn.123735; unknown cDNA 174 LOC690911
Rn.25022; similar to Msx2-interacting protein (SPEN homolog) 175
Pex6 Rn.10675; Peroxisomal biogenesis factor 6 176 RGD1311424
Rn.57800; similar to hypothetical protein FLJ38348 (predicted) 177
AI013238 Rn.135595; unknown cDNA 178 BI288719 Rn.45106; unknown
cDNA 179 Evp1 Rn.19832; envoplakin (predicted)
180 SERPINE2 Rn.2271; Hs.38449; serine (or cysteine) proteinase
inhibitor clade E member 2 181 C20orf160 Rn.6807; Hs.382157;
C20orf160 predicted; cystein type endopeptidase 182 AI072137
Rn.33396; Transcribed locus 183 LOC338328 Rn.7294; Hs.426410; high
density lipoprotein binding protein; RGD1564237_predicted 184 PTPRR
Rn.6277; Hs.506076; protein tyrosine phosphatase receptor type R
185 LYPLA3 Rn.93631; Hs.632199; Lysophospholipase 3 186 CYYR1
Rn.1528; Hs.37445; cysteine-tyrosine-rich 1 membrane associated
protein 187 SOX17 Rn.7884; Hs.98367; SRY-box gene 17 188 LY6H
Rn.40119 189 SEMA3G Rn.32183; HS.59729; Semaphorin 3G 190 C1QTNF1
Rn.53880; Hs.201398; C1q and tumor necrosis factor related protein
1 191 ADCY4 Rn.1904; Hs.443428; adenylate cyclase 4 192 RBP7
Rn.13092; Hs.422688; retinol binding protein 7;
RGD1562168_predicted 193 ADRB3 Rn.10100; Hs.2549; adrenergic
receptor beta-3 194 NR1H3 Rn.11209; Hs.438863; nuclear receptor
subfamily, group H, member 3 195 TMEFF1 Rn.162809; Hs.657066;
transmembrane protein with EGF-like and two follistatin-like
domains 1 196 TIMP-4 Rn.155651; Hs.591665; Tissue inhibitor of
metalloproteinase 4 197 CYP4F8 (human) Rn.10170; Hs.268554;
cytochrome P450, family 4, subfamily F, polypeptide 8 198 FOLR1
Rn.6912; Hs.73769; folate receptor 1 199 SCD2 Rn.83595; Hs.558396;
stearoyl-CoA desaturase 2 200 KIAA2022 Rn.62924; Hs.124128; DNA
polymerase activity 201 GK Rn.44654; Hs.1466; glycerol kinase; Gyk
202 OCLN Rn.31429; Hs.592605; occluding 203 SPINT2 Rn.3857;
Hs.31439; serine peptidase inhibitor, Kunitz type, 2 204 RBM24
Rn.164640; Hs.519904; RNA binding motif protein 24 205 SLC25A13
Rn.14686; Hs.489190; solute carrier family 25, member 13 (citrin)
206 TPMT Rn.112598; Hs.444319; thiopurine S- methyltransferase 207
KRT18 Rn.103924; Hs.406013; keratin 18; keratin complex 1, acidic,
gene 18; Krt1-18 208 Unknown Rn.153497 209 C2orf40 Rn.16593;
Hs.43125; chromosome 2 open reading frame 40 210 LOC440335
Rn.137175; Hs.390599; hypothetical gene supported by BC022385;
RGD1563547; RGE1563547 (predicted) 211 BEXL1 Rn.9287; Hs.184736;
brain expressed X-linked-like 1; BI289546; brain expressed X-linked
4 212 CYB561 Rn.14673; Hs.355264; cytochrome b-561 213 AMOT
Rn.149241; Hs.528051; angiomotin 214 SQLE Rn.33239; Hs.71465;
squalene epoxidase 215 ANKRD6 Rn.45844; Hs.656539; ankyrin repeat
domain 6 216 CCDC8 Rn.171055; Hs.97876; coiled-coil domain
containing 8 217 KRT8 Rn.11083; Hs.533782; keratin 8 218 WWC1 (Mus
musculus) Rn.101912; Hs.484047; WW and C2 domain containing 1;
RGD1308329; similar to KIAA0869 protein (predicted) 219 PFKP
Rn.2278; Hs.26010; phosphofructokinase 220 PEBP1 Rn.29745;
Hs.433863; phosphatidylethanolamine binding protein 1 221 SLC7A1
Rn.9439; Hs.14846; solute carrier family 7 (cationic amino acid
transport, y+ system), member 1 222 GSTM1 Rn.625; Hs.301961;
glutathione S-transferase M1; glutathione metabolism, mu 1 223 CCL5
Rn.8019; Hs.514821; chemokine (C-C motif) ligand 5 224 STEAP1
Rn.51773; Hs.61635; six transmembrane epithelial antigen of the
prostate 1 225 IAH1 Rn.8209; HS.656852; isoamyl acetate-hydrolyzing
esterase 1 homolog (S. cerevisiae) 226 GNA14 Rn.35127; Hs.657795;
guanine nucleotide binding protein (G protein), alpha 14 227 TMEM64
Rn.164935; Hs.567759; transmembrane protein 64 228 CCL11 Rn.10632;
Hs.54460; chemokine (C-C motif) ligand 11 229 CNN1 Rn.31788;
Hs.465929; Calponin 1 230 GGH Rn.10260; Hs.78619; gamma-glutamyl
hydrolase 231 TPM3 Rn.17580; Hs.645521; tropomyosin 3 232 PCDH7
Rn.25383; Hs.570785; protocadherin 7 233 FHL2 Rn.3849; Hs.443687;
Four and a half LIM domains 2 234 COL11A1 Rn.260; Hs.523446;
Collagen, type XI, alpha 1 235 EMB Rn.16221; Hs.645309; Embigin
homolog (mouse) 236 ISG15 Rn.198318; Hs.458485; ISG15
ubiquitin-like modifier 237 CRYAB Rn.98208; Hs.408767; crystalline,
alpha B 238 ACADSB Rn.44423; Hs.81934; Acyl-Coenzyme A
dehydrogenase 239 Unknown Rn.7699; Rn.7699; IMAGE clone BC086433
240 ABCA1 Rn.3724; Hs.429294; ATP-binding cassette, subfamily A
(ABC1), member 1 241 ACSM3 Rn.88644; Hs.653192; Acyl-CoA synthetase
medium-chain family member 3 242 ACTA2 Rn.195319; Hs.500483; Actin,
alpha 2, smooth muscle, aorta 243 RAMP3 Rn.48672; Hs.25691;
receptor (G-protein coupled; calcitonin) activity modifying protein
3 244 DDEF1 Rn.63466; Hs.655552; development and differentiation
enhancing factor 1 245 NIPSNAP3A Rn.8287; Hs.591897; Nipsnap
homolog 3A (C. elegans) 246 Unknown Rn.9546 247 GPR64 Rn.57243;
Hs.146978; G protein-coupled receptor 64 248 SGCB Rn.98258;
Hs.428953; sarcoglycan, beta; AI413058; 43 kDa
dystrophin-associated glycoprotein (43DAG) 249 BM389408 Rn.146540;
Transcribed locus 250 RGD1310037_predicted Rn.199679; Transcribed
locus 251 CALML3 Rn.105124; Hs.239600; calmodulin-like 3 252
LOC645638 Rn.41321; Hs.463652; similar to WDNM1-like protein 253
Upk3b_predicted Rn.6638; transcribed locus 254 SCEL Rn.34468;
Hs.534699; sciellin 255 BNC1 Rn.26595; Hs.459153; Basonuclin 1;
BF411725 256 FGL2 Rn.64635; Hs.520989; fibrinogen-like 2 257 UPK1B
Rn.9134; Hs.271580; uroplakin 1B 258 CTDSPL Rn.37030; Hs.475963;
CTD (carboxy-terminal domain, RNA polymerase II, polypeptide A)
small phosphatase-like 259 PIK3R1 Rn.163585; Hs.132225;
phosphoinositide-3-kinase, regulatory subunit (p85 alpha) 260 POLA2
Rn.153998; Hs.201897; polymerase (DNA directed), alpha 2 (70 kD
subunit); AI175779 261 SPTBN1 Rn.93208; Hs.659362; spectrin, beta,
non- erythrocytic 1 262 RTEL1 Rn.98315; Hs.434878; regulator of
telomere elongation helicase 1 263 MSLN Rn.18607; Hs.08488;
mesothelin 264 ARVCF Rn.220; Hs.655877; armadillo repeat gene
deleted in velocardiofacial syndrome; Comt; catechol-O-
methyltransferase 265 ALB Rn.9174; Hs.418167; albumin 266 SLC6A4
Rn.1663; Hs.591192; solute carrier family 6 (neurotransmitter
transporter, serotonin), member 4 267 Unknown Rn.26537 268 BI302615
Rn.44072; Transcribed locus 269 Unknown Rn.199355 270 MRPL4
Rn.13113 271 GPR109A Rn.79620; Hs.524812; G protein-coupled
receptor 109A; BI296811 272 THBS1 Rn.185771; Hs.164226;
thrombospondin 1 273 ANGPTL4 Rn.119611; Hs.9613; angiopoietin-like
4 274 THBS2 Rn.165619; Hs.371147; thrombospondin 2 275 PCK1
Rn.104376; Hs.1872; phosphoenolpyruvate carboxykinase 1 276 UCP3
Rn.9902; Hs.101337; uncoupling protein 3 277 CYFIP2 Rn.44008;
Hs.519702; cytoplasmic FMR1 interacting protein 2 278 LOC646851
Rn.199989; hypothetical protein 279 DSP Rn.54711; Hs.519873;
desmoplakin 280 RNF128 Rn.7002; Hs.496542; ring finger protein 128
281 WDR78 Rn.22852; Hs.49421; WD repeat domain 78 282 SLC16A12
Rn.166976; Hs.530338; solute carrier family 16, member 12 283
GRAMD1B Rn.18035; Hs.144725; GRAM domain containing 1B 284 HPN
Rn.11139; Hs.182385; hepsin (transmembrane protease, serine 1) 285
RRAGD Rn.66516; Hs.485938; Ras-related GTP binding D 286 MDF1
Rn.43395; Hs.520119; MyoD family inhibitor 287 LTB4DH Rn.10656;
Hs.584864; leukotriene B4 12- hydroxydehydrogenase 288 CELSR2
Rn.2912; Hs.57652; cadherin, EGF LAG seven-pass G-type receptor 2
289 LRP4 Rn.21381; Hs.4930; low density lipoprotein
receptor-related protein 4 290 TPCN2 Rn.138237; Hs.131851; two pore
calcium channel protein 2 291 TMOD1 Rn.1646; Hs.494595;
tropomodulin 1 292 USP2 Rn.92548; Hs.524085; ubiquitin specific
peptidase 2 293 SLC16A6 Rn.54795; Hs.42645; solute carrier family
16, member 6 294 ATP1A1 Rn.2992; Hs.371889; ATPase, Na+/K+
transporting, alpha 1 polypeptide 295 CSRP2 Rn.94754; Hs.530904;
cysteine and glycine-rich protein 2 296 Unknown Rn.144632 297
SLC19A2 Rn.19386; Hs.30246; solute carrier family 19 (thiamine
transporter), member 2 298 HRSP12 Rn.6987; Hs.18426;
heat-responsive protein 12 299 Fkbp11 Rn.100569; RK506 binding
protein 11 300 Ace Rn.10149; angiotensin I converting enzyme
(peptidyl-dipeptidase A) I 301 Cyp4f4 (rat) Rn.10170; cytochrome
P450, family 5, subfamily 4, polypeptide 4 302 BI274837 Rn.101798;
transcribed locus 303 Hyou1 Rn.10542; hypoxia up-regulated 1 304
MI15 Rn.106040; myeloid/lymphoid or mixed-lineage leukemia 5
(trithorax homolog, Drosophila) 305 Tcf7 Rn.106335; transcription
factor 7, T-cell specific (predicted) 306 Arf3 Rn.106440;
ADP-ribosylation factor 3 307 Mia1 Rn.10660; melanoma inhibitory
activity 1 308 Sat Rn.107986; spermidine/spermine N1-acetyl
transferase (mapped) 309 Mpg Rn.11241; N-methylpurine-DNA
glycosylase 310 BE115368 Rn.118708; transcribed locus 311 BI281874
Rn.125724; Kelch-like 23 (Drosophila)(predicted) 312 Lcp1 Rn.14256;
lymphocyte cytosolic protein 1 313 RGD1306682 Rn.143893; similar to
RIKEN cDNA 1810046J19 (predicted) 314 AI502114 RN.148916;
ATP-binding cassette, sub-family A (ABC1), member 1 315 AA899202
Rn.14907; transcribed locus 316 BI275261 Rn.157564; transcribed
locus 317 AW532939 Rn.158403; transcribed locus 318 Isg20 Rn.16103;
interferon stimulated exonuclease 20 319 AI137294 Rn.161824;
similar to Mkrn1protein 320 BE107848 Rn.162933; similar to FYVE,
RhoGEF and PH domain containing 6 (predicted) 321 BM390584
Rn.163173; cDNA clone IMAGE: 7455180, containing frame-shift errors
322 Slc25a15 Rn.163331; solute carrier family 25 (mitochondrial
carrier; ornithine transporter) member 15 323 AA848795 Rn.163635;
transcribed locus 324 AI103213 Rn.164935; transcribed locus 325
Nans Rn.17006; N-acetylneuraminic acid synthase (sialic acid
synthase) (predicted) 326 BE108415 Rn.171133; transcribed locus 327
Pfn2 Rn.17153; profilin 2 328 Ube2n Rn.177520;
ubiguitin-conjugating enzyme E2N 329 BM384251 Rn.177573;
transcribed locus 330 Gga2 Rn.18248; Golgi associated, gamma
adaptin ear containing, ARF binding protein 2 331 BE106888
Rn.19198; cysteine-rich with EGF-like domains 2 332 AI070306
Rn.19710; transcribed locus 333 Reln Rn.198116; reelin 334 Glp2
Rn.1998318; interferon, alpha-inducible protein (clone IFI-15K)
(predicted) 335 Gpc4 Rn.19945; glypican 4 336 BF567145 Rn.200155;
transcribed locus 337 Manba Rn.20578; mannosidase, beta A,
lysosomal 338 BM386110 Rn.223; proliferating cell nuclear antigen
339 RGD1562142 Rn.23219; similar to homeotic protein Hox 2.2 --
mouse (predicted) 340 BG378045 Rn.23614; transcribed locus 341
AI146051 Rn.24020; transcribed locus 342 AI102873 Rn.2721;
transcribed locus 343 Rdx Rn.27421; radixin 344 Dnase 113 Rn.29996;
deoxyribonuclease I-like 3 345 Hexb Rn.3021; hexosaminidase B 346
Pls3 Rn.32103; plastin 3 (T-isoform) 347 RGD1566102_predicted
Rn.34703; transcribed locus 348 AI535113 Rn.34745; transcribed
locus 349 Pdia4 Rn.39305; protein disulfide isomerase associated 4
350 AW529628 Rn.43319; transcribed locus 351 BI292232 Rn.43415;
transcribed locus 352 Kcne3 Rn.44843; potassium voltage-gated
channel, Isk- related subfamily, member 3 353 St14 Rn.49170;
suppression of tumorigenicity 14 (colon carcinoma)
354 Mt1a Rn.54397; metallothionein 1a 355 St6gal1 Rn.54567;
betagalactoside alpha 2,6 sialyltransferase 1 356 Alcam Rn.5789;
activated leukocyte cell adhesion molecule 357 Maob Rn.6656;
monoamine oxidase B 358 AA891161 Rn.7257; transcribed locus 359
S1c17a5 Rn.74591; solute carrier family 17 (anion/sugar
transporter), member 5 360 RGD1306766 Rn.7655; similar to
hypothetical protein FLJ23514 361 Gja5 Rn.88300; gap junction
membrane channel protein alpha 5 362 RGD1566265_predicted Rn.8881;
similar to RIKEN cDNA 2610002M06 (predicted) 363 AI136703 Rn.92818;
transcribed locus 364 Mta3_predicted Rn.94848; metastasis
associated 3 (predicted) 365 Pctp Rn.9487; phosphatidylcholine
transfer protein 366 Map1b Rn.98152; microtubule-associated protein
1b 367 Tspan5 Rn.98240; tetraspanin 5 368 Got2 Rn.98650; glutamate
oxaloacetate transaminase 2, mitochondrial 369 BI285489 Rn.98850;
similar to myo-inositol 1-phosphate synthase A1 370 Zfp423 Rn.9981;
Zinc finger protein 423 371 Slc6a6 Rn.9968; solute carrier family 6
(neurotransmitter transporter, taurine), member 6 372 Agtr1a
Rn.9814; angiotensin II receptor, type 1 (AT1A) 373 Ppp1r1a
Rn.9756; protein phosphatase 1, regulatory (inhibitor) subunit 1A
374 Plin Rn.9737; perilipin 375 Dgat2 Rn.9523; diacylglycerol
O-acyltransferase homolog 2 (mouse) 376 Pcsk6 Rn.950; proprotein
convertase subtilisin/kexin type 6 377 BI281177 Rn.9403;
transcribed locus 378 AI599621 Rn.92531; Wilms tumor 1 379 Ceacam1
Rn.91235; CEA-related cell adhesion molecule 1 380 Gng11 Rn.892;
guanine nucleotide binding protein (G protein), gamma 11 381 Cdh11
Rn.8900; cadherin 11 382 Fmo1 Rn.867; flavin containing
monooxygenase 1 383 Cbr3_predicted Rn.8624; carbonyl reductase 3
(predicted) 384 BE113281 Rn.85462; quaking homolog, KH domain RNA
binding (mouse) 385 Cidea_predicted Rn.8171; cell death-inducing
DNA fragmentation factor, alpha subunit-like effector A (predicted)
386 Cav2 Rn.81070; caveolin 2 387 BI273836 Rn.79933; transcribed
locus 388 Mmrn2_predicted Rn.7966; multimerin 2 (predicted) 389
Agtr1 Rn.7965; angiotensin receptor-like 1 390 Gypc Rn.7693;
Glycophorin C (Gerbich blood group) 391 RGD1305719_predicted
Rn.76732; similar to putative N-acetyltransferase Camello 2
(predicted) 392 AI171656 Rn.7615; RGD1564859 (predicted) 393
Spsb1_predicted Rn.75037; SplA/ryanodine receptor domain and SOCS
box containing 1 (predicted) 394 Bcar3_predicted Rn.7383; breast
cancer anti-estrogen resistance 3 (predicted) 395 BE115406 Rn.7282;
similar to expressed sequence AA408877 396 Dlc1 Rn.7255; deleted in
liver cancer 1 397 AW915115 Rn.65477; transcribed locus 398 Cdkn2c
Rn.63865; cyclin-dependent kinase inhibitor 2C (p18, inhibits CDK4)
399 BF387865 Rn.63789; Transcribed locus 400 Tst Rn.6360;
Thiosulfate sulfurtransferase 401 Mbp Rn.63285; Myelin basic
protein 402 RGD1311474 Rn.6288; Similar to transmembrane protein
induced by tumor necrosis factor alpha 403 Pfk1 Rn.59431; Mesoderm
specific transcript 404 BI297693 Rn.57310; Similar to protein of
unknown function (predicted) 405 Agpat2_predicted Rn.55456;
1-acylglycerol-3-phosphate O- acyltransferase 2 (lysophosphatidic
acid acyltransferase, beta) (predicted) 406 Ilvb1_predicted
Rn.54315; Synapse defective 1, Rho GTPase, homolog 1 (C. elegans)
(predicted) 407 Ptpns1 Rn.53971; Protein tyrosine phosphatase, non-
receptor type substrate 1 408 Col4a1 Rn.53801; Procollagen, type
IV, alpha 1 409 Ccl2 Rn.4772; Chemokine (C-C motif) ligand 2 410
Gprc5b_predicted Rn.47330; G protein-coupled receptor, family C,
group 5, member B (predicted) 411 AI071994 Rn.44861; Dickkopf
homolog 4 (Xenopus laevis) (predicted) 412 BF414285 Rn.44465;
Chemokine-like receptor 1 413 Gpd1 Rn.44452; Glycerol-3-phosphate
dehydrogenase 1 (soluble) 414 Acacb Rn.44359; Transcribed locus 415
AI412164 Rn.44086; Transcribed locus 416 BF283694 Rn.44024;
Transcribed locus 417 Ankrd5_predicted Rn.44014; Ankyrin repeat
domain 5 (predicted) 418 AI144739 Rn.43251; Similar to KIAA0303
(predicted) 419 BG661061 Rn.41321; WDNM1 homolog 420 Prkar2b
Rn.4075; Protein kinase, cAMP dependent regulatory, type II beta
421 BI290794 Rn.40729; Transcribed locus 422 BM384701 Rn.40541; PE
responsive protein c64 423 RGD1565118_predicted Rn.39037; Similar
to mKIAA0843 protein (predicted) 424 Cd248_predicted Rn.38806;
CD248 antigen, endosialin (predicted) 425 Acaa2 Rn.3786;
Acetyl-Coenzyme A acyltransferase 2 (mitochondrial
3-oxoacyl-Coenzyme A thiolase) 426 BM390128 Rn.36545; Tenascin XA
427 RGD1309578 Rn.35367; Similar to Aa2-174 428 Inhbb Rn.35074;
Inhibin beta-B 429 AA943681 Rn.3504; Response gene to complement 32
430 BI274428 Rn.34454; Transcribed locus 431 Gpm6a Rn.34370;
Glycoprotein m6a 432 Cbr1 Rn.3425; Carbonyl reductase 1 433 Slc1a3
Rn.34134; Solute carrier family 1 (glial high affinity glutamate
transporter), member 3 434 AI179450 Rn.34019; Transcribed locus 435
RGD1560062_predicted Rn.32891; Similar to Laminin alpha-4 chain
precursor (predicted) 436 Phyhd1 Rn.32623; Phytanoyl-CoA
dioxygenase domain containing 1 437 Rgl1_predicted Rn.28005; Ral
guanine nucleotide dissociation stimulator, -like 1 (predicted) 438
Grifin Rn.26894; Galectin-related inter-fiber protein 439 BG381647
Rn.26832; Transcribed locus 440 Ccl7 Rn.26815; Chemokine (C-C
motif) ligand 7 441 AI548615 Rn.26537; Transcribed locus 442 Per2
Rn.25935; Period homolog 2 (Drosophila) 443 Dgat1 Rn.252;
Diacylglycerol O-acyltransferase 1 444 Gda Rn.24783; Transcribed
locus 445 Psme1 Rn.2472; Proteasome (prosome, macropain) 28
subunit, alpha 446 Tm4sf1_predicted Rn.24712; Transmembrane 4
superfamily member 1 (predicted) 447 Slc22a3 Rn.24231; Solute
carrier family 22, member 3 448 AI228291 Rn.2361; Similar to
CG3740-PA 449 Rasip1_predicted Rn.23451; Ras interacting protein 1
(predicted) 450 Pparg Rn.23443; Peroxisome proliferator activated
receptor gamma 451 BG378238 Rn.23273; Transcribed locus 452
Abca8a_predicted Rn.22789; ATP-binding cassette, sub-family A
(ABC1), member 8a (predicted) 453 BF290937 Rn.22733; Transcribed
locus 454 Sox18 Rn.22446; SRY-box containing gene 18 455 AI230554
Rn.22441; Carbonic anhydrase VB, mitochondrial 456 Col4a2_predicted
Rn.2237; Procollagen, type IV, alpha 2 (predicted) 457 BF547294
Rn.22135; Protein tyrosine phosphatase, receptor type, M 458 Id1
Rn.2113; Inhibitor of DNA binding 1 459 Sulf1 Rn.20664; Transcribed
locus 460 AI411941 Rn.20633; Fibronectin type III domain containing
1 461 AI385260 Rn.20514; Unknown (protein for MGC: 72614) 462
RGD1562428_predicted Rn.199567; Transcribed locus 463 Aoc3
Rn.198327; Amine oxidase, copper containing 3 464 AI599365
Rn.19608; Transcribed locus 465 RGD1305061 Rn.196026; Similar to
RIKEN cDNA 2700055K07 466 BF282889 Rn.19393; Transcribed locus 467
RGD1311800 Rn.1935; Similar to genethonin 1 468 Daf1 Rn.18841;
decay accelerating factor 1 469 AI030806 Rn.18599; Transcribed
locus 470 BM386662 Rn.18571; Tumor suppressor candidate 5 471
BF283405 Rn.18479; Transcribed locus 472 BI277619 Rn.18388;
Transcribed locus 473 Anxa1 Rn.1792; Annexin A1 474 Phlda3
Rn.17905; Pleckstrin homology-like domain, family A, member 3 475
Zdhhc2 Rn.17310; Zinc finger, DHHC domain containing 2 476 AI101500
Rn.17209; Transcribed locus 477 AW525722 Rn.168623; Transcribed
locus Transcribed locus 478 AI600020 Rn.168403; Transcribed locus
479 Hdgfrp2 Rn.167154; Transcribed locus 480 Degs1 Rn.167052;
Transcribed locus 481 BM389225 Rn.1664; Transcribed locus 482
AI407050 Rn.165854; Transcribed locus 483 BF291140 Rn.165750;
Transcribed locus 484 AI176379 Rn.165711; Transcribed locus 485
BF403558 Rn.165637; Transcribed locus 486 AI008140 Rn.165579;
Transcribed locus 487 AW536030 Rn.165356; Similar to liver-specific
bHLH-Zip transcription factor 488 Sdpr Rn.165134; Transcribed locus
489 AI385201 Rn.164647; Transcribed locus 490 Tgfbr2 Rn.164421;
Transcribed locus 491 AW535515 Rn.164403; Transcribed locus 492
Gata6 Rn.164357; Transcribed locus 493 RGD1566234_predicted
Rn.164243; Transcribed locus 494 Acaca Rn.163753; Acetyl-coenzyme A
carboxylase alpha 495 RGD1311037 Rn.163715; Transcribed locus 496
AA926305 Rn.163580; Transcribed locus 497 Efemp1 Rn.163265;
Epidermal growth factor-containing fibulin-like extracellular
matrix protein 1 498 Aps Rn.163202; Adaptor protein with pleckstrin
homology and src homology 2 domains 499 Vnn1 Rn.16319; Vanin 1 500
Lpin1 Rn.162853; Lipin 1 501 Ppp1r3c Rn.162528; Protein phosphatase
1, regulatory (inhibitor) subunit 3C 502 Twist1 Rn.161904; Twist
gene homolog 1 (Drosophila) 503 C6 Rn.16145; Complement component 6
504 Cabc1 Rn.160865; Chaperone, ABC1 activity of bc1 complex like
(S. pombe) 505 Vegfb Rn.160277; Transcribed locus 506 Ehd2
Rn.16016; EH-domain containing 2 507 Dpyd Rn.158382;
Dihydropyrimidine dehydrogenase 508 Nnmt_predicted Rn.15755;
Nicotinamide N-methyltransferase (predicted) 509 BI289692 Rn.15749;
Transcribed locus 510 Chpt1 Rn.154718; Choline phosphotransferase 1
511 BI295900 Rn.15413; Dihydrolipoamide S-acetyltransferase (E2
component of pyruvate dehydrogenase complex) 512 AW917217
Rn.153603; CCAAT/enhancer binding protein (C/EBP), alpha 513
AA942745 Rn.149118; Transcribed locus 514 BI283648 Rn.148951;
Hypothetical protein LOC691485 515 BF393275 Rn.148773; Transcribed
locus 516 AI555775 Rn.147356; Transcribed locus 517 Tgif Rn.144418;
Transcribed locus 518 Cldn15_predicted Rn.144007; Transcribed locus
519 AI578098 Rn.137828; Similar to CD209 antigen 520 Cyp2e1
Rn.1372; Cytochrome P450, family 2, subfamily e, polypeptide 1 521
Tm4sf2_mapped Rn.13685; Transmembrane 4 superfamily member 2
(mapped) 522 Mdh1 Rn.13492; Malate dehydrogenase 1, NAD (soluble)
523 Slc2a4 Rn.1314; Solute carrier family 2 (facilitated glucose
transporter), member 4 524 Cmkor1 Rn.12959; Chemokine orphan
receptor 1 525 AW528864 Rn.129539; Transcribed locus 526 Dnd1
Rn.12947; Similar to KIAA0564 protein (predicted) 527 AW528112
Rn.119594; Transcribed locus 528 BF397229 Rn.11817; Transcribed
locus 529 Sfxn1 Rn.115752; Sideroflexin 1 530 Hrasls3 Rn.11377;
HRAS like suppressor 3 531 Pla2g2a Rn.11346; Phospholipase A2,
group IIA (platelets, synovial fluid) 532 Ebf1 Rn.11257; Early
B-cell factor 1 533 Sdc2 Rn.11127; Syndecan 2 534 Aqp7 Rn.11111;
Aquaporin 7 535 Pc Rn.11094; Pyruvate carboxylase 536 Bhlhb3
Rn.10784; Basic helix-loop-helix domain containing, class B3 537
AI602542 Rn.107412; Transcribed locus 538 Maf Rn.10726; V-maf
musculoaponeurotic fibrosarcoma oncogene homolog (avian) 539 Cpa3
Rn.10700; Carboxypeptidase A3 540 Mcpt1 Rn.10698; Mast cell
protease 1 541 RGD1309821_predicted Rn.106115; Similar to KIAA1161
protein (predicted) 542 Acvr1c Rn.10580; Activin A receptor, type
IC 543 Ppp2r5a_predicted Rn.104461; Protein phosphatase 2,
regulatory subunit B (B56), alpha isoform (predicted) 544 Pde3b
Rn.10322; Phosphodiesterase 3B 545 Pxmp2 Rn.10292; Peroxisomal
membrane protein 2 546 P2rx5 Rn.10257; Purinergic receptor P2X,
ligand-gated ion channel, 5 547 Cma1 Rn.10182; Chymase 1, mast cell
548 Pfkfb1 Rn.10115; 6-phosphofructo-2-kinase/fructose-2,6-
biphosphatase 1
[0063] From among the 548 T2DBMARKERS discovered to date, the
present inventors have discovered one particular T2DBMARKER, a
peptide fragment from the Cohen diabetic (CD) rat model having a
molecular weight of about 4.2 kD, that is homologous to human
SERPINA1 and SERPINA3, and which may exhibit activity as an
anti-diabetic agent (SEQ ID NO: 1). In the CD rat model, the
sensitive strain (CDs) develops Diabetes within 30 days when
maintained on a high sucrose/copper-poor diet (HSD), whereas the
resistant strain (CDr) retains normal blood glucose levels. When
maintained indefinitely on regular rodent diet (RD), neither strain
develop symptoms of T2D. The peptide fragment was found in the
serum of CDr-RD and CDr-HSD, but not in the serum of CDs-RD or
CDs-HSD (Example 1), suggesting that the peptide is only found in
rats that have not progressed to a diabetic phenotype. The present
inventors have further studied this peptide and found that the
peptide exhibits strong kinase inhibitory activity. Other preferred
T2DBMARKERS include any of the peptide sequences described herein,
such as, for example, SEQ ID NO: 2, and SEQ ID NO: 3, or any
sequences derived from human serpin proteins, e.g., SERPINA1 and
SERPINA3 and which have been determined to be peptide inhibitors of
kinases implicated in Diabetes. These, too, are considered to be
"T2DBMARKERS" in the context of the present invention.
[0064] Serpins are a superfamily of proteins classified into 16
clades designated "A-P". The systematic name of each serpin is,
"SERPINXy," where X is the clade and y is the number within the
clade. To date, thirty-six (36) serpins have been identified in
humans. While serpins are named for their ability to inhibit serine
proteases of the chymotrypsin family, some are capable of
cross-class inhibition of proteases from the subtilisin, papain and
caspase families. In addition, some serpins lack protease
inhibitory activity and serve other roles, such as hormone
transporters, molecular chaperones or catalysts for DNA
condensation. Serpins are typically composed of 330-500 amino
acids, but can have large N--, C-terminal or internal insertion
loops. Serpins can also be post-translationally modified by
glycosylation, sulfation, phosphorylation and oxidation to alter
their function. Despite a low overall primary sequence identity for
the family, serpins share a highly conserved three-dimensional fold
comprised of a bundle of 9 .alpha.-helices, a .beta.-sandwich
composed of three P-sheets, and a reactive site loop (RSL) composed
of 20 amino acids (Rau, J. C. et al. (2007) J. Thromb. Hemostasis 5
(Suppl. 1): 102-115).
[0065] In the normal native state of a serpin, the RSL is exposed,
however, this state is not the most stable. An increase in
thermodynamic stability is achieved through the incorporation of
the RSL into one of the .beta.-sheets, triggered either through
strand extension to form the "latent" state, or through proteolytic
nicking anywhere near a scissile bond (the cleaved state). This
metastability of the native serpin is critical for protease
inhibition. A minimalist kinetic scheme is composed of two steps:
the formation of the encounter complex (also known as the Michaelis
complex) where the sequence of the RSL is recognized by the
protease as a substrate; and the formation of a final covalent
complex, where the protease is trapped in an inactive state. The
rates of formation and dissociation of the reversible Michaelis
complex, along with co-localization in tissues, determines the
specificity of the serpin-protease interaction. While the obligate
RSL-active site contacts contribute significantly to the formation
of the Michaelis complexes, exosite interactions may also be
involved.
[0066] Over 70 serpin structures have been determined, and these
data, along with a large amount of biochemical and biophysical
information, reveal that inhibitory serpins are `suicide` or
`single use` inhibitors that use a unique and extensive
conformational change to inhibit proteases. This conformational
mobility renders serpins heat-labile and vulnerable to mutations
that promote misfolding, spontaneous conformational change,
formation of inactive serpin polymers and serpin deficiency. In
humans, several conformational diseases or `serpinopathies` linked
to serpin polymerization have been identified, including emphysema
(SERPINA1 (antitrypsin) deficiency) (Lomas, D. A. et al. (1992)
Nature 357: 605-607), thrombosis (SERPINC1 (antithrombin)
deficiency) (Bruce, D. et al. (1994) J. Clin. Invest. 94:
2265-2274) and angioedema (SERPING1 (C1 esterase inhibitor)
deficiency) (Aulak, K. S. et al., (1988) Biochem. J. 253: 615-618).
Accumulation of serpin polymers in the endoplasmic reticulum of
serpin-secreting cells can also result in disease, most notably
cirrhosis (SERPINA1 polymerization) (Lomas, D. A. et al. (1992)
Nature 357: 605-607) and familial dementia (SERPINI1 (neuroserpin)
polymerization) (Davis, R. L. et al., (1999) Nature 401: 376-379).
Other serpin-related diseases are caused by null mutations or
(rarely) point mutations. In humans, the majority (27 out of the 36
heretofore identified) of serpins are inhibitory. Clade A serpins
include inflammatory response molecules such as SERPINA1
(antitrypsin) and SERPINA3 (antichymotrypsin) as well as the
non-inhibitory hormone-transport molecules SERPINA6
(corticosteroid-binding globulin) and SERPINA7 (thyroxine-binding
globulin). Clade B includes inhibitory molecules that function to
prevent inappropriate activity of cytotoxic apoptotic proteases
(SERPINB6, also called P16, and SERPINB9, also called P19) and
inhibit papain-like enzymes (SERPINB3, squamous cell carcinoma
antigen-1) as well as the non-inhibitory molecule SERPINB5
(maspin). SERPINB5 does not undergo the characteristic serpin-like
conformational change and functions to prevent metastasis in breast
cancer and other cancers through an incompletely characterized
mechanism.
[0067] The present invention is based in part on the discovery that
peptides derived from human SERPINA1 and SERPINA3 may serve as
inhibitors of kinases believed to be implicated in type 2 Diabetes
Mellitus. Thus, the present invention is directed to peptide
inhibitors of kinases and useful implications of these peptides in
the treatment of type 2 Diabetes Mellitus, pre-diabetic conditions,
and other diabetes-related conditions disclosed herein. The peptide
inhibitors of the invention include the amino acid sequences
disclosed herein, containing one or more of the motifs "FNRPFL",
"FMS/GKVT/VNP", "R[S/K]XXPP" or "SXXPP" where F=phenylalanine,
N=asparagine, R=arginine, P=proline, L=leucine, M=methionine,
S=serine, G=glycine, K=lysine, V=valine, and X=any amino acid. The
peptides of the invention have been shown to inhibit kinases in
vitro and in vivo.
[0068] Protein kinases are enzymes that phosphorylate protein
substrates and are key players in signal transduction events from
outside the cell to the cytoplasm. Protein kinases are involved in
many events relating to the life and death of cells, including
mitosis, differentiation, and apoptosis. As such, protein kinases
have been considered as favorable drug targets. However, inhibition
of many kinases could lead to cell death or other manifestations of
cell abnormalities, because their activity is so crucial to the
well-being of the cell. Although this is a desirable effect for
anticancer drugs, it is a major drawback for most other
therapeutics. The present invention relates in part to peptide
inhibitors of protein kinases implicated in Diabetes, such as,
without limitation, members of the mitogen-activated protein kinase
(MAPK) family, such as p70S6K, protein kinase .beta. isoforms, such
as PKB.beta., protein kinase C isoforms, such as PKC.zeta., and
serum and glucocorticoid induced protein kinase (SGK).
[0069] The peptide inhibitors of the invention can be used to
inhibit the activity of kinases involved in type 2 Diabetes
Mellitus, pre-diabetic conditions (such as, for example, metabolic
syndrome, impaired glucose tolerance, insulin resistance, or
impaired fasting glycemia), or complications relating to type 2
Diabetes Mellitus. The peptide inhibitors are useful for treating
type 2 Diabetes Mellitus or a pre-diabetic condition in a subject
or preventing type 2 diabetes or pre-diabetic conditions in a
subject. The peptide inhibitors are also useful therapeutic or
research tools in the areas of immunology, hematologic deficiencies
and malignancies, metabolism, or any field of study where serpins
have been shown to be important.
Methods of Detecting Biomarkers
[0070] Levels of T2DBMARKERS can be determined at the protein or
nucleic acid level using any method known in the art. T2DBMARKER
amounts can be detected, inter alia, electrophoretically (such as
by agarose gel electrophoresis, sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Tris-HCl
polyacrylamide gels, non-denaturing protein gels, two-dimensional
gel electrophoresis (2DE), and the like), immunochemically (i.e.,
radioimmunoassay, immunoblotting, immunoprecipitation,
immunofluorescence, enzyme-linked immunosorbent assay), by
"proteomics technology", or by "genomic analysis." For example, at
the nucleic acid level, Northern and Southern hybridization
analysis, as well as ribonuclease protection assays using probes
which specifically recognize one or more of these sequences can be
used to determine gene expression. Alternatively, expression can be
measured using reverse-transcription-based PCR assays (RT-PCR),
e.g., using primers specific for the differentially expressed
sequence of genes. Expression can also be determined at the protein
level, e.g., by measuring the levels of peptides encoded by the
gene products described herein, or activities thereof. Such methods
are well known in the art and include, e.g., immunoassays based on
antibodies to proteins encoded by the genes, aptamers or molecular
imprints. Any biological material can be used for the
detection/quantification of the protein or its activity.
Alternatively, a suitable method can be selected to determine the
activity of proteins encoded by the marker genes according to the
activity of each protein analyzed.
[0071] "Proteomics technology" includes, but is not limited to,
surface enhanced laser desorption ionization (SELDI),
matrix-assisted laser desorption ionization-time of flight
(MALDI-TOF), high performance liquid chromatography (HPLC), liquid
chromatography with or without mass spectrometry (LC/MS), tandem
LC/MS, protein arrays, peptide arrays, and antibody arrays.
[0072] "Genome analysis" can comprise, for example, polymerase
chain reaction (PCR), real-time PCR (such as by Light Cycler.RTM.,
available from Roche Applied Sciences), serial analysis of gene
expression (SAGE), Northern blot analysis, and Southern blot
analysis.
[0073] Microarray technology can be used as a tool for analyzing
gene or protein expression, comprising a small membrane or solid
support (such as but not limited to microscope glass slides,
plastic supports, silicon chips or wafers with or without fiber
optic detection means, and membranes including nitrocellulose,
nylon, or polyvinylidene fluoride). The solid support can be
chemically (such as silanes, streptavidin, and numerous other
examples) or physically derivatized (for example, photolithography)
to enable binding of the analyte of interest, usually nucleic
acids, proteins, or metabolites or fragments thereof. The nucleic
acid or protein can be printed (i.e., inkjet printing), spotted, or
synthesized in situ. Deposition of the nucleic acid or protein of
interest can be achieved by xyz robotic microarrayers, which
utilize automated spotting devices with very precise movement
controls on the x-, y-, and z-axes, in combination with pin
technology to provide accurate, reproducible spots on the arrays.
The analytes of interest are placed on the solid support in an
orderly or fixed arrangement so as to facilitate easy
identification of a particularly desired analyte. A number of
microarray formats are commercially available from, inter alia,
Affymetrix, ArrayIt, Agilent Technologies, Asper Biotech, BioMicro,
CombiMatrix, GenePix, Nanogen, and Roche Diagnostics.
[0074] The nucleic acid or protein of interest can be synthesized
in the presence of nucleotides or amino acids tagged with one or
more detectable labels. Such labels include, for example,
fluorescent dyes and chemiluminescent labels. In particular, for
microarray detection, fluorescent dyes such as but not limited to
rhodamine, fluorescein, phycoerythrin, cyanine dyes like Cy3 and
Cy5, and conjugates like streptavidin-phycoerythrin (when nucleic
acids or proteins are tagged with biotin) are frequently used.
Detection of fluorescent signals and image acquisition are
typically achieved using confocal fluorescence laser scanning or
photomultiplier tube, which provide relative signal intensities and
ratios of analyte abundance for the nucleic acids or proteins
represented on the array. A wide variety of different scanning
instruments are available, and a number of image acquisition and
quantification packages are associated with them, which allow for
numerical evaluation of combined selection criteria to define
optimal scanning conditions, such as median value, inter-quartile
range (IQR), count of saturated spots, and linear regression
between pair-wise scans (r.sup.2 and P). Reproducibility of the
scans, as well as optimization of scanning conditions, background
correction, and normalization, are assessed prior to data
analysis.
[0075] Normalization refers to a collection of processes that are
used to adjust data means or variances for effects resulting from
systematic non-biological differences between arrays, subarrays (or
print-tip groups), and dye-label channels. An array is defined as
the entire set of target probes on the chip or solid support. A
subarray or print-tip group refers to a subset of those target
probes deposited by the same print-tip, which can be identified as
distinct, smaller arrays of proves within the full array. The
dye-label channel refers to the fluorescence frequency of the
target sample hybridized to the chip. Experiments where two
differently dye-labeled samples are mixed and hybridized to the
same chip are referred to in the art as "dual-dye experiments",
which result in a relative, rather than absolute, expression value
for each target on the array, often represented as the log of the
ratio between "red" channel and "green channel." Normalization can
be performed according to ratiometric or absolute value methods.
Ratiometric analyses are mainly employed in dual-dye experiments
where one channel or array is considered in relation to a common
reference. A ratio of expression for each target probe is
calculated between test and reference sample, followed by a
transformation of the ratio into log.sub.2(ratio) to symmetrically
represent relative changes. Absolute value methods are used
frequently in single-dye experiments or dual-dye experiments where
there is no suitable reference for a channel or array. Relevant
"hits" are defined as expression levels or amounts that
characterize a specific experimental condition. Usually, these are
nucleic acids or proteins in which the expression levels differ
significantly between different experimental conditions, usually by
comparison of the expression levels of a nucleic acid or protein in
the different conditions and analyzing the relative expression
("fold change") of the nucleic acid or protein and the ratio of its
expression level in one set of samples to its expression in another
set.
[0076] Data obtained from microarray experiments can be analyzed by
any one of numerous statistical analyses, such as clustering
methods and scoring methods. Clustering methods attempt to identify
targets (such as nucleic acids and/or proteins) that behave
similarly across a range of conditions or samples. The motivation
to find such targets is driven by the assumption that targets that
demonstrate similar patterns of expression share common
characteristics, such as common regulatory elements, common
functions, or common cellular origins.
[0077] Hierarchical clustering is an agglomerative process in which
single-member clusters are fused to bigger and bigger clusters. The
procedure begins by computing a pairwise distance matrix between
all the target molecules, the distance matrix is explored for the
nearest genes, and they are defined as a cluster. After a new
cluster is formed by agglomeration of two clusters, the distance
matrix is updated to reflect its distance from all other clusters.
Then, the procedure searches for the nearest pair of clusters to
agglomerate, and so on. This procedure results in a hierarchical
dendrogram in which multiple clusters are fused to nodes according
to their similarity, resulting in a single hierarchical tree.
Hierarchical clustering software algorithms include Cluster and
Treeview.
[0078] K-means clustering is an iterative procedure that searches
for clusters that are defined in terms of their "center" points or
means. Once a set of cluster centers is defined, each target
molecule is assigned to the cluster it is closest to. The
clustering algorithm then adjusts the center of each cluster of
genes to minimize the sum of distances of target molecules in each
cluster to the center. This results in a new choice of cluster
centers, and target molecules can be reassigned to clusters. These
iterations are applied until convergence is observed.
Self-organizing maps (SOMs) are related in part to the k-means
procedure, in that the data is assigned to a predetermined set of
clusters. However, unlike k-means, what follows is an iterative
process in which gene expression vectors in each cluster are
"trained" to find the best distinctions between the different
clusters. In other words, a partial structure is imposed on the
data and then this structure is iteratively modified according to
the data. SOM is included in many software packages, such as, for
instance, GeneCluster. Other clustering methods include
graph-theoretic clustering, which utilizes graph-theoretic and
statistical techniques to identify tight groups of highly similar
elements (kernels), which are likely to belong to the same true
cluster. Several heuristic procedures are then used to expand the
kernels into the full clustering. An example of software utilizing
graph-theoretic clustering includes CLICK in combination with the
Expander visualization tool.
[0079] Data obtained from high-throughput expression analyses can
be scored using statistical methods such as parametric and
non-parametric methods. Parametric approaches model expression
profiles within a parametric representation and ask how different
the parameters of the experimental groups are. Examples of
parametric methods include, without limitation, t-tests, separation
scores, and Bayesian t-tests. Non-parametric methods involve
analysis of the data, wherein no a priori assumptions are made
about the distribution of expression profiles in the data, and the
degree to which the two groups of expression measurements are
distinguished is directly examined. Another method uses the TNOM,
or the threshold number of misclassifications, which measures the
success in separation two groups of samples by a simple threshold
over the expression values.
[0080] SAGE (serial analysis of gene expression) can also be used
to systematically determine the levels of gene expression. In SAGE,
short sequence tags within a defined position containing sufficient
information to uniquely identify a transcript are used, followed by
concatenation of tags in a serial fashion. See, for example,
Velculescu V. E. et al, (1995) Science 270: 484-487. Polyadenylated
RNA is isolated by oligo-dT priming, and cDNA is then synthesized
using a biotin-labeled primer. The cDNA is subsequently cleaved
with an anchoring restriction endonucleases, and the 3'-terminal
cDNA fragments are bound to streptavidin-coated beads. An
oligonucleotide linker containing recognition sites for a tagging
enzyme is linked to the bound cDNA. The tagging enzyme can be a
class II restriction endonucleases that cleaves the DNA at a
constant number of bases 3' to the recognition site, resulting in
the release of a short tag and the linker from the beads after
digestion with the enzyme. The 3' ends of the released tags plus
linkers are then blunt-ended and ligated to one another to form
linked ditags that are approximately 100 base pairs in length. The
ditags are then subjected to PCR amplification, after which the
linkers and tags are released by digestion with the anchoring
restriction endonucleases. Thereafter, the tags (usually ranging in
size from 25-30-mers) are gel purified, concatenated, and cloned
into a sequence vector. Sequencing the concatemers enables
individual tags to be identified and the abundance of the
transcripts for a given cell or tissue type can be determined.
[0081] The T2DBMARKER proteins, polypeptides (including the peptide
inhibitors of the invention), mutations, and polymorphisms thereof
can be detected in any manner known to those skilled in the art. Of
particular utility are two-dimensional gel electrophoresis, which
separates a mixture of proteins (such as found in biological
samples such as serum) in one dimension according to the
isoelectric point (such as, for example, a pH range from 5-8), and
according to molecular weight in a second dimension.
Two-dimensional liquid chromatography is also advantageously used
to identify or detect T2DBMARKER proteins, polypeptides, mutations,
and polymorphisms of the invention, and one specific example, the
ProteomeLab PF 2D Protein Fractionation System is detailed in the
Examples. The PF 2D system resolves proteins in one dimension by
isoelectric point and by hydrophobicity in the second dimension.
Another advantageous method of detecting proteins, polypeptides,
mutations, and polymorphisms include SELDI (disclosed herein) and
other high-throughput proteomic arrays.
[0082] T2DBMARKER proteins, polypeptides, mutations, and
polymorphisms can be typically detected by contacting a sample from
the subject with an antibody which binds the T2DBMARKER protein,
polypeptide, mutation, or polymorphism and then detecting the
presence or absence of a reaction product. The antibody may be
monoclonal, polyclonal, chimeric, or a fragment of the foregoing,
as discussed in detail herein, and the step of detecting the
reaction product may be carried out with any suitable immunoassay.
In a particularly preferred embodiment, the T2DBMARKER proteins,
polypeptides, mutations, and polymorphisms can be detected with an
isolated antibody of the present invention, as disclosed elsewhere
in this disclosure. The isolated antibody provided by the invention
can comprise, for example, a human constant region (as defined
herein) and an antigen-binding region that binds to one or more
T2DBMARKERS set forth in Table 1, preferably at least one,
preferably two, three, four, five, six, seven, eight, nine, ten or
more amino acid residues of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID
NO: 3. The sample from the subject is typically a biological fluid
as described above, and may be the same sample of biological fluid
used to conduct the method described above.
[0083] Immunoassays carried out in accordance with the present
invention may be homogeneous assays or heterogeneous assays. In a
homogeneous assay, the immunological reaction usually involves the
specific antibody (e.g., anti-T2DBMARKER protein antibody), a
labeled analyte, and the sample of interest. The signal arising
from the label is modified, directly or indirectly, upon the
binding of the antibody to the labeled analyte. Both the
immunological reaction and detection of the extent thereof can be
carried out in a homogeneous solution. Immunochemical labels which
may be employed include free radicals, radioisotopes, fluorescent
dyes, enzymes, bacteriophages, or coenzymes.
[0084] In a heterogeneous assay approach, the reagents are usually
the sample, the antibody, and means for producing a detectable
signal. Samples as described above may be used. The antibody can be
immobilized on a support, such as a bead (such as protein A
agarose, protein G agarose, latex, polystyrene, magnetic or
paramagnetic beads), plate or slide, and contacted with the
specimen suspected of containing the antigen in a liquid phase. The
support is then separated from the liquid phase and either the
support phase or the liquid phase is examined for a detectable
signal employing means for producing such signal. The signal is
related to the presence of the analyte in the sample. Means for
producing a detectable signal include the use of radioactive
labels, fluorescent labels, or enzyme labels. For example, if the
antigen to be detected contains a second binding site, an antibody
which binds to that site can be conjugated to a detectable group
and added to the liquid phase reaction solution before the
separation step. The presence of the detectable group on the solid
support indicates the presence of the antigen in the test sample.
Examples of suitable immunoassays are oligonucleotides,
immunoblotting, immunoprecipitation, immunofluorescence methods,
chemiluminescence methods, electrochemiluminescence or
enzyme-linked immunoassays.
[0085] Those skilled in the art will be familiar with numerous
specific immunoassay formats and variations thereof which may be
useful for carrying out the method disclosed herein. See generally
E. Maggio, Enzyme-Immunoassay, (1980) (CRC Press, Inc., Boca Raton,
Fla.); see also U.S. Pat. No. 4,727,022 to Skold et al. titled
"Methods for Modulating Ligand-Receptor Interactions and their
Application," U.S. Pat. No. 4,659,678 to Forrest et al. titled
"Immunoassay of Antigens," U.S. Pat. No. 4,376,110 to David et al.,
titled "Immunometric Assays Using Monoclonal Antibodies," U.S. Pat.
No. 4,275,149 to Litman et al., titled "Macromolecular Environment
Control in Specific Receptor Assays," U.S. Pat. No. 4,233,402 to
Maggio et al., titled "Reagents and Method Employing Channeling,"
and U.S. Pat. No. 4,230,767 to Boguslaski et al., titled
"Heterogenous Specific Binding Assay Employing a Coenzyme as
Label."
[0086] Antibodies, such as those provided by the present invention,
can be conjugated to a solid support suitable for a diagnostic
assay (e.g., beads such as protein A or protein G agarose,
microspheres, plates, slides or wells formed from materials such as
latex or polystyrene) in accordance with known techniques, such as
passive binding. Antibodies as described herein may likewise be
conjugated to detectable labels or groups such as radiolabels
(e.g., .sup.35S, .sup.125I, .sup.131I), enzyme labels (e.g.,
horseradish peroxidase, alkaline phosphatase), and fluorescent
labels (e.g., fluorescein, Alexa, green fluorescent protein) in
accordance with known techniques.
[0087] Antibodies can also be useful for detecting
post-translational modifications of T2DBMARKER proteins,
polypeptides, mutations, and polymorphisms, such as tyrosine
phosphorylation, threonine phosphorylation, serine phosphorylation,
glycosylation (e.g., O-GlcNAc). Such antibodies specifically detect
the phosphorylated amino acids in a protein or proteins of
interest, and can be used in immunoblotting, immunofluorescence,
and ELISA assays described herein. These antibodies are well-known
to those skilled in the art, and commercially available.
Post-translational modifications can also be determined using
metastable ions in reflector matrix-assisted laser desorption
ionization-time of flight mass spectrometry (MALDI-TOF) (Wirth, U.
et al. (2002) Proteomics 2(10): 1445-51).
[0088] For T2DBMARKER proteins, polypeptides, mutations, and
polymorphisms known to have enzymatic activity, the activities can
be determined in vitro using enzyme assays known in the art. Such
assays include, without limitation, kinase assays (such as those
exemplified in Example X herein), phosphatase assays, reductase
assays, among many others. Modulation of the kinetics of enzyme
activities can be determined by measuring the rate constant KM
using known algorithms, such as the Hill plot, Michaelis-Menten
equation, linear regression plots such as Lineweaver-Burk analysis,
and Scatchard plot.
[0089] Using sequence information provided by the database entries
for the T2DBMARKER sequences, expression of the T2DBMARKER
sequences can be detected (if present) and measured using
techniques well known to one of ordinary skill in the art. For
example, sequences within the sequence database entries
corresponding to T2DBMARKER sequences, or within the sequences
disclosed herein, can be used to construct probes for detecting
T2DBMARKER RNA sequences in, e.g., Northern blot hybridization
analyses or methods which specifically, and, preferably,
quantitatively amplify specific nucleic acid sequences. As another
example, the sequences can be used to construct primers for
specifically amplifying the T2DBMARKER sequences in, e.g.,
amplification-based detection methods such as reverse-transcription
based polymerase chain reaction (RT-PCR). When alterations in gene
expression are associated with gene amplification, deletion,
polymorphisms, and mutations, sequence comparisons in test and
reference populations can be made by comparing relative amounts of
the examined DNA sequences in the test and reference cell
populations.
[0090] Expression of the genes disclosed herein can be measured at
the RNA level using any method known in the art. For example,
Northern hybridization analysis using probes which specifically
recognize one or more of these sequences can be used to determine
gene expression. Alternatively, expression can be measured using
reverse-transcription-based PCR assays (RT-PCR), e.g., using
primers specific for the differentially expressed sequences.
[0091] Alternatively, T2DBMARKER protein and nucleic acid
metabolites or fragments can be measured. The term "metabolite"
includes any chemical or biochemical product of a metabolic
process, such as any compound produced by the processing, cleavage
or consumption of a biological molecule (e.g., a protein, nucleic
acid, carbohydrate, or lipid). Metabolites can be detected in a
variety of ways known to one of skill in the art, including the
refractive index spectroscopy (RI), ultra-violet spectroscopy (UV),
fluorescence analysis, radiochemical analysis, near-infrared
spectroscopy (near-IR), nuclear magnetic resonance spectroscopy
(NMR), light scattering analysis (LS), mass spectrometry, pyrolysis
mass spectrometry, nephelometry, dispersive Raman spectroscopy, gas
chromatography combined with mass spectrometry, liquid
chromatography combined with mass spectrometry, matrix-assisted
laser desorption ionization-time of flight (MALDI-TOF) combined
with mass spectrometry, surface-enhanced laser desorption
ionization (SELDI), ion spray spectroscopy combined with mass
spectrometry, capillary electrophoresis, NMR and IR detection.
(See, WO 04/056456 and WO 04/088309, each of which are hereby
incorporated by reference in their entireties) In this regard,
other T2DBMARKER analytes can be measured using the above-mentioned
detection methods, or other methods known to the skilled
artisan.
Kits
[0092] The invention also includes a T2DBMARKER-detection reagent,
e.g., nucleic acids that specifically identify one or more
T2DBMARKER nucleic acids by having homologous nucleic acid
sequences, such as oligonucleotide sequences, complementary to a
portion of the T2DBMARKER nucleic acids or antibodies to proteins
encoded by the T2DBMARKER nucleic acids packaged together in the
form of a kit. The kits of the present invention allow one of skill
in the art to generate the reference and subject expression
profiles disclosed herein. The kits of the invention can also be
used to advantageously carry out any of the methods provided in
this disclosure. The oligonucleotides can be fragments of the
T2DBMARKER genes. For example the oligonucleotides can be 200, 150,
100, 50, 25, 10 or less nucleotides in length. The
T2DBMARKER-detection reagents can also comprise, inter alia,
antibodies or fragments of antibodies, and aptamers. The kit may
contain in separate containers a nucleic acid or antibody (either
already bound to a solid matrix or packaged separately with
reagents for binding them to the matrix), control formulations
(positive and/or negative), and/or a detectable label. Instructions
(e.g., written, tape, VCR, CD-ROM, etc.) for carrying out the assay
detecting one or more T2DBMARKERS of the invention may be included
in the kit. The assay may for example be in the form of a Northern
blot hybridization or a sandwich ELISA as known in the art.
Alternatively, the kit can be in the form of a microarray as known
in the art.
[0093] Diagnostic kits for carrying out the methods described
herein are produced in a number of ways. Preferably, the kits of
the present invention comprise a control (or reference) sample
derived from a subject having normal glucose levels. Alternatively,
the kits can comprise a control sample derived from a subject who
has been diagnosed with or identified as suffering from type 2
Diabetes or a pre-diabetic condition. In one embodiment, the
diagnostic kit comprises (a) an antibody (e.g., fibrinogen ccC
domain peptide) conjugated to a solid support and (b) a second
antibody of the invention conjugated to a detectable group. The
reagents may also include ancillary agents such as buffering agents
and protein stabilizing agents, e.g., polysaccharides and the like.
The diagnostic kit may further include, where necessary, other
members of the signal-producing system of which system the
detectable group is a member (e.g., enzyme substrates), agents for
reducing background interference in a test, control reagents,
apparatus for conducting a test, and the like. Alternatively, a
test kit contains (a) an antibody of the invention, and (b) a
specific binding partner for the antibody conjugated to a
detectable group. The test kit may be packaged in any suitable
manner, typically with all elements in a single container,
optionally with a sheet of printed instructions for carrying out
the test.
[0094] For example, T2DBMARKER detection reagents can be
immobilized on a solid matrix such as a porous strip to form at
least one T2DBMARKER detection site. The measurement or detection
region of the porous strip may include a plurality of sites
containing a nucleic acid. A test strip may also contain sites for
negative and/or positive controls. Alternatively, control sites can
be located on a separate strip from the test strip. Optionally, the
different detection sites may contain different amounts of
immobilized nucleic acids, e.g., a higher amount in the first
detection site and lesser amounts in subsequent sites. Upon the
addition of test sample, the number of sites displaying a
detectable signal provides a quantitative indication of the amount
of T2DBMARKERS present in the sample. The detection sites may be
configured in any suitably detectable shape and are typically in
the shape of a bar or dot spanning the width of a test strip.
[0095] Alternatively, the kit contains a nucleic acid substrate
array comprising one or more nucleic acid sequences. The nucleic
acids on the array specifically identify one or more nucleic acid
sequences represented by T2DBMARKERS 1-548. In various embodiments,
the expression of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40,
50, or more of the T2DBMARKERS 1-548 can be identified by virtue of
binding to the array. The substrate array can be on, e.g., a solid
substrate, e.g., a "chip" as described in U.S. Pat. No. 5,744,305.
Alternatively, the substrate array can be a solution array, e.g.,
xMAP (Luminex, Austin, Tex.), Cyvera (Illumina, San Diego, Calif.),
CellCard (Vitra Bioscience, Mountain View, Calif.) and Quantum
Dots' Mosaic (Invitrogen, Carlsbad, Calif.).
[0096] The skilled artisan can routinely make antibodies, nucleic
acid probes, e.g., oligonucleotides, aptamers, siRNAs, anti sense
oligonucleotides, against any of the T2DBMARKERS in Table 1. The
Examples presented herein describe generation of monoclonal
antibodies in mice, as well as generation of polyclonal hyperimmune
serum from rabbits. Such techniques are well-known to those of
ordinary skill in the art.
Peptides, Proteins, and Nucleic Acids of the Invention
[0097] As used herein, a "protein," "polypeptide," or "peptide"
generally refers, but is not limited to, a protein of greater than
about 200 amino acids, up to a full length sequence translated from
a gene; a polypeptide of greater than about 100 amino acids; and/or
a peptide of from about 3 to about 100 amino acids. For
convenience, the terms "protein," "polypeptide" and "peptide" are
used interchangeably herein.
[0098] The size of at least one protein or peptide may comprise,
but is not limited to, 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, 30,
31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,
48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,
65, 66, 67, 68, 69, 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, 100, about 110, about 120, about 130, about 140, about 150,
about 160, about 170, about 180, about 190, about 200, about 210,
about 220, about 230, about 240, about 250, about 275, about 300,
about 325, about 350, about 375, about 400, about 425, about 450,
about 475, about 500, about 525, about 550, about 575, about 600,
about 625, about 650, about 675, about 700, about 725, about 750,
about 775, about 800, about 825, about 850, about 875, about 900,
about 925, about 950, about 975, about 1000, about 1100, about
1200, about 1300, about 1400, about 1500, about 1750, about 2000,
about 2250, about 2500 or greater amino acid residues.
[0099] The peptides of the invention can be isolated, synthetic, or
recombinant peptides that can be about 7 to 100 amino acids in
length, comprising one or more of the motifs "FNRPFL",
"FMS/GKVT/VNP", "R[S/K]XXPP" or "SXXPP", preferably of 100 amino
acids or less. Peptide inhibitors of about 7-100 amino acid
residues or greater are believed to be sufficient to inhibit kinase
activity. A peptide of the invention may be 50, 30, 20, 10 or 5
amino acids or less, including all intervening peptide lengths. The
peptide may comprise 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, or 18 contiguous amino acids of one or more peptide sequences
identified herein. The peptide inhibitors can be about 10-85 amino
acid residues in length. Inhibitors of 12-60 amino acid residues in
length are preferred, with a length of 12-50 amino acids being more
preferred, and 12-40 amino acids most preferred. A peptide
inhibitor of the invention may include, but is not limited to, one
or more of the amino acid sequences provided in SEQ ID NO: 1, SEQ
ID NO:2, and SEQ ID NO:3. The peptide inhibitors of the present
invention have been shown to strongly inhibit kinases implicated in
Diabetes (including, without limitation, mitogen activated protein
(MAP) kinases such as p70S6K, protein kinase .beta. isoforms such
as PKBP, protein kinase C isoforms such as PKC4, and SGK) with
IC.sub.50 values ranging from 0.3 to 2 .mu.M, as measured by in
vitro kinase assay (see Example 6).
[0100] As used herein, an "amino acid residue" refers to any
naturally occurring amino acid, any amino acid derivative or any
amino acid mimic known in the art. In certain embodiments, the
residues of the protein or peptide are sequential, without any
non-amino acid interrupting the sequence of amino acid residues. In
other embodiments, the sequence may comprise one or more non-amino
acid moiety. In particular embodiments, the sequence of residues of
the protein or peptide may be interrupted by one or more non-amino
acid moieties.
[0101] Accordingly, the term "protein, polypeptide, or peptide"
encompasses amino acid sequences comprising at least one of the 20
common amino acids found in naturally occurring proteins, or at
least one modified or unusual amino acid, including but not limited
to Aad, 2-Aminoadipic acid; EtAsn, N-Ethylasparagine; Baad,
3-Aminoadipic acid, Hyl, Hydroxylysine; Bala, .beta.-alanine,
.beta.-Amino-propionic acid; AHyl, allo-Hydroxylysine; Abu,
2-Aminobutyric acid; 3Hyp, 3-Hydroxyproline; 4Abu, 4-Aminobutyric
acid, piperidinic acid; 4Hyp, 4-Hydroxyproline; Acp, 6-Aminocaproic
acid, Ide, Isodesmosine; Ahe, 2-Aminoheptanoic acid; AIle,
allo-Isoleucine; Aib, 2-Aminoisobutyric acid; MeGly,
N-Methylglycine, sarcosine; Baib, 3-Aminoisobutyric acid; MeIle,
N-Methylisoleucine; Apm, 2-Aminopimelic acid; MeLys,
6-N-Methyllysine; Dbu, 2,4-Diaminobutyric acid; MeVal,
N-Methylvaline; Des, Desmosine; Nva, Norvaline; Dpm,
2,2'-Diaminopimelic acid; Nle, Norleucine; Dpr,
2,3-Diaminopropionic acid; Orn, Ornithine; and EtGly,
N-Ethylglycine.
[0102] The amino acid residues described herein are preferably in
the "L" isomeric form. However, residues in the "D" isomeric form
can be substituted for any L-amino acid residue, as long as the
peptide inhibitors retain the ability to inhibit kinases. This
definition includes, unless otherwise specifically indicated,
chemically-modified amino acids, including amino acid analogs (such
as penicillamine, 3-mercapto-D-valine), naturally-occurring
non-proteogenic amino acids (such as norleucine), and
chemically-synthesized compounds that have properties known in the
art to be characteristic of an amino acid. The term "proteogenic"
indicates that the amino acid can be incorporated into a protein in
a cell through metabolic pathways well-known to those skilled in
the art.
[0103] Proteins or peptides may be made by any technique known to
those of skill in the art, including the expression of proteins,
polypeptides or peptides through standard molecular biological
techniques, the isolation of proteins or peptides from natural
sources, or the chemical synthesis of proteins or peptides. The
nucleotide and protein, polypeptide and peptide sequences
corresponding to various genes have been previously disclosed, and
may be found at computerized databases known to those of ordinary
skill in the art. One such database is the National Center for
Biotechnology Information's Genbank and GenPept databases, which
are well known to those skilled in the art. The coding regions for
known genes may be amplified and/or expressed using the techniques
disclosed herein or as would be know to those of ordinary skill in
the art. Alternatively, various commercial preparations of
proteins, polypeptides and peptides are known to those of skill in
the art.
[0104] The peptide can be a peptide "mimetic". Thus, one aspect of
the present invention provides for peptidomimetics which mimic the
structural features of the critical amino acid motif "FNRPFL",
"FMS/GKVT/VNP", "R[S/K]XXPP" or "SXXPP". Although most kinase
inhibitors are expected to be peptides, other non-peptide
inhibitors of kinases can be identified. The peptidomimetics that
are non-peptide in nature can be designed and synthesized by
standard organic chemical methods. The peptidomimetics that are
non-peptide in nature can be even more advantageous in therapeutic
use, by displaying properties such as resistance to degradation,
cell permeability, and the ability to be formulated for oral
administration.
[0105] Peptidomimetics are small molecules that can bind to
proteins by mimicking certain structural aspects of peptides and
proteins. See, for example, Johnson et al., 1993, incorporated
herein by reference. The underlying rationale behind the use of
peptide mimetics is that the peptide backbone of proteins exists
chiefly to orient amino acid side chains in such a way as to
facilitate molecular interactions, such as those of antibody and
antigen. A peptide mimetic is expected to permit molecular
interactions similar to the natural molecule. These principles may
be used to engineer second generation molecules having many of the
natural properties of the targeting peptides disclosed herein, but
with altered and even improved characteristics. They are used
extensively as agonists and antagonists of protein and peptide
ligands of cellular receptors and as substrates and substrate
analogs for enzymes. Some examples include, without limitation,
morphine alkaloids (such as, for example, naturally-occurring
endorphin analogs), penicillins (semi-synthetic), and HIV protease
inhibitors (synthetic). Such compounds can comprise structural
features that mimic a peptide or a protein and as such are
recognized and bound by other proteins. Binding the peptidomimetic
either induces the binding protein to carry out the normal function
caused by such binding (agonist) or disrupts such function
(antagonist, inhibitor).
[0106] One goal in the design of peptide mimetics has been to
reduce the susceptibility of mimetics to cleavage and inactivation
by peptidases. In one approach, such as disclosed by Sherman et al
(1990), one or more amide bonds have been replaced in an
essentially isosteric manner by a variety of chemical functional
groups. This stepwise approach has met with some success in that
active analogs have been obtained. In some instances, these analogs
have been shown to possess longer biological half-lives than their
naturally-occurring counterparts. In another approach, a variety of
uncoded or modified amino acids, such as D-amino acids and N-methyl
amino acids, have been used to modify peptides. In yet other
approaches, a presumed bioactive conformation can be stabilized by
a covalent modification, such as cyclization or by incorporation of
.gamma.-lactam or other types of bridges. See, e.g., Veber et al
(1978) and Thorsett et al (1983). Another approach by Rich (1986)
involves designing peptide mimics through the application of the
transition state analog concept in enzyme inhibitor design. For
example, it is known that the secondary alcohol of statine mimics
the tetrahedral transition state of the sessile amide bond of the
pepsin substrate. Nicolaou et al (1990) disclosed non-peptide
somatostatin mimics.
[0107] U.S. Pat. No. 5,552,534 discloses non-peptide compounds that
can mimic or inhibit the chemical and/or biological activity of a
variety of peptides. These non-peptide compounds can be produced by
appending to certain core species, such as the tetrahydropyranyl
ring, chemical functional groups which cause the compounds to be at
least partially crossreactive with the peptide. Compounds which
mimic or inhibit peptides can be, in varying degrees, crossreactive
with each other. Other techniques for preparing peptidomimetics are
disclosed in, without limitation, U.S. Pat. Nos. 5,550,251 and
5,288,707.
[0108] Protein phosphorylation plays a crucial part in the
biochemical control of cellular activity. Phosphorylation usually
means formation of a phosphate ester bond between a phosphate
(PO.sub.4) group and an amino acid containing a hydroxyl (OH) group
(such as tyrosine, serine and threonine). Many phosphorylation
sites in proteins act as recognition elements for binding to other
proteins, and those binding events activate or deactivate signaling
and other pathways. Protein phosphorylation thus acts as a switch
to turn biochemical signaling on and off. Phosphopeptide mimetics
are a subclass of peptidomimetics that contain analogs of
phosphorylated tyrosine, serine and threonine. Phosphate esters may
be hydrolyzed by various enzymes, thus turning off a
phosphorylation signal. Phosphopeptide mimetics, however, usually
contain non-hydrolyzable analogs to prevent inactivation (Burke et
al, 1994a; Burke et al, 1996a; Chen et al, 1995; Wiemann et al,
2000; Shapiro et al, 1997; Otaka et al, 1995; Otaka et al, 2000).
General examples of phosphopeptide mimetics in the art include SH2
domain analogs (Burke et al, 1994a; Fu et al, 1998; Gao et al,
2000; Mikol et al, 1995; Ye et al, 1995), transcription factor
NF-(kappa)B analog (McKinsey et al, 1997), P53 analog (Higashimoto
et al, 2000) and protein-tyrosine phosphatase inhibitors (Burke et
al, 1994b; Burke et al, 1996b; Groves et al, 1998; Kole et al,
1995; Kole et al, 1997; Roller et al, 1998).
[0109] Commercially available software packages can be used to
design small peptides and/or peptidomimetics containing,
phosphoserine or phosphothreonine analogs, preferably
non-hydrolyzable analogs, as specific antagonists/inhibitors.
Suitable commercially available software for analyzing crystal
structure, designing and optimizing small peptides and
peptidomimetics include, but are not limited to: Macromolecular
X-ray Crystallography QUANTA Environment (Molecular Simulations,
Inc.); TeXsan, BioteX, and SQUASH (Molecular Structure
Corporation); and Crystallographica (Oxford Cryostsystems).
[0110] The peptide inhibitors of the present invention also include
salts and chemical derivatives of the peptides. "Chemical
derivative" can refer to a peptide of the invention having one or
more residues chemically derivatized by reaction of a functional
side group. Such derivatized molecules can include, for example,
those molecules in which free amino groups have been derivatized to
form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy
groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl
groups. Free carboxyl groups may be derivatized to form salts,
methyl and ethyl esters or other types of esters or hydrazides.
Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl
derivatives. Also included as chemical derivatives are those
peptides that contain one or more naturally occurring amino acid
derivatives of the twenty standard amino acids. For example,
4-hydroxyproline may be substituted for proline; 5-hydroxylysine
may be substituted for lysine; 3-methylhistidine may be substituted
for histidine; homoserine may be substituted for serine; and
ornithine may be substituted for lysine. The chemical
derivatization does not comprehend changes in functional groups
which change one amino acid to another.
[0111] Some useful modifications are designed to increase the
stability of the peptide inhibitor in solution and, therefore,
serve to prolong the half-life of the peptide inhibitor in
solutions, particularly biological fluids, such as blood, plasma or
serum, by blocking proteolytic activity in the blood. A peptide
inhibitor can have a stabilizing group at one or both termini.
Typical stabilizing groups include amido, acetyl, benzyl, phenyl,
tosyl, alkoxycarbonyl, alkyl carbonyl, benzyloxycarbonyl and the
like end group modifications. Additional modifications include
using a "L" amino acid in place of a "D" amino acid at the termini,
cyclization of the peptide inhibitor, and amide rather than amino
or carboxy termini to inhibit exopeptidase activity.
[0112] A peptide inhibitor of the invention may or may not be
glycosylated. The peptide inhibitors are not glycosylated, for
example, when produced directly by peptide synthesis techniques or
are produced in a prokaryotic cell transformed with a recombinant
polynucleotide. Peptide molecules produced in eukaryotic expression
systems (such as, for example, Saccharomyces cerevisiae-based
expression systems, baculovirus-based expression systems utilizing
for example, Sf9 insect cells, and mammalian expression systems)
are typically glycosylated.
[0113] The peptide inhibitors of the invention can be produced by
well known chemical procedures, such as solution or solid-phase
peptide synthesis, or semi-synthesis in solution beginning with
protein fragments coupled through conventional solution methods, as
described by Dugas et al (1981). Alternatively, a peptide inhibitor
of the invention can be synthesized by using well known methods,
including recombinant methods and chemical synthesis.
[0114] A peptide inhibitor of the invention can be chemically
synthesized, for example, by the solid phase peptide synthesis of
Merrifield et al (1964). Alternatively, a peptide inhibitor of the
invention can be synthesized using standard solution methods (see,
for example, Bodanszky, 1984). Newly synthesized peptides can be
purified, for example, by high performance liquid chromatography
(HPLC), and can be characterized using, for example, mass
spectrometry or amino acid sequence analysis.
[0115] The peptide inhibitors of the invention can be particularly
useful when they are maintained in a constrained secondary
conformation. The terms "constrained secondary structure,"
"stabilized" and "conformationally stabilized" indicate that the
peptide bonds comprising the peptide are not able to rotate freely
but instead are maintained in a relatively fixed structure. A
method for constraining the secondary structure of a newly
synthesized linear peptide is to cyclize the peptide using any of
various methods well known in the art. For example, a cyclized
peptide inhibitor of the invention can be prepared by forming a
peptide bond between non-adjacent amino acid residues as described,
for example, by Schiller et al (1985). Peptides can be synthesized
on the Merrifield resin by assembling the linear peptide chain
using N.alpha.-Fmoc-amino acids and Boc and tertiary-butyl
proteins. Following the release of the peptide from the resin, a
peptide bond can be formed between the amino and carboxy
termini.
[0116] A newly synthesized linear peptide can also be cyclized by
the formation of a bond between reactive amino acid side chains.
For example, a peptide containing a cysteine-pair can be
synthesized, with a disulfide bridge, can be formed by oxidizing a
dilute aqueous solution of the peptide with K.sub.3Fe(CN).sub.6.
Alternatively, a lactam such as an .di-elect
cons.-(.gamma.-glutamyl)-lysine bond can be formed between lysine
and glutamic acid residues, a lysinonorleucine bond can be formed
between lysine and leucine residues or a dityrosine bond can be
formed between two tyrosine residues. Cyclic peptides can be
constructed to contain, for example, four lysine residues, which
can form the heterocyclic structure of desmosine (see, for example,
Devlin, 1997). Methods for forming these and other bonds are well
known in the art and are based on well-known rules of chemical
reactivity (Morrison et al, 1992).
[0117] Alternatively, the peptide inhibitor of the invention can be
produced recombinantly. Systems for cloning and expressing
polypeptide of the invention include various microorganisms and
cells that are well known in recombinant technology. These include,
for example, various strains of E. coli, Bacillus, Streptomyces,
and Saccharomyces, as well as mammalian, yeast and insect cells.
The peptide inhibitor of the invention can be produced as a peptide
or fusion protein. Suitable vectors for producing the peptide
inhibitor are known and available from private and public
laboratories and depositories and from commercial vendors. See
Sambrook et al, (1989). Recipient cells capable of expressing the
gene product are then transfected. The transfected recipient cells
are cultured under conditions that permit expression of the
recombinant gene products, which are recovered from the culture.
Host mammalian cells, such as Chinese Hamster ovary cells (CHO) or
COS-1 cells, can be used. These hosts can be used in connection
with poxvirus vectors, such as vaccinia or swinepox. Suitable
non-pathogenic viruses that can be engineered to carry the
synthetic gene into the cells of the host include poxviruses, such
as vaccinia, adenovirus, retroviruses and the like. A number of
such non-pathogenic viruses are commonly used for human gene
therapy, and as carrier for other vaccine agents, and are known and
selectable by one of skill in the art. The selection of other
suitable host cells and methods for transformation, culture,
amplification, screening and product production and purification
can be performed by one of skill in the art by reference to known
techniques (see, e.g., Gething et al, 1981).
[0118] The isolated, synthetic, or recombinant peptide may be
attached to a macromolecular complex. The macromolecular complex
can be, without limitation, a virus, a bacteriophage, a bacterium,
a liposome, a microparticle, a nanoparticle (e.g., a gold
nanoparticle), a magnetic bead, a yeast cell, a mammalian cell, a
cell or a microdevice. These are representative examples only and
macromolecular complexes within the scope of the present invention
can include virtually any complex that can be attached to a peptide
inhibitor and administered to a subject. The isolated, synthetic,
or recombinant peptide may also be attached to a eukaryotic
expression vector, more preferably a gene therapy vector.
[0119] The isolated peptide can be attached to a solid support,
such as, for example, magnetic beads, Sepharose beads, agarose
beads, a nitrocellulose membrane, a nylon membrane, a column
chromatography matrix, a high performance liquid chromatography
(HPLC) matrix or a fast performance liquid chromatography (FPLC)
matrix.
Other embodiments concern fusion proteins. These molecules
generally have all or a substantial portion of the peptides of the
invention, linked at the N- or C-terminus, to all or a portion of a
second polypeptide or protein. For example, fusions may employ
leader sequences from other species to permit the recombinant
expression of a protein in a heterologous host. Another useful
fusion includes the addition of an immunologically active domain,
such as an antibody epitope, to facilitate purification of the
fusion protein. Inclusion of a cleavage site at or near the fusion
junction will facilitate removal of the extraneous polypeptide
after purification. Other useful fusions include linking of
functional domains, such as, for example, active sites from
enzymes, glycosylation domains, cellular targeting signals or
transmembrane regions.
[0120] The fusion proteins of the instant invention can comprise a
peptide of the invention linked to a therapeutic protein or
peptide. Examples of proteins or peptides that may be incorporated
into a fusion protein include, but are not limited to, cytostatic
proteins, cytocidal proteins, pro-apoptosis agents, anti-angiogenic
agents, hormones, cytokines, growth factors, peptide drugs,
antibodies, Fab fragments antibodies, antigens, receptor proteins,
enzymes, lectins, MHC proteins, cell adhesion proteins and binding
proteins. These examples are not meant to be limiting and it is
contemplated that within the scope of the present invention
virtually any protein or peptide could be incorporated into a
fusion protein comprising a targeting peptide. Methods of
generating fusion proteins are well known to those of skill in the
art. Such proteins can be produced, for example, by chemical
attachment using bifunctional cross-linking reagents, by de novo
synthesis of the complete fusion protein, or by attachment of a DNA
sequence encoding the targeting peptide to a DNA sequence encoding
the second peptide or protein, followed by expression of the intact
fusion protein.
[0121] In certain embodiments, it may be desirable to couple
specific bioactive agents to one or more targeting moieties for
targeted delivery to an organ, tissue or cell type. Such agents
include, but are not limited to, cytokines, chemokines,
pro-apoptosis factors and anti-angiogenic factors. The term
"cytokine" is a generic term for proteins released by one cell
population that act on another cell as intercellular mediators.
[0122] Examples of such cytokines are lymphokines, monokines,
growth factors and traditional polypeptide hormones. Included among
the cytokines are growth hormones such as human growth hormone,
N-methionyl human growth hormone, and bovine growth hormone;
parathyroid hormone; thyroxine; insulin; proinsulin; relaxin;
prorelaxin; glycoprotein hormones such as follicle stimulating
hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing
hormone (LH); hepatic growth factor; prostaglandin, fibroblast
growth factor; prolactin; placental lactogen, OB protein; tumor
necrosis factor-.alpha. and -.beta.; mullerian-inhibiting
substance; mouse gonadotropin-associated peptide; inhibin; activin;
vascular endothelial growth factor; integrin; thrombopoietin (TPO);
nerve growth factors such as NGF-.beta.; platelet-growth factor;
transforming growth factors (TGFs) such as TGF-.alpha. and
TGF-.beta.; insulin-like growth factor-I and -II; erythropoietin
(EPO); osteoinductive factors; interferons such as
interferon-.alpha., -..beta., and -.gamma.; colony stimulating
factors (CSFs) such as macrophage-CSF (M-CSF);
granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);
interleukins (ILs) such as IL-1, IL-1.alpha., IL-2, IL-3, IL-4,
IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-13, IL-14,
IL-15, IL-16, IL-17, IL-18, LIF, G-CSF, GM-CSF, M-CSF, EPO,
kit-ligand or FLT-3, angiostatin, thrombospondin, endostatin, tumor
necrosis factor and LT. As used herein, the term cytokine includes
proteins from natural sources or from recombinant cell culture and
biologically active equivalents of the native sequence
cytokines.
[0123] Chemokines generally act as chemoattractants to recruit
immune effector cells to the site of chemokine expression. It may
be advantageous to express a particular chemokine gene in
combination with, for example, a cytokine gene, to enhance the
recruitment of other immune system components to the site of
treatment. Chemokines include, but are not limited to, RANTES,
MCAF, MIP1-alpha, MIP1-Beta, and IP-10. The skilled artisan will
recognize that certain cytokines are also known to have
chemoattractant effects and could also be classified under the term
chemokines.
[0124] In certain embodiments, the targeting moieties of the
present invention may be attached to imaging agents of use for
imaging and diagnosis of various diseased organs, tissues or cell
types. Many appropriate imaging agents are known in the art, as are
methods for their attachment to proteins or peptides (see, e.g.,
U.S. Pat. Nos. 5,021,236 and 4,472,509, both incorporated herein by
reference). Certain attachment methods involve the use of a metal
chelate complex employing, for example, an organic chelating agent
such a DTPA attached to the protein or peptide (U.S. Pat. No.
4,472,509). Proteins or peptides also may be reacted with an enzyme
in the presence of a coupling agent such as glutaraldehyde or
periodate. Conjugates with fluorescein markers are prepared in the
presence of these coupling agents or by reaction with an
isothiocyanate.
[0125] Non-limiting examples of paramagnetic ions of potential use
as imaging agents include chromium (III), manganese (II), iron
(III), iron (II), cobalt (II), nickel (II), copper (II), neodymium
(III), samarium (III), ytterbium (III), gadolinium (III), vanadium
(II), terbium (III), dysprosium (III), holmium (III) and erbium
(III), with gadolinium being particularly preferred. Ions useful in
other contexts, such as X-ray imaging, include but are not limited
to lanthanum (III), gold (III), lead (II), and especially bismuth
(III).
[0126] Radioisotopes of potential use as imaging or therapeutic
agents include .sup.211astatine, .sup.14carbon, .sup.51chromium,
.sup.36chlorine, .sup.57cobalt, .sup.58cobalt, .sup.67copper,
.sup.152Eu, .sup.67gallium, .sup.3hydrogen, .sup.123iodine,
.sup.125iodine, .sup.131iodine, .sup.111indium, .sup.59iron,
.sup.32phosphorus, .sup.186rhenium, .sup.188rhenium,
.sup.75selenium, .sup.35sulphur, .sup.99mtechnicium and
.sup.90yttrium. .sup.125I is often being preferred for use in
certain embodiments, and .sup.99mtechnicium and .sup.111indium are
also often preferred due to their low energy and suitability for
long range detection.
[0127] Radioactively labeled proteins or peptides of the present
invention may be produced according to well-known methods in the
art. For instance, they can be iodinated by contact with sodium or
potassium iodide and a chemical oxidizing agent such as sodium
hypochlorite, or an enzymatic oxidizing agent, such as
lactoperoxidase. Proteins or peptides according to the invention
may be labeled with technetium-99m by ligand exchange process, for
example, by reducing pertechnate with stannous solution, chelating
the reduced technetium onto a Sephadex column and applying the
peptide to this column or by direct labeling techniques, e.g., by
incubating pertechnate, a reducing agent such as SNCl.sub.2, a
buffer solution such as sodium-potassium phthalate solution, and
the peptide. Intermediary functional groups that are often used to
bind radioisotopes that exist as metallic ions to peptides are
diethylenetriaminepenta-acetic acid (DTPA) and ethylene
diaminetetra-acetic acid (EDTA). Also contemplated for use are
fluorescent labels, including rhodamine, fluorescein isothiocyanate
and renographin.
[0128] In certain embodiments, the claimed proteins or peptides may
be linked to a secondary binding ligand or to an enzyme (an enzyme
tag) that will generate a colored product upon contact with a
chromogenic substrate. Examples of suitable enzymes include urease,
alkaline phosphatase, (horseradish) hydrogen peroxidase and glucose
oxidase. Preferred secondary binding ligands are biotin and avidin
or streptavidin compounds. The use of such labels is well known to
those of skill in the art in light and is described, for example,
in U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;
4,277,437; 4,275,149 and 4,366,241; each incorporated herein by
reference.
[0129] In still further embodiments, a targeting moiety may be
operatively coupled to a nanoparticle. Nanoparticles include, but
are not limited to colloidal gold and silver nanoparticles. Metal
nanoparticles exhibit colors in the visible spectral region. It is
believed that these colors are the result of excitation of surface
plasmon resonances in the metal particles and are extremely
sensitive to particle size, shape, and aggregation state;
dielectric properties of the surrounding medium; adsorption of ions
on the surface of the particles (For examples, see U.S. Patent
Application Publication No. 20040023415, which is incorporated
herein by reference).
[0130] Bifunctional cross-linking reagents have been extensively
used for a variety of purposes including preparation of affinity
matrices, modification and stabilization of diverse structures,
identification of ligand and receptor binding sites, and structural
studies. Homobifunctional reagents that carry two identical
functional groups proved to be highly efficient in inducing
cross-linking between identical and different macromolecules or
subunits of a macromolecule, and linking of polypeptide ligands to
their specific binding sites. Heterobifunctional reagents contain
two different functional groups. By taking advantage of the
differential reactivities of the two different functional groups,
cross-linking can be controlled both selectively and sequentially.
The bifunctional cross-linking reagents can be divided according to
the specificity of their functional groups, e.g., amino,
sulfhydryl, guanidino, indole, carboxyl specific groups. Of these,
reagents directed to free amino groups have become especially
popular because of their commercial availability, ease of synthesis
and the mild reaction conditions under which they can be applied. A
majority of heterobifunctional cross-linking reagents contains a
primary amine-reactive group and a thiol-reactive group.
[0131] Exemplary methods for cross-linking ligands to liposomes are
described in U.S. Pat. Nos. 5,603,872 and 5,401,511, each
specifically incorporated herein by reference in its entirety.
Various ligands can be covalently bound to liposomal surfaces
through the cross-linking of amine residues. Liposomes, in
particular, multilamellar vesicles (MLV) or unilamellar vesicles
such as microemulsified liposomes (MEL) and large unilamellar
liposomes (LUVET), each containing phosphatidylethanolamine (PE),
have been prepared by established procedures. The inclusion of PE
in the liposome provides an active functional residue, a primary
amine, on the liposomal surface for cross-linking purposes. Ligands
such as epidermal growth factor (EGF) have been successfully linked
with PE-liposomes. Ligands are bound covalently to discrete sites
on the liposome surfaces. The number and surface density of these
sites are dictated by the liposome formulation and the liposome
type. The liposomal surfaces may also have sites for non-covalent
association. To form covalent conjugates of ligands and liposomes,
cross-linking reagents have been studied for effectiveness and
biocompatibility. Cross-linking reagents include glutaraldehyde
(GAD), bifunctional oxirane (OXR), ethylene glycol diglycidyl ether
(EGDE), and a water soluble carbodiimide, preferably
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). Through the
complex chemistry of cross-linking, linkage of the amine residues
of the recognizing substance and liposomes is established.
[0132] In another example, heterobifunctional cross-linking
reagents and methods of using the cross-linking reagents are
described (U.S. Pat. No. 5,889,155, specifically incorporated
herein by reference in its entirety). The cross-linking reagents
combine a nucleophilic hydrazide residue with an electrophilic
maleimide residue, allowing coupling in one example, of aldehydes
to free thiols. The cross-linking reagent can be modified to
cross-link various functional groups.
[0133] In certain embodiments a protein or peptide may be isolated
or purified. Protein purification techniques are well known to
those of skill in the art. These techniques involve, at one level,
the homogenization and crude fractionation of the cells, tissue or
organ to polypeptide and non-polypeptide fractions. The protein or
polypeptide of interest may be further purified using
chromatographic and electrophoretic techniques to achieve partial
or complete purification (or purification to homogeneity).
Analytical methods particularly suited to the preparation of a pure
peptide are ion-exchange chromatography, gel exclusion
chromatography, polyacrylamide gel electrophoresis, affinity
chromatography, immunoaffinity chromatography and isoelectric
focusing. An example of receptor protein purification by affinity
chromatography is disclosed in U.S. Pat. No. 5,206,347, the entire
text of which is incorporated herein by reference. A particularly
efficient method of purifying peptides is fast performance liquid
chromatography (FPLC) or even high performance liquid
chromatography (HPLC).
[0134] A purified protein or peptide is intended to refer to a
composition, isolatable from other components, wherein the protein
or peptide is purified to any degree relative to its
naturally-obtainable state. An isolated or purified protein or
peptide, therefore, also refers to a protein or peptide free from
the environment in which it may naturally occur. Generally,
"purified" will refer to a protein or peptide composition that has
been subjected to fractionation to remove various other components,
and which composition substantially retains its expressed
biological activity. Where the term "substantially purified" is
used, this designation will refer to a composition in which the
protein or peptide forms the major component of the composition,
such as constituting about 50%, about 60%, about 70%, about 80%,
about 90%, about 95%, or more of the proteins in the
composition.
[0135] Various methods for quantifying the degree of purification
of the protein or peptide are known to those of skill in the art in
light of the present disclosure. These include, for example,
determining the specific activity of an active fraction, or
assessing the amount of polypeptides within a fraction by SDS/PAGE
analysis. A preferred method for assessing the purity of a fraction
is to calculate the specific activity of the fraction, to compare
it to the specific activity of the initial extract, and to thus
calculate the degree of purity therein, assessed by a "-fold
purification number." The actual units used to represent the amount
of activity will, of course, be dependent upon the particular assay
technique chosen to follow the purification, and whether or not the
expressed protein or peptide exhibits a detectable activity.
[0136] Various techniques suitable for use in protein purification
are well known to those of skill in the art. These include, for
example, precipitation with ammonium sulfate, polyethylene glycol
(PEG), antibodies and the like, or by heat denaturation, followed
by: centrifugation; chromatography steps such as ion exchange, gel
filtration, reverse phase, hydroxyapatite and affinity
chromatography; isoelectric focusing; gel electrophoresis; and
combinations of these and other techniques. As is generally known
in the art, it is believed that the order of conducting the various
purification steps may be changed, or that certain steps may be
omitted, and still result in a suitable method for the preparation
of a substantially purified protein or peptide.
[0137] There is no general requirement that the protein or peptide
always be provided in their most purified state. Indeed, it is
contemplated that less substantially purified products will have
utility in certain embodiments. Partial purification may be
accomplished by using fewer purification steps in combination, or
by utilizing different forms of the same general purification
scheme. For example, it is appreciated that a cation-exchange
column chromatography performed utilizing an HPLC apparatus will
generally result in a greater "-fold" purification than the same
technique utilizing a low pressure chromatography system. Methods
exhibiting a lower degree of relative purification may have
advantages in total recovery of protein product, or in maintaining
the activity of an expressed protein.
[0138] Affinity chromatography is a chromatographic procedure that
relies on the specific affinity between a substance to be isolated
and a molecule to which it can specifically bind. This is a
receptor-ligand type of interaction. The column material is
synthesized by covalently coupling one of the binding partners to
an insoluble matrix. The column material is then able to
specifically adsorb the substance from the solution. Elution occurs
by changing the conditions to those in which binding will not occur
(e.g., altered pH, ionic strength, temperature, etc.). The matrix
should be a substance that itself does not adsorb molecules to any
significant extent and that has a broad range of chemical, physical
and thermal stability. The ligand should be coupled in such a way
as to not affect its binding properties. The ligand should also
provide relatively tight binding. And it should be possible to
elute the substance without destroying the sample or the
ligand.
[0139] The invention also concerns isolated nucleic acids encoding
the peptide inhibitors described herein. A "nucleic acid" as used
herein includes single-stranded and double-stranded molecules, as
well as DNA, RNA, chemically modified nucleic acids and nucleic
acid analogs. It is contemplated that a nucleic acid within the
scope of the present invention may be of almost any size,
determined in part by the length of the encoded protein or peptide.
Nucleic acids according to the present invention may encode a
peptide/peptide inhibitor, a targeting antibody, a therapeutic
polypeptide a fusion protein or other protein or peptide. The
nucleic acid may be derived from genomic DNA, complementary DNA
(cDNA) or synthetic DNA.
[0140] In certain aspects, the nucleic acids may be 300 nucleotides
or less in length. In still further embodiments the nucleic acids
may be 270, 240, 210, 180, 150, 120, 90, 60, 30 or even 9
nucleotides in length. Exemplary non-limiting nucleic acid
sequences include those that encode the peptides provided in SEQ ID
NO:1, SEQ ID NO:2, or SEQ ID NO:3.
[0141] It is contemplated that the peptides, antibodies, and fusion
proteins of the invention may be encoded by any nucleic acid
sequence that encodes the appropriate amino acid sequence. The
design and production of nucleic acids encoding a desired amino
acid sequence is well known to those of skill in the art, using
standardized codon tables. In preferred embodiments, the codons
selected for encoding each amino acid may be modified to optimize
expression of the nucleic acid in the host cell of interest.
[0142] There are a number of ways in which gene therapy vectors may
introduced into cells. One or more isolated nucleic acid can be
incorporated into a eukaryotic or a prokaryotic expression vector.
The vector can be, without limitation, a plasmid, a cosmid, a yeast
artificial chromosome (YAC), a bacterial artificial chromosome
(BAC), a virus or a bacteriophage. The isolated nucleic acid can
also be operatively linked to a leader sequence that localizes the
expressed peptide to the extracellular surface of a host cell, or
to a specific organelle within the host cell (such as, for example,
localization to the nucleus of a cell, via a nuclear localization
sequence, or NLS). In certain embodiments of the invention, the
gene therapy vector comprises a virus. The ability of certain
viruses to enter cells via receptor-mediated endocytosis, to
integrate into host cell genome or be maintained episomally, and
express viral genes stably and efficiently have made them
attractive candidates for the transfer of foreign genes into
mammalian cells (Ridgeway, 1988; Nicolas and Rubinstein, 1988.;
Baichwal and Sugden, 1986; Temin, 1986). Preferred gene therapy
vectors are generally viral vectors. DNA viruses used as gene
therapy vectors include the papovaviruses (e.g., simian virus 40,
bovine papilloma virus, and polyoma) (Ridgeway, 1988; Baichwal and
Sugden, 1986) and adenoviruses (Ridgeway, 1988; Baichwal and
Sugden, 1986).
[0143] Other gene transfer vectors may be constructed from
retroviruses. (Coffin, 1990.) In order to construct a retroviral
vector, a nucleic acid encoding protein of interest is inserted
into the viral genome in the place of certain viral sequences to
produce a virus that is replication-defective. In order to produce
virions, a packaging cell line containing the gag, pol, and env
genes, but without the LTR and packaging components, is constructed
(Mann et al., 1983). When a recombinant plasmid containing a cDNA,
together with the retroviral LTR and packaging sequences is
introduced into this cell line (by calcium phosphate precipitation
for example), the packaging sequence allows the RNA transcript of
the recombinant plasmid to be packaged into viral particles, which
are then secreted into the culture media (Nicolas and Rubenstein,
1988; Temin, 1986; Mann et al., 1983). The media containing the
recombinant retroviruses is then collected, optionally
concentrated, and used for gene transfer. Retroviral vectors are
capable of infecting a broad variety of cell types. However,
integration and stable expression require the division of host
cells (Paskind et al., 1975).
[0144] Other viral vectors may be employed as targeted gene therapy
vectors. Vectors derived from viruses such as vaccinia virus
(Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988),
adeno-associated virus (AAV) (Ridgeway, 1988; Baichwal and Sugden,
1986; Hermonat and Muzycska, 1984), and herpes viruses may be
employed.
[0145] In a further embodiment of the invention, gene therapy
construct may be entrapped in a liposome. Liposome-mediated nucleic
acid delivery and expression of foreign DNA in vitro has been very
successful. Wong et al., (1980) demonstrated the feasibility of
liposome-mediated delivery and expression of foreign DNA in
cultured chick embryo, HeLa, and hepatoma cells. Nicolau et al.,
(1987) accomplished successful liposome-mediated gene transfer in
rats after intravenous injection.
[0146] Gene therapy vectors of the invention may comprise various
transgenes, which are typically encoded DNA or RNA of an expression
vector. DNA may be in form of cDNA, in vitro polymerized DNA,
plasmid DNA, parts of a plasmid DNA, genetic material derived from
a virus, linear DNA, vectors (P1, PAC, BAC, YAC, artificial
chromosomes), expression cassettes, chimeric sequences, recombinant
DNA, chromosomal DNA, an oligonucleotide, anti-sense DNA, or
derivatives of these groups. RNA may be in the form of
oligonucleotide RNA, tRNA (transfer RNA), snRNA (small nuclear
RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), in vitro
polymerized RNA, recombinant RNA, chimeric sequences, anti-sense
RNA, siRNA (small interfering RNA), ribozymes, or derivatives of
these groups. An anti-sense polynucleotide is a polynucleotide that
interferes with the function of DNA and/or RNA. Antisense
polynucleotides include, but are not limited to: morpholinos,
2'-O-methyl polynucleotides, DNA, RNA and the like. SiRNA comprises
a double stranded structure typically containing 15-50 base pairs
and preferably 21-25 base pairs and having a nucleotide sequence
identical or nearly identical to an expressed target gene or RNA
within the cell. Interference may result in suppression of
expression. The polynucleotide can also be a sequence whose
presence or expression in a cell alters the expression or function
of cellular genes or RNA. In addition, DNA and RNA may be single,
double, triple, or quadruple stranded.
Antibodies
[0147] The present invention also provides antibodies that are
capable of binding to one or more T2DBMARKERS presented in Table 1,
such as the peptide inhibitors of the invention, and preferably,
antibodies that are capable of binding to one or more amino acids
of SEQ ID NO: 1, 2, or 3. The term "antibody" as used in the
context of the present invention includes polyclonal antibodies,
monoclonal antibodies (mAbs), chimeric antibodies, anti-idiotypic
(anti-Id) antibodies, that can be labeled in soluble or bound form,
as well as fragments, regions, or derivatives thereof, provided by
any known technique, such as, but not limited to, enzymatic
cleavage, peptide synthesis, or recombinant techniques.
[0148] Polyclonal antibodies are heterogeneous populations of
antibody molecules derived from the sera of animals immunized with
an antigen. A monoclonal antibody contains a substantially
homogeneous population of antibodies specific to antigens, which
population contains substantially similar epitope binding sites.
MAbs may be human, murine, monkey, rat, hamster, rabbit, or chicken
in origin and obtained by methods known to those skilled in the
art. See, for example Kohler and Milstein, Nature 256:495-497
(1975); U.S. Pat. No. 4,376,110; Ausubel et al., eds., Current
Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley
Interscience, N.Y., (1987, 1992); and Harlow and Lane ANTIBODIES. A
Laboratory Manual Cold Spring Harbor Laboratory (1988); Colligan et
al., eds., Current Protocols in Immunology, Greene Publishing
Assoc. and Wiley Interscience, N.Y., (1992, 1993), the contents of
which references are incorporated entirely herein by reference.
[0149] Such antibodies may be of any immunoglobulin class including
IgG, IgM, IgE, IgD, IgA, GILD and any subclass thereof. A hybridoma
producing a mAb of the present invention may be cultivated in
vitro, in situ or in vivo. Production of high titers of mAbs in
vivo or in situ makes this a preferred method of production.
[0150] Chimeric antibodies are molecules different portions of
which are derived from different animal species, such as those
having variable region derived from a murine mAb and a human
immunoglobulin constant region, which are primarily used to reduce
immunogenicity in application and to increase yields in production,
for example, where murine mabs have higher yields from hybridomas
but higher immunogenicity in humans, such that human/murine
chimeric mAbs are used. Chimeric antibodies and methods for their
production are known in the art (Cabilly et al., Proc. Natl. Acad.
Sci. USA 81:3273-3277 (1984); Morrison et al., Proc. Natl. Acad.
Sci. USA 81:6851-6855 (1984); Boulianne et al., Nature 312:643-646
(1984); Cabilly et al., European Patent Application 125023
(published Nov. 14, 1984); Neuberger et al., Nature 314:268-270
(1985); Taniguchi et al., European Patent Application 171496
(published Feb. 19, 1985); Morrison et al., European Patent
Application 173494 (published Mar. 5, 1986); Neuberger et al., PCT
Application WO 86/01533, (published Mar. 13, 1986); Kudo et al.,
European Patent Application 184187 (published Jun. 11, 1986);
Morrison et al., European Patent Application 173494 (published Mar.
5, 1986); Sahagan et al., J. Immunol. 137:1066-1074 (1986);
Robinson et al., International Patent Publication No.
PCT/US86/02269 (published 7 May 1987); Liu et al., Proc. Natl.
Acad. Sci. USA 84:3439-3443 (1987); Sun et al., Proc. Natl. Acad.
Sci. USA 84:214-218 (1987); Better et al., Science 240:1041-1043
(1988); and Harlow and Lane Antibodies: a Laboratory Manual Cold
Spring Harbor Laboratory (1988)). These references are entirely
incorporated herein by reference.
[0151] An anti-idiotypic (anti-Id) antibody is an antibody which
recognizes unique determinants generally associated with the
antigen-binding site of an antibody. An Id antibody can be prepared
by immunizing an animal of the same species and genetic type (e.g.,
mouse strain) as the source of the mAb with the mAb to which an
anti-Id is being prepared. The immunized animal will recognize and
respond to the idiotypic determinants of the immunizing antibody by
producing an antibody to these idiotypic determinants (the anti-Id
antibody). See, for example, U.S. Pat. No. 4,699,880, which is
herein entirely incorporated by reference.
[0152] The anti-Id antibody may also be used as an "immunogen" to
induce an immune response in yet another animal, producing a
so-called anti-anti-Id antibody. The anti-anti-Id may be
epitopically identical to the original mAb which induced the
anti-Id. Thus, by using antibodies to the idiotypic determinants of
a mAb, it is possible to identify other clones expressing
antibodies of identical specificity.
[0153] Antibodies of the present invention can include at least one
of a heavy chain constant region (H.sub.c), a heavy chain variable
region (H.sub.v), a light chain variable region (L.sub.v) and a
light chain constant region (L.sub.c), wherein a polyclonal Ab,
monoclonal Ab, fragment and/or regions thereof include at least one
heavy chain variable region (H.sub.v) or light chain variable
region (L.sub.v) which binds a portion of SEQ ID NO: 1, SEQ ID NO:
2, or SEQ ID NO: 3.
Preferred methods for determining mAb specificity and affinity by
competitive inhibition can be found in Harlow, et al., Antibodies:
A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y., 1988), Colligan et al., eds., Current
Protocols in Immunology, Greene Publishing Assoc. and Wiley
Interscience, N.Y., (1992, 1993), and Muller, Meth. Enzymol.
92:589-601 (1983), which references are entirely incorporated
herein by reference.
[0154] The techniques to raise antibodies of the present invention
to small peptide sequences that recognize and bind to those
sequences in the free or conjugated form or when presented as a
native sequence in the context of a large protein are well known in
the art. Such antibodies include murine, murine-human and
human-human antibodies produced by hybridoma or recombinant
techniques known in the art.
[0155] As used herein, the term "antigen binding region" refers to
that portion of an antibody molecule which contains the amino acid
residues that interact with an antigen and confer on the antibody
its specificity and affinity for the antigen. The antibody region
includes the "framework" amino acid residues necessary to maintain
the proper conformation of the antigen-binding residues.
[0156] As used herein, the term "chimeric antibody" includes
monovalent, divalent or polyvalent immunoglobulins. A monovalent
chimeric antibody is a dimer (HL) formed by a chimeric H chain
associated through disulfide bridges with a chimeric L chain. A
divalent chimeric antibody is tetramer (H.sub.2L.sub.2) formed by
two HL dimers associated through at least one disulfide bridge. A
polyvalent chimeric antibody can also be produced, for example, by
employing a C.sub.H region that aggregates (e.g., from an IgM H
chain, or p chain).
[0157] Murine and chimeric antibodies, fragments and regions of the
present invention comprise individual heavy (H) and/or light (L)
immunoglobulin chains. A chimeric H chain comprises an antigen
binding region derived from the H chain of a non-human antibody
specific for one or more T2DBMARKERS or preferably, SEQ ID NO: 1,
SEQ ID NO: 2, or SEQ ID NO: 3, which is linked to at least a
portion of a human H chain C region (C.sub.H), such as CH.sub.1 or
CH.sub.2.
[0158] A chimeric L chain according to the present invention,
comprises an antigen binding region derived from the L chain of a
non-human antibody specific for one or more T2DBMARKERS or
preferably, SEQ ID NO: 1, 2, or 3, linked to at least a portion of
a human L chain C region (C.sub.L). Antibodies, fragments or
derivatives having chimeric H chains and L chains of the same or
different variable region binding specificity, can also be prepared
by appropriate association of the individual polypeptide chains,
according to known method steps, e.g., according to Ausubel,
Harlow, and Colligan, the contents of which references are
incorporated entirely herein by reference. With this approach,
hosts expressing chimeric H chains (or their derivatives) are
separately cultured from hosts expressing chimeric L chains (or
their derivatives), and the immunoglobulin chains are separately
recovered and then associated. Alternatively, the hosts can be
co-cultured and the chains allowed to associate spontaneously in
the culture medium, followed by recovery of the assembled
immunoglobulin, fragment or derivative.
[0159] The hybrid cells are formed by the fusion of a non-human
anti-T2DBMARKER or anti-SEQ ID NO: 1 (e.g., anti-D3 as disclosed in
the Examples) antibody-producing cell, typically a spleen cell of
an animal immunized against either natural or recombinant
T2DBMARKERS or SEQ ID NO: 1, 2, or 3, or a peptide fragment of any
one or more of the T2DBMARKERS or SEQ ID NO: 1, 2, or 3.
Alternatively, the non-human antibody-producing cell can be a B
lymphocyte obtained from the blood, spleen, lymph nodes or other
tissue of an animal immunized with one or more T2DBMARKERS, or the
full or partial amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2,
or SEQ ID NO: 3.
[0160] The second fusion partner, which provides the immortalizing
function, can be a lymphoblastoid cell or a plasmacytoma or myeloma
cell, which is not itself an antibody producing cell, but is
malignant. Preferred fusion partner cells include the hybridoma
SP2/0-Ag14, abbreviated as SP2/0 (ATCC CRL1581) and the myeloma
P3X63Ag8 (ATCC TIB9), or its derivatives. See, e.g, Ausubel,
Harlow, and Colligan, the contents of which are incorporated
entirely herein by reference.
[0161] The antibody-producing cell contributing the nucleotide
sequences encoding the antigen-binding region of the chimeric
antibody of the present invention can also be produced by
transformation of a non-human, such as a primate, or a human cell.
For example, a B lymphocyte which produces an antibody of the
invention can be infected and transformed with a virus such as
Epstein-Barr virus to yield an immortal antibody producing cell
(Kozbor et al., Immunol. Today 4:72-79 (1983)). Alternatively, the
B lymphocyte can be transformed by providing a transforming gene or
transforming gene product, as is well-known in the art. See, e.g,
Ausubel infra, Harlow infra, and Colligan infra, the contents of
which references are incorporated entirely herein by reference.
[0162] Monoclonal antibodies obtained by cell fusions and
hybridomas are accomplished by standard procedures well known to
those skilled in the field of immunology. Fusion partner cell lines
and methods for fusing and selecting hybridomas and screening for
mAbs are well known in the art. See, e.g, Ausubel, Harlow, and
Colligan, the contents of which are incorporated entirely herein by
reference.
[0163] The mAbs of the present invention can be produced in large
quantities by injecting hybridoma or transfectoma cells secreting
the antibody into the peritoneal cavity of mice and, after
appropriate time, harvesting the ascites fluid which contains a
high titer of the mAb, and isolating the mAb therefrom. For such in
vivo production of the mAb with a non-murine hybridoma (e.g., rat
or human), hybridoma cells are preferably grown in irradiated or
athymic nude mice. Alternatively, the antibodies can be produced by
culturing hybridoma or transfectoma cells in vitro and isolating
secreted mAb from the cell culture medium or recombinantly, in
eukaryotic or prokaryotic cells.
[0164] The invention also provides for "derivatives" of the murine
or chimeric antibodies, fragments, regions or derivatives thereof,
which term includes those proteins encoded by truncated or modified
genes to yield molecular species functionally resembling the
immunoglobulin fragments. The modifications include, but are not
limited to, addition of genetic sequences coding for cytotoxic
proteins such as plant and bacterial toxins. The fragments and
derivatives can be produced from any of the hosts of this
invention. Alternatively, antibodies, fragments and regions can be
bound to cytotoxic proteins or compounds in vitro, to provide
cytotoxic antibodies which would selectively kill cells having
receptors corresponding to one or more T2DBMARKERS.
[0165] Fragments include, for example, Fab, Fab', F(ab').sub.2 and
Fv. These fragments lack the Fc fragment of intact antibody, clear
more rapidly from the circulation, and can have less non-specific
tissue binding than an intact antibody (Wahl et al., J. Nucl. Med.
24:316-325 (1983)). These fragments are produced from intact
antibodies using methods well known in the art, for example by
proteolytic cleavage with enzymes such as papain (to produce Fab
fragments) or pepsin (to produce F(ab').sub.2 fragments).
[0166] The identification of these antigen binding region and/or
epitopes recognized by mAbs of the present invention provides the
information necessary to generate additional monoclonal antibodies
with similar binding characteristics and therapeutic or diagnostic
utility that parallel the embodiments of this application.
[0167] Recombinant murine or chimeric murine-human or human-human
antibodies that bind an epitope included in the amino acid
sequences residues of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3
can be provided according to the present invention using known
techniques based on the teaching provided herein. See, e.g.,
Ausubel et al., eds. Current Protocols in Molecular Biology, Wiley
Interscience, N.Y. (1987, 1992, 1993); and Sambrook et al.
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press (1989), the entire contents of which are
incorporated herein by reference.
[0168] The DNA encoding an antibody of the present invention can be
genomic DNA or cDNA which encodes at least one of the heavy chain
constant region (H.sub.c), the heavy chain variable region
(H.sub.v), the light chain variable region (L.sub.v) and the light
chain constant regions (L.sub.c). A convenient alternative to the
use of chromosomal gene fragments as the source of DNA encoding the
murine V region antigen-binding segment is the use of cDNA for the
construction of chimeric immunoglobulin genes, e.g., as reported by
Liu et al. (Proc. Natl. Acad. Sci., USA 84:3439 (1987) and J.
Immunology 139:3521 (1987), which references are hereby entirely
incorporated herein by reference. The use of cDNA requires that
gene expression elements appropriate for the host cell be combined
with the gene in order to achieve synthesis of the desired protein.
The use of cDNA sequences is advantageous over genomic sequences
(which contain introns), in that cDNA sequences can be expressed in
bacteria or other hosts which lack appropriate RNA splicing
systems.
[0169] For example, a cDNA encoding a murine V region
antigen-binding segment capable of binding to one or more
T2DBMARKERS, for example, SEQ ID NO: 1, 2, or 3, can be provided
using known methods. Probes that bind a portion of a DNA sequence
encoding the antibodies of the present invention can be used to
isolate DNA from hybridomas expressing antibodies, fragments or
regions, as presented herein, according to the present invention,
by known methods.
[0170] Oligonucleotides representing a portion of the variable
region are useful for screening for the presence of homologous
genes and for the cloning of such genes encoding variable or
constant regions of antibodies according to the invention. Such
probes preferably bind to portions of sequences which encode light
chain or heavy chain variable regions which bind an epitope of one
or more T2DBMARKERS, especially an epitope of at least 5 amino
acids of residues 1-38 of SEQ ID NO:1, or at least 5 amino acids of
SEQ ID NOs 2 or 3. Such techniques for synthesizing such
oligonucleotides are well known and disclosed by, for example, Wu,
et al., Prog. Nucl. Acid. Res. Molec. Biol. 21:101-141 (1978), and
Ausubel et al., eds. Current Protocols in Molecular Biology, Wiley
Interscience (1987, 1993), the entire contents of which are herein
incorporated by reference.
[0171] Because the genetic code is degenerate, more than one codon
can be used to encode a particular amino acid (Watson, et al.).
Using the genetic code, one or more different oligonucleotides can
be identified, each of which would be capable of encoding the amino
acid. The probability that a particular oligonucleotide will, in
fact, constitute the actual XXX-encoding sequence can be estimated
by considering abnormal base pairing relationships and the
frequency with which a particular codon is actually used (to encode
a particular amino acid) in eukaryotic or prokaryotic cells
expressing an antibody of the invention or a fragment thereof. Such
"codon usage rules" are disclosed by Lathe, et al., J. Molec. Biol.
183:1-12 (1985). Using the "codon usage rules" of Lathe, a single
oligonucleotide, or a set of oligonucleotides, that contains a
theoretical "most probable" nucleotide sequence capable of encoding
preferred variable or constant region sequences is identified.
[0172] Although occasionally an amino acid sequence can be encoded
by only a single oligonucleotide, frequently the amino acid
sequence can be encoded by any of a set of similar
oligonucleotides. Importantly, whereas all of the members of this
set contain oligonucleotides which are capable of encoding the
peptide fragment and, thus, potentially contain the same
oligonucleotide sequence as the gene which encodes the peptide
fragment, only one member of the set contains the nucleotide
sequence that is identical to the nucleotide sequence of the gene.
Because this member is present within the set, and is capable of
hybridizing to DNA even in the presence of the other members of the
set, it is possible to employ the unfractionated set of
oligonucleotides in the same manner in which one would employ a
single oligonucleotide to clone the gene that encodes the
protein.
[0173] The oligonucleotide, or set of oligonucleotides, containing
the theoretical "most probable" sequence capable of encoding an
antibody of the present invention or fragment including a variable
or constant region is used to identify the sequence of a
complementary oligonucleotide or set of oligonucleotides which is
capable of hybridizing to the "most probable" sequence, or set of
sequences. An oligonucleotide containing such a complementary
sequence can be employed as a probe to identify and isolate the
variable or constant region gene (Sambrook et al., infra).
[0174] A suitable oligonucleotide, or set of oligonucleotides,
which is capable of encoding a fragment of the variable or constant
region (or which is complementary to such an oligonucleotide, or
set of oligonucleotides) is identified (using the above-described
procedure), synthesized, and hybridized by means well known in the
art, against a DNA or, more preferably, a cDNA preparation derived
from cells which are capable of expressing antibodies or variable
or constant regions thereof. Single stranded oligonucleotide
molecules complementary to the "most probable" variable or constant
anti-T2DBMARKER region peptide coding sequences can be synthesized
using procedures which are well known to those of ordinary skill in
the art (Belagaje, et al., J. Biol. Chem. 254:5765-5780 (1979);
Maniatis, et al., In: Molecular Mechanisms in the Control of Gene
Expression, Nierlich, et al., Eds., Acad. Press, NY (1976); Wu, et
al., Prog. Nucl. Acid Res. Molec. Biol. 21:101-141 (1978); Khorana,
Science 203:614-625 (1979)). Additionally, DNA synthesis can be
achieved through the use of automated synthesizers. Techniques of
nucleic acid hybridization are disclosed by Sambrook et al.
(infra), and by Hayrnes, et al. (In: Nucleic Acid Hybridization, A
Practical Approach, IRL Press, Washington, D.C. (1985)), which
references are herein incorporated by reference.
[0175] In an alternative way of cloning a polynucleotide encoding a
variable or constant region, a library of expression vectors is
prepared by cloning DNA or, more preferably, cDNA (from a cell
capable of expressing an antibody or variable or constant region)
into an expression vector. The library can then be screened for
members capable of expressing a protein which competitively
inhibits the binding of an antibody, and which has a nucleotide
sequence that is capable of encoding polypeptides that have the
same amino acid sequence as the antibodies of the present invention
or fragments thereof. In this embodiment, DNA, or more preferably
cDNA, is extracted and purified from a cell which is capable of
expressing an antibody or fragment. The purified cDNA is fragmented
(by shearing, endonuclease digestion, etc.) to produce a pool of
DNA or cDNA fragments. DNA or cDNA fragments from this pool are
then cloned into an expression vector in order to produce a genomic
library of expression vectors whose members each contain a unique
cloned DNA or cDNA fragment such as in a lambda phage library,
expression in prokaryotic cell (e.g., bacteria) or eukaryotic
cells, (e.g., mammalian, yeast, insect or, fungus). See, e.g.,
Ausubel, Harlow, Colligan; Nyyssonen et al. Bio/Technology
11:591-595 (Can 1993); Marks et al., Bio/Technology 11:1145-1149
(October 1993). Once a nucleic acid encoding such variable or
constant regions is isolated, the nucleic acid can be appropriately
expressed in a host cell, along with other constant or variable
heavy or light chain encoding nucleic acid, in order to provide
recombinant MAbs that bind one or more T2DBMARKERS with inhibitory
activity. Such antibodies preferably include a murine or human
variable region which contains a framework residue having
complementarity determining residues which are responsible for
antigen binding. Preferably, a variable light or heavy chain
encoded by a nucleic acid as described above binds an epitope of at
least 5 amino acids included within residues 1-38 of SEQ ID NO: 1,
or an epitope of at least 5 amino acids of SEQ ID NOs 2 or 3.
[0176] Human genes which encode the constant (C) regions of the
murine and chimeric antibodies, fragments and regions of the
present invention can be derived from a human fetal liver library,
by known methods. Human C regions genes can be derived from any
human cell including those which express and produce human
immunoglobulins. The human C.sub.H region can be derived from any
of the known classes or isotypes of human H chains, including
.gamma., .mu., .alpha., .delta. or .di-elect cons., and subtypes
thereof, such as G1, G2, G3 and G4. Since the H chain isotype is
responsible for the various effector functions of an antibody, the
choice of C.sub.H region will be guided by the desired effector
functions, such as complement fixation, or activity in
antibody-dependent cellular cytotoxicity (ADCC). Preferably, the
C.sub.H region is derived from gamma 1 (IgG1), gamma 3 (IgG3),
gamma 4 (IgG4), or .mu. (IgM). The human C.sub.L region can be
derived from either human L chain isotype, kappa (.kappa.) or
lambda (.lamda.).
[0177] Genes encoding human immunoglobulin C regions are obtained
from human cells by standard cloning techniques (Sambrook, et al.
(Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring
Harbor Press, Cold Spring Harbor, N.Y. (1989) and Ausubel et al.,
eds. Current Protocols in Molecular Biology (1987-1993)). Human C
region genes are readily available from known clones containing
genes representing the two classes of L chains, the five classes of
H chains and subclasses thereof. Chimeric antibody fragments, such
as F(ab').sub.2 and Fab, can be prepared by designing a chimeric H
chain gene which is appropriately truncated. For example, a
chimeric gene encoding an H chain portion of an F(ab').sub.2
fragment would include DNA sequences encoding the CH.sub.1 domain
and hinge region of the H chain, followed by a translational stop
codon to yield the truncated molecule.
[0178] Generally, the murine, human or murine and chimeric
antibodies, fragments and regions of the present invention are
produced by cloning DNA segments encoding the H and L chain
antigen-binding regions of an antibody, and joining these DNA
segments to DNA segments encoding C.sub.H and C.sub.L regions,
respectively, to produce murine, human or chimeric
immunoglobulin-encoding genes.
[0179] A fused chimeric gene can be created which comprises a first
DNA segment that encodes at least the antigen-binding region of
non-human origin, such as a functionally rearranged V region with
joining (J) segment, linked to a second DNA segment encoding at
least a part of a human C region. Therefore, cDNA encoding the
antibody V and C regions, the method of producing the chimeric
antibody according to the present invention involves several steps,
involving isolation of messenger RNA (mRNA) from the cell line
producing an antibody of the invention and from optional additional
antibodies supplying heavy and light constant regions; cloning and
cDNA production therefrom; preparation of a full length cDNA
library from purified mRNA from which the appropriate V and/or C
region gene segments of the L and H chain genes can be identified
with appropriate probes, sequenced, and made compatible with a C or
V gene segment from another antibody for a chimeric antibody;
constructing complete H or L chain coding sequences by linkage of
the cloned specific V region gene segments to cloned C region gene;
expressing and producing L and H chains in selected hosts,
including prokaryotic and eukaryotic cells to provide
murine-murine, human-murine, human-human or human murine
antibodies.
[0180] One common feature of all immunoglobulin H and L chain genes
and their encoded mRNAs is the J region. H and L chain J regions
have different sequences, but a high degree of sequence homology
exists (greater than 80%) among each group, especially near the C
region. This homology is exploited in this method and consensus
sequences of H and L chain J regions can be used to design
oligonucleotides for use as primers for introducing useful
restriction sites into the J region for subsequent linkage of V
region segments to human C region segments.
[0181] C region cDNA vectors prepared from human cells can be
modified by site-directed mutagenesis to place a restriction site
at the analogous position in the human sequence. For example, one
can clone the complete human kappa chain C (C.sub.k) region and the
complete human gamma-1 C region (C.sub..gamma.1). In this case, the
alternative method based upon genomic C region clones as the source
for C region vectors would not allow these genes to be expressed in
bacterial systems where enzymes needed to remove intervening
sequences are absent. Cloned V region segments are excised and
ligated to L or H chain C region vectors. Alternatively, the human
C.sub..gamma.1 region can be modified by introducing a termination
codon thereby generating a gene sequence which encodes the H chain
portion of an Fab molecule. The coding sequences with linked V and
C regions are then transferred into appropriate expression vehicles
for expression in appropriate hosts, prokaryotic or eukaryotic.
[0182] Two coding DNA sequences are said to be "operably linked" if
the linkage results in a continuously translatable sequence without
alteration or interruption of the triplet reading frame. A DNA
coding sequence is operably linked to a gene expression element if
the linkage results in the proper function of that gene expression
element to result in expression of the coding sequence.
[0183] Expression vehicles include plasmids or other vectors, which
are used for carrying a functionally complete human C.sub.H or
C.sub.L chain sequence having appropriate restriction sites
engineered so that any V.sub.H or V.sub.L chain sequence with
appropriate cohesive ends can be easily inserted therein. Human
C.sub.H or C.sub.L chain sequence-containing vehicles thus serve as
intermediates for the expression of any desired complete H or L
chain in any appropriate host.
[0184] A chimeric antibody, such as a mouse-human or human-human,
will typically be synthesized from genes driven by the chromosomal
gene promoters native to the mouse H and L chain V regions used in
the constructs; splicing usually occurs between the splice donor
site in the mouse J region and the splice acceptor site preceding
the human C region and also at the splice regions that occur within
the human C region; polyadenylation and transcription termination
occur at native chromosomal sites downstream of the human coding
regions.
[0185] A nucleic acid sequence encoding at least one antibody or Ab
fragment of the present invention may be recombined with vector DNA
in accordance with conventional techniques, including blunt-ended
or staggered-ended termini for ligation, restriction enzyme
digestion to provide appropriate termini, filling in of cohesive
ends as appropriate, alkaline phosphatase treatment to avoid
undesirable joining, and ligation with appropriate ligases.
Techniques for such manipulations are disclosed, e.g., by Ausubel,
infra, Sambrook, infra, entirely incorporated herein by reference,
and are well known in the art.
[0186] A nucleic acid molecule, such as DNA, is said to be "capable
of expressing" a polypeptide if it contains nucleotide sequences
which contain transcriptional and translational regulatory
information and such sequences are "operably linked" to nucleotide
sequences which encode the polypeptide. An operable linkage is a
linkage in which the regulatory DNA sequences and the DNA sequence
sought to be expressed are connected in such a way as to permit
gene expression of antibodies or Ab fragments in recoverable
amounts. The precise nature of the regulatory regions needed for
gene expression may vary from organism to organism, as is well
known in the analogous art. See, e.g., Sambrook, supra and Ausubel
supra.
[0187] The present invention accordingly encompasses the expression
of antibodies or Ab fragments, in either prokaryotic or eukaryotic
cells, although eukaryotic expression is preferred. Preferred hosts
are bacterial or eukaryotic hosts including bacteria, yeast,
insects, fungi, bird and mammalian cells either in vivo, or in
situ, or host cells of mammalian, insect, bird or yeast origin. It
is preferable that the mammalian cell or tissue is of human,
primate, hamster, rabbit, rodent, cow, pig, sheep, horse, goat, dog
or cat origin, but any other mammalian cell may be used.
[0188] Further, by use of, for example, the yeast ubiquitin
hydrolase system, in vivo synthesis of ubiquitin-transmembrane
polypeptide fusion proteins can be achieved. The fusion proteins
produced thereby may be processed in vivo or purified and processed
in vitro, allowing synthesis of an antibody or Ab fragment of the
present invention with a specified amino terminus sequence.
Moreover, problems associated with retention of initiation
codon-derived methionine residues in direct yeast (or bacterial)
expression may be avoided. Sabin et al., Bio/Technol. 7(7): 705-709
(1989); Miller et al., Bio/Technol. 7(7):698-704 (1989).
[0189] Any of a series of yeast gene expression systems
incorporating promoter and termination elements from the actively
expressed genes coding for glycolytic enzymes produced in large
quantities when yeast are grown in mediums rich in glucose can be
utilized to obtain the antibodies or Ab fragments of the present
invention. Known glycolytic genes can also provide very efficient
transcriptional control signals. For example, the promoter and
terminator signals of the phosphoglycerate kinase gene can be
utilized.
[0190] Production of antibodies or Ab fragments or functional
derivatives thereof in insects can be achieved, for example, by
infecting the insect host with a baculovirus engineered to express
a transmembrane polypeptide by methods known to those of skill. See
Ausubel et al., eds. Current Protocols in Molecular Biology Wiley
Interscience, 16.8-16.11 (1987, 1993).
[0191] In a preferred embodiment, the introduced nucleotide
sequence will be incorporated into a plasmid or viral vector
capable of autonomous replication in the recipient host. Any of a
wide variety of vectors may be employed for this purpose. See,
e.g., Ausubel et al., sections 1.5, 1.10, 7.1, 7.3, 8.1, 9.6, 9.7,
13.4, 16.2, 16.6, and 16.8-16.11. Factors of importance in
selecting a particular plasmid or viral vector include: the ease
with which recipient cells that contain the vector may be
recognized and selected from those recipient cells which do not
contain the vector; the number of copies of the vector which are
desired in a particular host; and whether it is desirable to be
able to "shuttle" the vector between host cells of different
species.
[0192] Preferred prokaryotic vectors known in the art include
plasmids such as those capable of replication in E. coli (such as,
for example, pBR322, ColE1, pSC11, pACYC 184, .pi.VX). Such
plasmids are, for example, disclosed by Maniatis, T., et al.
(Molecular Cloning, A Laboratory Manual, Second Edition, Cold
Spring Harbor Press, Cold Spring Harbor, N.Y. (1989); Ausubel,
infra. Bacillus plasmids include pC194, pC221, pT127, etc. Such
plasmids are disclosed by Gryczan, T. (In: The Molecular Biology of
the Bacilli, Academic Press, NY (1982), pp. 307-329). Suitable
Streptomyces plasmids include pIJ101 (Kendall, K. J., et al., J.
Bacteriol. 169:4177-4183 (1987)), and streptomyces bacteriophages
such as .phi.C31 (Chater, K. F., et al., In: Sixth International
Symposium on Actinomycetales Biology, Akademiai Kaido, Budapest,
Hungary (1986), pp. 45-54). Pseudomonas plasmids are reviewed by
John, J. F., et al. (Rev. Infect. Dis. 8:693-704 (1986)), and
Izaki, K. (Jpn. J. Bacteriol. 33:729-742 (1978); and Ausubel et
al., supra).
[0193] Alternatively, gene expression elements useful for the
expression of cDNA encoding antibodies, antibody fragments, or
peptides include, but are not limited to (a) viral transcription
promoters and their enhancer elements, such as the SV40 early
promoter (Okayama, et al., Mol. Cell. Biol. 3:280 (1983)), Rous
sarcoma virus LTR (Gorman, et al., Proc. Natl. Acad. Sci., USA
79:6777 (1982)), and Moloney murine leukemia virus LTR (Grosschedl,
et al., Cell 41:885 (1985)); (b) splice regions and polyadenylation
sites such as those derived from the SV40 late region (Okayarea et
al., infra); and (c) polyadenylation sites such as in SV40 (Okayama
et al., infra).
[0194] Immunoglobulin cDNA genes can be expressed as described by
Liu et al., infra, and Weidle et al., Gene 51:21 (1987), using as
expression elements the SV40 early promoter and its enhancer, the
mouse immunoglobulin H chain promoter enhancers, SV40 late region
mRNA splicing, rabbit S-globin intervening sequence, immunoglobulin
and rabbit S-globin polyadenylation sites, and SV40 polyadenylation
elements.
[0195] For immunoglobulin genes comprised of part cDNA, part
genomic DNA (Whittle et al., Protein Engineering 1:499 (1987)), the
transcriptional promoter can be human cytomegalovirus, the promoter
enhancers can be cytomegalovirus and mouse/human immunoglobulin,
and mRNA splicing and polyadenylation regions can be the native
chromosomal immunoglobulin sequences. For example, for expression
of cDNA genes in rodent cells, the transcriptional promoter is a
viral LTR sequence, the transcriptional promoter enhancers are
either or both the mouse immunoglobulin heavy chain enhancer and
the viral LTR enhancer, the splice region contains an intron of
greater than 31 bp, and the polyadenylation and transcription
termination regions are derived from the native chromosomal
sequence corresponding to the immunoglobulin chain being
synthesized. cDNA sequences encoding other proteins can also be
combined with the above-recited expression elements to achieve
expression of the proteins in mammalian cells.
[0196] Each fused gene can be assembled in, or inserted into, an
expression vector. Recipient cells capable of expressing the
chimeric immunoglobulin chain gene product are then transfected
singly with the sequence encoding the antibody, or chimeric H or
chimeric L chain-encoding gene, or are co-transfected with a
chimeric H and a chimeric L chain gene. The transfected recipient
cells are cultured under conditions that permit expression of the
incorporated genes and the expressed immunoglobulin chains or
intact antibodies or fragments are recovered from the culture. The
fused genes encoding the antibodies or chimeric H and L chains, or
portions thereof, can be assembled in separate expression vectors
that are then used to co-transfect a recipient cell.
[0197] Each vector can contain two selectable genes, a first
selectable gene designed for selection in a bacterial system and a
second selectable gene designed for selection in a eukaryotic
system, wherein each vector has a different pair of genes. This
strategy results in vectors which first direct the production, and
permit amplification, of the fused genes in a bacterial system. The
genes so produced and amplified in a bacterial host are
subsequently used to co-transfect a eukaryotic cell, and allow
selection of a co-transfected cell carrying the desired transfected
genes.
[0198] Examples of selectable genes for use in a bacterial system
are the gene that confers resistance to ampicillin and the gene
that confers resistance to chloramphenicol. Preferred selectable
genes for use in eukaryotic transfectants include the xanthine
guanine phosphoribosyl transferase gene (designated gpt) and the
phosphotransferase gens from Tn5 (designated neo). Selection of
cells expressing gpt is based on the fact that the enzyme encoded
by this gene utilizes xanthine as a substrate for purine nucleotide
synthesis, whereas the analogous endogenous enzyme cannot. In a
medium containing mycophenolic acid, which blocks the conversion of
inosine monophosphate to xanthine monophosphate, and xanthine, only
cells expressing the gpt gene can survive. The product of the neo
blocks the inhibition of protein synthesis by the antibiotic G418
and other antibiotics of the neomycin class.
[0199] The two selection procedures can be used simultaneously or
sequentially to select for the expression of immunoglobulin chain
genes introduced on two different DNA vectors into a eukaryotic
cell. It is not necessary to include different selectable markers
for eukaryotic cells; an H and an L chain vector, each containing
the same selectable marker can be co-transfected. After selection
of the appropriately resistant cells, the majority of the clones
will contain integrated copies of both H and L chain vectors and/or
antibody fragments. Alternatively, the fused genes encoding the
chimeric H and L chains can be assembled on the same expression
vector.
[0200] For transfection of the expression vectors and production of
the chimeric antibody, the preferred recipient cell line is a
myeloma cell. Myeloma cells can synthesize, assemble and secrete
immunoglobulins encoded by transfected immunoglobulin genes and
possess the mechanism for glycosylation of the immunoglobulin. A
particularly preferred recipient cell is the recombinant
Ig-producing myeloma cell SP2/0 (ATCC #CRL 8287). SP2/0 cells
produce only immunoglobulin encoded by the transfected genes.
Myeloma cells can be grown in culture or in the peritoneal cavity
of a mouse, where secreted immunoglobulin can be obtained from
ascites fluid. Other suitable recipient cells include lymphoid
cells such as B lymphocytes of human or non-human origin, hybridoma
cells of human or non-human origin, or interspecies heterohybridoma
cells.
[0201] The expression vector carrying a chimeric antibody
construct, antibody, or antibody fragment of the present invention
can be introduced into an appropriate host cell by any of a variety
of suitable means, including such biochemical means as
transformation, transfection, conjugation, protoplast fusion,
calcium phosphate-precipitation, and application with polycations
such as diethylaminoethyl (DEAE) dextran, and such mechanical means
as electroporation, direct microinjection, and microprojectile
bombardment (Johnston et al., Science 240:1538 (1988)). A preferred
way of introducing DNA into lymphoid cells is by electroporation
(Potter et al., Proc. Natl. Acad. Sci. USA 81:7161 (1984);
Yoshikawa, et al., Jpn. J. Cancer Res. 77:1122-1133). In this
procedure, recipient cells are subjected to an electric pulse in
the presence of the DNA to be incorporated. Typically, after
transfection, cells are allowed to recover in complete medium for
about 24 hours, and are then seeded in 96-well culture plates in
the presence of the selective medium. G418 selection is performed
using about 0.4 to 0.8 mg/ml G418. Mycophenolic acid selection
utilizes about 6 .mu.g/ml plus about 0.25 mg/ml xanthine. The
electroporation technique is expected to yield transfection
frequencies of about 10.sup.-5 to about 10.sup.-4 for Sp2/0 cells.
In the protoplast fusion method, lysozyme is used to strip cell
walls from catarrhal harboring the recombinant plasmid containing
the chimeric antibody gene. The resulting spheroplasts can then be
fused with myeloma cells with polyethylene glycol.
[0202] The immunoglobulin genes of the present invention can also
be expressed in nonlymphoid mammalian cells or in other eukaryotic
cells, such as yeast, or in prokaryotic cells, in particular
bacteria. Yeast provides substantial advantages over bacteria for
the production of immunoglobulin H and L chains. Yeasts carry out
post-translational peptide modifications including glycosylation. A
number of recombinant DNA strategies now exist which utilize strong
promoter sequences and high copy number plasmids which can be used
for production of the desired proteins in yeast. Yeast recognizes
leader sequences of cloned mammalian gene products and secretes
peptides bearing leader sequences (i.e., pre-peptides) (Hitzman, et
al., 11th International Conference on Yeast, Genetics and Molecular
Biology, Montpelier, France, Sep. 13-17, 1982).
[0203] Yeast gene expression systems can be routinely evaluated for
the levels of production, secretion and the stability of antibody
and assembled murine and chimeric antibodies, fragments and regions
thereof. Any of a series of yeast gene expression systems
incorporating promoter and termination elements from the actively
expressed genes coding for glycolytic enzymes produced in large
quantities when yeasts are grown in media rich in glucose can be
utilized. Known glycolytic genes can also provide very efficient
transcription control signals. For example, the promoter and
terminator signals of the phosphoglycerate kinase (PGK) gene can be
utilized. A number of approaches can be taken for evaluating
optimal expression plasmids for the expression of cloned
immunoglobulin cDNAs in yeast (see Glover, ed., DNA Cloning, Vol.
11, pp 45-66, IRL Press, 1985).
[0204] Bacterial strains can also be utilized as hosts for the
production of antibody molecules or peptides described by this
invention, E. coli K.sub.12 strains such as E. coli W3110 (ATCC
27325), and other enterobacteria such as Salmonella typhimurium or
Serratia marcescens, and various Pseudomonas species can be used.
Plasmid vectors containing replicon and control sequences which are
derived from species compatible with a host cell are used in
connection with these bacterial hosts. The vector carries a
replication site, as well as specific genes which are capable of
providing phenotypic selection in transformed cells. A number of
approaches can be taken for evaluating the expression plasmids for
the production of murine and chimeric antibodies, fragments and
regions or antibody chains encoded by the cloned immunoglobulin
cDNAs in bacteria (see Glover, ed., DNA Cloning, Vol. 1, IRL Press,
1985, Ausubel, infra, Sambrook, infra, Colligan, infra).
[0205] Preferred hosts are mammalian cells, grown in vitro or in
vivo. Mammalian cells provide post-translational modifications to
immunoglobulin protein molecules including leader peptide removal,
folding and assembly of H and L chains, glycosylation of the
antibody molecules, and secretion of functional antibody protein.
Mammalian cells which can be useful as hosts for the production of
antibody proteins, in addition to the cells of lymphoid origin
described above, include cells of fibroblast origin, such as Vero
(ATCC CRL 81) or CHO-K1 (ATCC CRL 61).
[0206] Many vector systems are available for the expression of
cloned antibodies, H and L chain genes, or antibody fragments in
mammalian cells (see Glover, ed., DNA Cloning, Vol. II, pp 143-238,
IRL Press, 1985). Different approaches can be followed to obtain
complete H.sub.2L.sub.2 antibodies. As discussed above, it is
possible to co-express H and L chains in the same cells to achieve
intracellular association and linkage of H and L chains into
complete tetrameric H.sub.2L.sub.2 antibodies and/or antibodies
and/or antibody fragments of the invention. The co-expression can
occur by using either the same or different plasmids in the same
host. Genes for both H and L chains and/or antibodies and/or
antibody fragments can be placed into the same plasmid, which can
then be transfected into cells, thereby selecting directly for
cells that express both chains. Alternatively, cells can be
transfected first with a plasmid encoding one chain, for example
the L chain, followed by transfection of the resulting cell line
with an H chain plasmid containing a second selectable marker. Cell
lines producing antibodies and/or H.sub.2L.sub.2 molecules and/or
antibody fragments via either route could be transfected with
plasmids encoding additional copies of peptides, H, L, or H plus L
chains in conjunction with additional selectable markers to
generate cell lines with enhanced properties, such as higher
production of assembled H.sub.2L.sub.2 antibody molecules or
enhanced stability of the transfected cell lines.
[0207] In addition to monoclonal or chimeric antibodies, the
present invention is also directed to an anti-idiotypic (anti-Id)
antibody specific for the antibodies of the invention. An anti-Id
antibody is an antibody which recognizes unique determinants
generally associated with the antigen-binding region of another
antibody. The antibody specific for one or more T2DBMARKERS, or any
of SEQ ID NO: 1, 2, or 3 is termed the idiotypic or Id antibody.
The anti-Id can be prepared by immunizing an animal of the same
species and genetic type (e.g. mouse strain) as the source of the
Id antibody with the Id antibody or the antigen-binding region
thereof. The immunized animal will recognize and respond to the
idiotypic determinants of the immunizing antibody and produce an
anti-Id antibody. The anti-Id antibody can also be used as an
"immunogen" to induce an immune response in yet another animal,
producing a so-called anti-anti-Id antibody. The anti-anti-Id can
be epitopically identical to the original antibody which induced
the anti-Id. Thus, by using antibodies to the idiotypic
determinants of a mAb, it is possible to identify other clones
expressing antibodies of identical specificity.
[0208] Accordingly, mAbs generated against one or more T2DBMARKERS
according to the present invention can be used to induce anti-Id
antibodies in suitable animals, such as BALB/c mice. Spleen cells
from such immunized mice can be used to produce anti-Id hybridomas
secreting anti-Id mAbs. Further, the anti-Id InAbs can be coupled
to a carrier such as keyhole limpet hemocyanin (KLH) and used to
immunize additional BALB/c mice. Sera from these mice will contain
anti-anti-Id antibodies that have the binding properties of the
original mAb specific for an epitope of a T2DBMARKER, or
preferably, an epitope containing within amino acid residues 1-38
of SEQ ID NO: 1, or within SEQ ID NO: 2, or SEQ ID NO: 3.
[0209] Other aspects of the invention provide antibodies to
T2DMARKER peptides, proteins, polypeptides or antibody idiotopes
thereof that are linked to at least one agent to form an antibody
conjugate. To increase the efficacy of antibody molecules as
diagnostic or therapeutic agents, it is conventional to link or
covalently bind or complex at least one desired molecule or moiety.
A reporter molecule is defined as any moiety which may be detected
using an assay. Non-limiting examples of reporter molecules which
have been conjugated to antibodies include enzymes, radiolabels,
haptens, fluorescent labels, phosphorescent molecules,
chemiluminescent molecules, chromophores, luminescent molecules,
photoaffinity molecules, colored particles or ligands, such as
biotin.
[0210] Examples of antibody conjugates are those conjugates in
which the antibody is linked to a detectable label. "Detectable
labels" are compounds and/or elements that can be detected due to
their specific functional properties, and/or chemical
characteristics, the use of which allows the antibody to which they
are attached to be detected, and/or further quantified if desired.
An example of such a detectable label is gold nanoparticles.
Another such example is the formation of a conjugate comprising an
antibody linked to a cytotoxic or anti-cellular agent, and may be
termed "immunotoxins".
Pharmaceutical Compositions and Methods of Treatment
[0211] The invention provides pharmaceutical compositions
comprising an effective amount, or a therapeutically effective
amount, of one or more T2DBMARKERS disclosed herein, preferably the
peptide and kinase inhibitors of the invention in a
pharmaceutically acceptable carrier or diluent, for administration
to a subject, such as a human patient. The phrases "pharmaceutical
or pharmacologically acceptable" refers to molecular entities and
compositions that do not produce an adverse, allergic or other
untoward reaction when administered to an animal, such as, for
example, a human, as appropriate. Moreover, for animal (e.g.,
human) administration, it will be understood that preparations
should meet sterility, pyrogenicity, general safety and purity
standards as required by FDA Office of Biological Standards.
[0212] The term "treating" or "treatment" in its various
grammatical forms in relation to the present invention refers to
preventing (i.e. chemoprevention), curing, reversing, attenuating,
alleviating, minimizing, suppressing or halting the deleterious
effects of a disease state, disease progression, disease causative
agent (e.g., bacteria or viruses) or other abnormal condition. For
example, treatment may involve alleviating a symptom (i.e., not
necessarily all symptoms) of a disease or attenuating the
progression of a disease.
[0213] As used herein, the term "therapeutically effective amount"
is intended to qualify the amount or amounts of T2DBMARKERS or
other diabetes-modulating agents that will achieve a desired
biological response. In the context of the present invention, the
desired biological response can be partial or total inhibition,
delay or prevention of the progression of type 2 Diabetes,
pre-diabetic conditions, and complications associated with type 2
Diabetes or pre-diabetic conditions; inhibition, delay or
prevention of the recurrence of type 2 Diabetes, pre-diabetic
conditions, or complications associated with type 2 Diabetes or
pre-diabetic conditions; or the prevention of the onset or
development of type 2 Diabetes, pre-diabetic conditions, or
complications associated with type 2 Diabetes or pre-diabetic
conditions (chemoprevention) in a subject, for example a human.
[0214] Therapeutically effective amount" as used herein can also
refer to an amount that is effective to obtain the desired
therapeutic result. The term "an effective amount" of, for example,
the peptide kinase inhibitors of the invention refers to an amount
that is effective to induce an inhibition of kinase activity, which
can be kinase activity from one or more kinases implicated in type
2 Diabetes Mellitus or pre-diabetic conditions as defined herein.
The inhibitory amount may be determined directly by measuring the
inhibition of kinase activity, or, for example, where the desired
effect is an effect on an activity downstream of a particular
kinase activity in a pathway that includes one or more kinases
involved in Diabetes or a pre-diabetic condition, the inhibition
may be measured by measuring a downstream effect. Thus, the
inhibition of kinase activity will depend in part on the nature of
the inhibited pathway or process that involves kinase activity, and
on the effects that inhibition of kinase activity has in a given
biological context.
[0215] The amount of the inhibitor that will constitute an
inhibitory amount will vary depending on such parameters as the
inhibitor and its potency, the half-life of the inhibitor in the
body, the rate of progression of the disease or biological
condition being treated, the responsiveness of the condition to the
dose of treatment or pattern of administration, the formulation,
the attending physician's assessment of the medical situation, and
other relevant factors, and in general the health of the patient,
and other considerations such as prior administration of other
therapeutics, or co-administration of any therapeutic that will
have an effect on the inhibitory activity of the inhibitor or that
will have an effect on kinase activity, or a pathway mediated by
kinase activity. It is expected that the inhibitory amount will
fall in a relatively broad range that can be determined through
routine trials.
[0216] The T2DBMARKERS, preferably included as part of a
pharmaceutical composition, can be administered by any known
administration method known to a person skilled in the art. The
mode of administration can depend on the disease condition or the
injury being treated. In particular, the peptide inhibitors of the
invention can be administered in an amount and by a route of
administration that blocks about 50% or greater of kinase
phosphorylation activity, as measured by in vitro kinase assay (see
Example X). Examples of routes of administration include but are
not limited to oral, nasal, ophthalmic, parenteral,
intraperitoneal, intravenous, intravascular, intraarterial,
intraventricular, intraepidural, intratumor, intraorbital,
intracapsule, intraperitoneal, intracistern, transdermal, topical,
sublingual, intramuscular, rectal, transbuccal, intranasal,
liposomal, via inhalation, vaginal, mucosal, intraoccular, via
local delivery by catheter or stent, by depot injection, by
erodible implants, subcutaneous, intraadiposal, intraarticular,
intrathecal, or in a slow release dosage form. The T2DBMARKERS or
pharmaceutical compositions comprising the T2DBMARKERS can be
administered in accordance with any dose and dosing schedule that
achieves a dose effective to treat disease.
[0217] As examples, T2DBMARKERS or pharmaceutical compositions
comprising T2DBMARKERS of the invention can be administered in such
oral forms as tablets, capsules (each of which includes sustained
release or timed release formulations), pills, powders, granules,
elixirs, tinctures, suspensions, syrups, and emulsions. Likewise,
the T2DBMARKERS or pharmaceutical compositions comprising
T2DBMARKERS can be administered by intravenous (e.g., bolus or
infusion), intraperitoneal, subcutaneous, intramuscular, or other
routes using forms well known to those of ordinary skill in the
pharmaceutical arts.
[0218] T2DBMARKERS and pharmaceutical compositions comprising
T2DBMARKERS can also be administered in the form of a depot
injection or implant preparation, which may be formulated in such a
manner as to permit a sustained release of the active ingredient.
The active ingredient can be compressed into pellets or small
cylinders and implanted subcutaneously or intramuscularly as depot
injections or implants. Implants may employ inert materials such as
biodegradable polymers or synthetic silicones, for example,
Silastic, silicone rubber or other polymers manufactured by the
Dow-Corning Corporation.
[0219] T2DBMARKERS or pharmaceutical compositions comprising
T2DBMARKERS can also be administered in the form of liposome
delivery systems, such as small unilamellar vesicles, large
unilamellar vesicles and multilamellar vesicles. Liposomes can be
formed from a variety of phospholipids, such as cholesterol,
stearylamine, phosphatidylethanolamines, or phosphatidylcholines.
Liposomal preparations of diabetes-modulating agents may also be
used in the methods of the invention.
[0220] T2DBMARKERS or pharmaceutical compositions comprising
T2DBMARKERS can also be delivered by the use of monoclonal
antibodies as individual carriers to which the compound molecules
are coupled.
[0221] T2DBMARKERS or pharmaceutical compositions comprising
T2DBMARKERS can also be prepared with soluble polymers as
targetable drug carriers. Such polymers can include
polyvinylpyrrolidone, pyran copolymer,
polyhydroxy-propyl-methacrylamide-phenol,
polyhydroxyethyl-aspartamide-phenol, or
polyethyleneoxide-polylysine substituted with palmitoyl residues.
Furthermore, T2DBMARKERS or pharmaceutical compositions comprising
T2DBMARKERS can be prepared with biodegradable polymers useful in
achieving controlled release of a drug, for example, polylactic
acid, polyglycolic acid, copolymers of polylactic and polyglycolic
acid, polyepsilon caprolactone, polyhydroxy butyric acid,
polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates
and cross linked or amphipathic block copolymers of hydrogels.
[0222] The T2DBMARKERS or pharmaceutical compositions comprising
T2DBMARKERS can also be administered in intranasal form via topical
use of suitable intranasal vehicles, or via transdermal routes,
using those forms of transdermal skin patches well known to those
of ordinary skill in that art. To be administered in the form of a
transdermal delivery system, the dosage administration will, or
course, be continuous rather than intermittent throughout the
dosage regime.
[0223] Suitable pharmaceutically acceptable salts of the agents
described herein and suitable for use in the method of the
invention, are conventional non-toxic salts and can include a salt
with a base or an acid addition salt such as a salt with an
inorganic base, for example, an alkali metal salt (e.g., lithium
salt, sodium salt, potassium salt, etc.), an alkaline earth metal
salt (e.g., calcium salt, magnesium salt, etc.), an ammonium salt;
a salt with an organic base, for example, an organic amine salt
(e.g., triethylamine salt, pyridine salt, picoline salt,
ethanolamine salt, triethanolamine salt, dicyclohexylamine salt,
N,N'-dibenzylethylenediamine salt, etc.) etc.; an inorganic acid
addition salt (e.g., hydrochloride, hydrobromide, sulfate,
phosphate, etc.); an organic carboxylic or sulfonic acid addition
salt (e.g., formate, acetate, trifluoroacetate, maleate, tartrate,
methanesulfonate, benzenesulfonate, p-toluenesulfonate, etc.); a
salt with a basic or acidic amino acid (e.g., arginine, aspartic
acid, glutamic acid, etc.) and the like.
[0224] In addition, this invention also encompasses pharmaceutical
compositions comprising any solid or liquid physical form of one or
more of the T2DBMARKERS of the invention. For example, the
T2DBMARKERS can be in a crystalline form, in amorphous form, and
have any particle size. The T2DBMARKER particles may be micronized,
or may be agglomerated, particulate granules, powders, oils, oily
suspensions or any other form of solid or liquid physical form.
[0225] For oral administration, the pharmaceutical compositions can
be liquid or solid. Suitable solid oral formulations include
tablets, capsules, pills, granules, pellets, and the like. Suitable
liquid oral formulations include solutions, suspensions,
dispersions, emulsions, oils, and the like.
[0226] Any inert excipient that is commonly used as a carrier or
diluent may be used in the formulations of the present invention,
such as for example, a gum, a starch, a sugar, a cellulosic
material, an acrylate, or mixtures thereof. The compositions may
further comprise a disintegrating agent and a lubricant, and in
addition may comprise one or more additives selected from a binder,
a buffer, a protease inhibitor, a surfactant, a solubilizing agent,
a plasticizer, an emulsifier, a stabilizing agent, a viscosity
increasing agent, a sweetener, a film forming agent, or any
combination thereof. Furthermore, the compositions of the present
invention may be in the form of controlled release or immediate
release formulations.
[0227] T2DBMARKERS can be administered as active ingredients in
admixture with suitable pharmaceutical diluents, excipients or
carriers (collectively referred to herein as "carrier" or "diluent"
materials or "pharmaceutically acceptable carriers or diluents")
suitably selected with respect to the intended form of
administration. As used herein, "pharmaceutically acceptable
carrier or diluent" is intended to include any and all solvents,
dispersion media, coatings, antibacterial and antifungal agents,
isotonic and absorption delaying agents, and the like, compatible
with pharmaceutical administration. Suitable carriers are described
in the most recent edition of Remington's Pharmaceutical Sciences,
a standard reference text in the field, which is incorporated
herein by reference.
[0228] For liquid formulations, pharmaceutically acceptable
carriers may be aqueous or non-aqueous solutions, suspensions,
emulsions or oils. Examples of non-aqueous solvents are propylene
glycol, polyethylene glycol, and injectable organic esters such as
ethyl oleate. Aqueous carriers include water, alcoholic/aqueous
solutions, emulsions, or suspensions, including saline and buffered
media. Examples of oils are those of petroleum, animal, vegetable,
or synthetic origin, for example, peanut oil, soybean oil, mineral
oil, olive oil, sunflower oil, and fish-liver oil. Solutions or
suspensions can also include the following components: a sterile
diluent such as water for injection, saline solution, fixed oils,
polyethylene glycols, glycerine, propylene glycol or other
synthetic solvents; antibacterial agents such as benzyl alcohol or
methyl parabens; antioxidants such as ascorbic acid or sodium
bisulfite; chelating agents such as ethylenediaminetetraacetic acid
(EDTA); buffers such as acetates, citrates or phosphates, and
agents for the adjustment of tonicity such as sodium chloride or
dextrose. The pH can be adjusted with acids or bases, such as
hydrochloric acid or sodium hydroxide.
[0229] Liposomes and non-aqueous vehicles such as fixed oils may
also be used. The use of such media and agents for pharmaceutically
active substances is well known in the art. Except insofar as any
conventional media or agent is incompatible with the active
compound, use thereof in the compositions is contemplated.
Supplementary active compounds can also be incorporated into the
compositions.
[0230] Solid carriers/diluents include, but are not limited to, a
gum, a starch (e.g., corn starch, pregelatinized starch), a sugar
(e.g., lactose, mannitol, sucrose, dextrose), a cellulosic material
(e.g., microcrystalline cellulose), an acrylate (e.g.,
polymethylacrylate), calcium carbonate, magnesium oxide, talc, or
mixtures thereof.
[0231] In addition, the compositions may further comprise binders
(e.g., acacia, cornstarch, gelatin, carbomer, ethyl cellulose, guar
gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose,
povidone), disintegrating agents (e.g., cornstarch, potato starch,
alginic acid, silicon dioxide, croscarmellose sodium, crospovidone,
guar gum, sodium starch glycolate, Primogel), buffers (e.g.,
tris-HCl, acetate, phosphate) of various pH and ionic strength,
additives such as albumin or gelatin to prevent absorption to
surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile
acid salts), protease inhibitors, surfactants (e.g., sodium lauryl
sulfate), permeation enhancers, solubilizing agents (e.g.,
glycerol, polyethylene glycerol); a glidant (e.g., colloidal
silicon dioxide), anti-oxidants (e.g., ascorbic acid, sodium
metabisulfite, butylated hydroxyanisole), stabilizers (e.g.,
hydroxypropyl cellulose, hydroxypropylmethyl cellulose), viscosity
increasing agents (e.g., carbomer, colloidal silicon dioxide, ethyl
cellulose, guar gum), sweeteners (e.g., sucrose, aspartame, citric
acid), flavoring agents (e.g., peppermint, methyl salicylate, or
orange flavoring), preservatives (e.g., Thimerosal, benzyl alcohol,
parabens), lubricants (e.g., stearic acid, magnesium stearate,
polyethylene glycol, sodium lauryl sulfate), flow-aids (e.g.,
colloidal silicon dioxide), plasticizers (e.g., diethyl phthalate,
triethyl citrate), emulsifiers (e.g., carbomer, hydroxypropyl
cellulose, sodium lauryl sulfate), polymer coatings (e.g.,
poloxamers or poloxamines), coating and film forming agents (e.g.,
ethyl cellulose, acrylates, polymethacrylates) and/or
adjuvants.
[0232] In one embodiment, the active compounds are prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes
targeted to infected cells with monoclonal antibodies to viral
antigens) comprised of neutral lipids, anionic lipids, cationic
lipids, or mixtures thereof can also be used as pharmaceutically
acceptable carriers. These can be prepared according to methods
known to those skilled in the art, for example, as described in
U.S. Pat. No. 4,522,811.
[0233] It is especially advantageous to formulate oral and
intravenous compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the subject to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved, and the limitations inherent in
the art of compounding such an active compound for the treatment of
individuals. The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration. A pharmaceutical composition typically contains an
amount of at least 0.1 weight % of active ingredient, i.e., a
peptide inhibitor or antibody of this invention, per weight of
total pharmaceutical composition. A weight % is a ratio by weight
of active ingredient to total composition. Thus, for example, 0.1
weight % is 0.1 grams of peptide inhibitor per 100 grams of total
composition.
[0234] The preparation of pharmaceutical compositions that contain
an active component is well understood in the art, for example, by
mixing, granulating, or tablet-forming processes. The active
therapeutic ingredient is often mixed with excipients that are
pharmaceutically acceptable and compatible with the active
ingredient. For oral administration, the active agents are mixed
with additives customary for this purpose, such as vehicles,
stabilizers, or inert diluents, and converted by customary methods
into suitable forms for administration, such as tablets, coated
tablets, hard or soft gelatin capsules, aqueous, alcoholic, or oily
solutions and the like as detailed above.
[0235] For intravenous administration, Glucuronic acid, L-lactic
acid, acetic acid, citric acid or any pharmaceutically acceptable
acid/conjugate base with reasonable buffering capacity in the pH
range acceptable for intravenous administration can be used as
buffers. Sodium chloride solution wherein the pH has been adjusted
to the desired range with either acid or base, for example,
hydrochloric acid or sodium hydroxide, can also be employed.
Typically, a pH range for the intravenous formulation can be in the
range of from about 5 to about 12.
[0236] Subcutaneous formulations can be prepared according to
procedures well known in the art at a pH in the range between about
5 and about 12, which include suitable buffers and isotonicity
agents. They can be formulated to deliver a daily dose of the
active agent in one or more daily subcutaneous administrations. The
choice of appropriate buffer and pH of a formulation, depending on
solubility of one or more T2DBMARKERS to be administered, is
readily made by a person having ordinary skill in the art. Sodium
chloride solution wherein the pH has been adjusted to the desired
range with either acid or base, for example, hydrochloric acid or
sodium hydroxide, can also be employed in the subcutaneous
formulation. Typically, a pH range for the subcutaneous formulation
can be in the range of from about 5 to about 12.
[0237] The compositions of the present invention can also be
administered in intranasal form via topical use of suitable
intranasal vehicles, or via transdermal routes, using those forms
of transdermal skin patches well known to those of ordinary skill
in that art. To be administered in the form of a transdermal
delivery system, the dosage administration will, or course, be
continuous rather than intermittent throughout the dosage
regime.
[0238] "Co-administration" as used herein means administration of a
pharmaceutical composition according to the invention in
combination with a second therapeutic agent. The second therapeutic
agent can be any therapeutic agent useful for treatment of the
patient's condition. For example, inhibition of kinases with a
diabetes-modulating drug as a second therapeutic agent used in
conjunction with the peptide inhibitors of the present invention is
contemplated. Additionally, for example, a first therapeutic agent
can be a peptide inhibitor of the invention and a second
therapeutic agent can be an antisense or ribozyme molecule against
one or more kinases that, when administered in a viral or nonviral
vector, will facilitate a transcriptional inhibition of that kinase
and which will complement the inhibitory activity of the small
molecule. Co-administration may be simultaneous, for example, by
administering a mixture of the therapeutic agents, or may be
accomplished by administration of the agents separately, such as
within a short time period. Co-administration also includes
successive administration of a peptide inhibitor of the invention
and one or more of another therapeutic agent. The second
therapeutic agent or agents may be administered before or after the
peptide inhibitor. The second therapeutic agent may also be an
inhibitor of kinases implicated in Diabetes or pre-diabetic
conditions, which has particular advantages when administered with
the first inhibitor. Dosage treatment may be a single dosing
schedule or a multiple dosing schedule.
[0239] A therapeutic agent may be a drug, a chemotherapeutic agent,
a radioisotope, a pro-apoptosis agent, an anti-angiogenic agent, a
hormone, a cytokine, a cytotoxic agent, a cytocidal agent, a
cytostatic agent, a peptide, a protein, an antibiotic, an antibody,
a Fab fragment of an antibody, a hormone antagonist, a nucleic acid
or an antigen. The anti-angiogenic agent is selected from the group
consisting of thrombospondin, angiostatin 5, pigment
epithelium-derived factor, angiotensin, laminin peptides,
fibronectin peptides, plasminogen activator inhibitors, tissue
metalloproteinase inhibitors, interferons, interleukin 12, platelet
factor 4, IP-10, Gro-.beta., thrombospondin, 2-methoxyoestradiol,
proliferin-related protein, carboxiamidotriazole, CMI 101,
Marimastat, pentosan polysuiphate, angiopoietin 2 (Regeneron),
interferon-alpha, herbimycin A, PNU 14515 6E, 16K prolactin
fragment, Linomide, thalidomide, pentoxifylline, genistein,
TNP-470, endostatin, paclitaxel, Docetaxel, polyamines, a
proteasome inhibitor, a kinase inhibitor, a signaling peptide,
accutin, cidofovir, vincristine, bleomycin, AGM-1470, platelet
factor 4 and minocycline. Whereas, the pro-apoptosis agent is
selected from the group consisting of etoposide, ceramide
sphingomyelin, Bax, Bid, Bik, Bad, caspase-3, caspase-8, caspase-9,
fas, fas ligand, fadd, fap-1, tradd, faf, rip, reaper, apoptin,
interleukin-2 converting enzyme or annexin V. Additional apoptotic
agents include gramicidin, magainin, mellitin, defensin, or
cecropin. Furthermore, a cytokine may be selected from the group
consisting of interleukin 1 (IL-1), IL-2, IL-5, IL-10, IL-11,
IL-12, IL-18, interferon-.gamma. (IF-.gamma.), IF-.alpha.,
IF-.beta., tumor necrosis factor-.alpha. (TNF-.alpha.), or GM-CSF
(granulocyte macrophage colony stimulating factor).
[0240] Examples of such therapeutics or agents frequently used in
Diabetes treatments, and may modulate the symptoms or risk factors
of Diabetes include, but are not limited to, sulfonylureas like
glimepiride, glyburide (also known in the art as glibenclamide),
glipizide, gliclazide; biguanides such as metformin; insulin
(including inhaled formulations such as Exubera), and insulin
analogs such as insulin lispro (Humalog), insulin glargine
(Lantus), insulin detemir, and insulin glulisine; peroxisome
proliferator-activated receptor-.gamma. (PPAR-.gamma.) agonists
such as the thiazolidinediones including troglitazone (Rezulin),
pioglitazone (Actos), rosiglitazone (Avandia), and isaglitzone
(also known as netoglitazone); dual-acting PPAR agonists such as
BMS-298585 and tesaglitazar; insulin secretagogues including
metglitinides such as repaglinide and nateglinide; analogs of
glucagon-like peptide-1 (GLP-1) such as exenatide (AC-2993) and
liraglutide (insulinotropin); inhibitors of dipeptidyl peptidase IV
like LAF-237; pancreatic lipase inhibitors such as orlistat;
.alpha.-glucosidase inhibitors such as acarbose, miglitol, and
voglibose; and combinations thereof, particularly metformin and
glyburide (Glucovance), metformin and rosiglitazone (Avandamet),
and metformin and glipizide (Metaglip). Such therapeutics or agents
have been prescribed for subjects diagnosed with Diabetes, one or
more complications related to Diabetes, or a pre-diabetic
condition, and may modulate the symptoms or risk factors of
Diabetes, one or more complications related to Diabetes, or a
pre-diabetic condition (herein, "diabetes-modulating agents").
[0241] The precise effective amount or therapeutically effective
amount of pharmaceutical compositions (including pharmaceutical
compositions comprising the peptide inhibitors disclosed herein)
applied or administered to humans can be determined by the
ordinarily-skilled artisan with consideration of individual
differences in age, weight, extent of cellular infiltration by
inflammatory cells and condition of the patient. The pharmaceutical
preparation of the invention should be administered to provide an
effective concentration of 5-100 .mu.M, preferably about 5
.mu.M.
[0242] The actual dosage amount of a composition of the present
invention administered to a subject can be determined by physical
and physiological factors such as body weight, severity of
condition, the type of disease being treated, previous or
concurrent therapeutic interventions, idiopathy of the patient and
on the route of administration. The practitioner responsible for
administration will, in any event, determine the concentration of
active ingredient(s) in a composition and appropriate dose(s) for
the individual subject. The total effective amount of a peptide
inhibitor of the invention can be administered to a subject as a
single dose, either as a bolus or by infusion over a relatively
short period of time, or can be administered using a fractionated
treatment protocol, in which the multiple doses are administered
over a more prolonged period of time.
[0243] In certain embodiments, pharmaceutical compositions may
comprise, for example, at least about 0.1% of an active compound.
In other embodiments, the an active compound may comprise between
about 2% to about 75% of the weight of the unit, or between about
25% to about 60%, for example, and any range derivable therein. In
other non-limiting examples, a dose may also comprise from about 1
.mu.g/kg/body weight, about 5 .mu.g/kg/body weight, about 10
.mu.g/kg/body weight, about 50 .mu.g/kg/body weight, about 100
.mu.g/kg/body weight, about 200 .mu.g/kg/body weight, about 350
.mu.g/kg/body weight, about 500 .mu.g/kg/body weight, about 1
mg/kg/body weight, about 5 mg/kg/body weight, about 10 mg/kg/body
weight, about 50 mg/kg/body weight, about 100 mg/kg/body weight,
about 200 mg/kg/body weight, about 350 mg/kg/body weight, about 500
mg/kg/body weight, to about 1000 mg/kg/body weight or more per
administration, and any range derivable therein. In non-limiting
examples of a derivable range from the numbers listed herein, a
range of about 5 mg/kg/body weight to about 100 mg/kg/body weight,
about 5 microgram/kg/body weight to about 500 milligram/kg/body
weight, can be administered, based on the numbers described
above.
[0244] The peptide inhibitors of the invention can be used to treat
a biological condition mediated by serpin activity. The biological
condition mediated by serpin activity includes type 2 Diabetes
Mellitus, also known in the art as non-insulin dependent Diabetes
mellitus. The biological condition further includes abnormal
bleeding, thrombosis and other coagulation disorders (such as, for
example, thrombocytopenia), defects in hemostasis and fibrinolysis,
inflammation and angiogenesis, cardiovascular disease (such as,
without limitation, atherosclerosis, atherothrombosis, coronary
artery disease, myocardial infarction), among others.
[0245] Potentiation of insulin signaling in vivo, which may result
from administration of the pharmaceutical compositions comprising
one or more peptide inhibitors of the invention, can be monitored
as a clinical endpoint. In principle, a way to look at insulin
potentiation in a patient is to perform an oral glucose tolerance
test. After fasting, glucose is given to a patient and the rate of
the disappearance of glucose from blood circulation (namely glucose
uptake by cells) is measured by assays well known in the art. Slow
rate (as compared to healthy subject) of glucose clearance will
indicate insulin resistance. The administration of pharmaceutical
compositions comprising one or more T2DBMARKERS, such as the
peptide inhibitors of the invention, to an insulin-resistant
subject increases the rate of glucose uptake as compared to a
non-treated subject. Peptide inhibitors may be administered to an
insulin resistant subject for a longer period of time, and the
levels of insulin, glucose, and leptin in blood circulation (which
are usually high) may be determined. Decrease in glucose levels
will indicate that the peptide inhibitor potentiated insulin
action. A decrease in insulin and leptin levels alone may not
necessarily indicate potentiation of insulin action, but rather
will indicate improvement of the disease condition by other
mechanisms.
[0246] The peptide inhibitors of the invention can be used to
therapeutically treat Diabetes or a pre-diabetic condition in a
patient with type 2 Diabetes or a pre-diabetic condition as defined
herein. A therapeutically effective amount of the inhibitor can be
administered to the patient, and clinical markers, for example
blood sugar level and/or IRS-1 phosphorylation, can be monitored.
The peptide inhibitors of the invention can further be used to
prevent type 2 Diabetes or a pre-diabetic condition in a
subject.
Treatment of Diabetes is determined by standard medical methods. A
goal of Diabetes treatment is to bring sugar levels down to as
close to normal as is safely possible. Commonly set goals are
80-120 milligrams per deciliter (mg/dl) before meals and 100-140
mg/dl at bedtime. A particular physician may set different targets
for the patent, depending on other factors, such as how often the
patient has low blood sugar reactions. Useful medical tests include
tests on the patient's blood and urine to determine blood sugar
level, tests for glycosylated hemoglobin level (HbAlc; a measure of
average blood glucose levels over the past 2-3 months, normal range
being 4-6%), tests for cholesterol and fat levels, and tests for
urine protein level. Such tests are standard tests known to those
of skill in the art (see, for example, American Diabetes
Association, 1998). A successful treatment program can also be
determined by having fewer patients in the program with
complications relating to Diabetes, such as diseases of the eye,
kidney disease, or nerve disease.
EXAMPLES
Example 1
Biomarker Identification in the Cohen Rat Model of Type 2
Diabetes
[0247] The Cohen diabetic (CD) rat is a well-known and versatile
animal model of Type 2 Diabetes, and is comprised of 2 rodent
strains that manifest many of the common features of Type 2
Diabetes (T2D) in humans. The sensitive strain (CDs) develops
Diabetes within 30 days when maintained on a high
sucrose/copper-poor diet (HSD), whereas the resistant strain (CDr)
retains normal blood glucose levels. When maintained indefinitely
on regular rodent diet (RD), neither strain develop symptoms of
T2D.
Sample Preparation
[0248] Serum, urine, and tissue samples (including splenic tissue,
pancreatic tissue, and liver tissue) were obtained from both CDr
and CDs rats that were fed either RD or HSD for 30 days. The
samples were flash-frozen and stored at -80.degree. C.
[0249] Whole protein extracts were prepared for each of the 4
experimental conditions, utilizing 10 individual organs per group.
Pancreatic tissues were processing using a mechanical shearing
device (Polytron). To preserve protein integrity in processed
samples, tissues were kept on dry ice until processing commenced
and all buffers and equipment were pre-chilled. Samples were also
kept on ice during the homogenization process.
[0250] T-Per buffer (Pierce) was pre-chilled on ice and two tablets
of Complete Protease Inhibitor (Roche Applied Sciences) were added
per 50 ml of buffer prior to use. Once protease inhibitors were
added, any unused buffer was discarded. T-Per buffer was used at 20
ml per gram of tissue. For each group, pancreatic samples were
weighed and the amount of lysis buffer required was calculated and
added to each tissue sample in a 50 ml tube. Each sample was
homogenized on ice for 10 seconds, followed by a 30 second rest
period to allow the sample to cool. If gross debris was still
apparent, the cycle was repeated until the homogenate was smooth.
The homogenization probe was inserted into the samples
approximately 1 cm from the bottom of the tube to minimize foaming.
When homogenization was complete, the extract was centrifuged at
10,000.times.g for 15 minutes at 4.degree. C.
[0251] Following centrifugation, the supernatant was harvested and
a bicinchoninic acid (BCA) assay was performed to determine the
total protein content. Table 2 provides the mean protein content of
the samples corresponding to CDr rats fed either RD or HSD, and CDs
rats fed either RD or HSD.
TABLE-US-00002 TABLE 2 Total Protein Content of Pancreatic Extracts
from Cohen Diabetic Rats Mean Protein Content (.mu.g/ml) Tissue
CDr-RD CDr-HSD CDs-RD CDs-HSD Pancreas 7969.2 6061.9 6876.4
3387.8
[0252] Supernatants were dispensed into aliquots and stored at
-80.degree. C. Pelleted material was also kept and stored at
-80.degree. C.
[0253] Protein expression profiling of the CDr and CDs phenotypes
was conducted on the pancreatic extracts using one-dimensional
SDS-PAGE. A sample of each extract containing 6 .mu.g of total
protein was prepared in sample buffer and loaded onto a 4-12%
acrylamide gel. Following completion of the electrophoretic run,
the gel was soaked with Coomassie stain for 1 hour and destained in
distilled water overnight. The resulting protein expression profile
allowed an empirical visual comparison of each extract. These
pancreatic extracts were then used for bi-directional immunological
contrasting, disclosed herein.
[0254] Since albumin, immunoglobulin and other abundant proteins
constitute about 95-97% of the total proteins in serum, the
detection of less abundant proteins and peptides markers are masked
if the whole serum were analyzed directly. Therefore, fractionation
of serum samples was necessary to reduce masking of low abundance
protein and to increase the number of peaks available for
analysis.
[0255] To increase the detection of a larger number of peaks as
well as to alleviate signal suppression effects on low abundance
proteins from high abundant proteins such as albumin,
immunoglobulin etc., the crude serum samples from CDr and CDs rats
fed RD or HSD were fractionated into six fractions. The
fractionation was carried out using anion exchange bead based serum
fractionate kit purchased from Ciphergen (Fremont, Calif.). In
brief, the serum samples were diluted with a 9M urea denaturant
solution; the diluted samples were then loaded onto a 96-well
filter microplate pre-filled with an anion exchange sorbent. Using
this process, samples were allowed to bind to the active surface of
the beads, and after 30 minutes incubation at 4.degree. C., the
samples were eluted using stepwise pH gradient buffers. The process
allowed the collection of 6 fractions including pH 9, pH 7, pH 5,
pH 4, pH 3 and an organic eluent. After the fractionation, the
serum samples were analyzed in the following formats on SELDI
chips.
SELDI (Surface Enhanced Laser Desorption Ionization)
[0256] SELDI Proteinchip.RTM. Technology (Ciphergen) is designed to
perform mass spectrometric analysis of protein mixtures retained on
chromatographic chip surfaces. The SELDI mass spectrometer produces
spectra of complex protein mixtures based on the mass/charge ratio
of the proteins in the mixture and their binding affinity to the
chip surface. Differentially expressed proteins are determined from
these protein profiles by comparing peak intensity. This technique
utilizes aluminum-based supports, or chips, engineered with
chemical modified surfaces (hydrophilic, hydrophobic,
pre-activated, normal-phase, immobilized metal affinity, cationic
or anionic), or biological (antibody, antigen binding fragments
(e.g., scFv), DNA, enzyme, or receptor) bait surfaces. These varied
chemical and biochemical surfaces allow differential capture of
proteins based on the intrinsic properties of the proteins
themselves. Tissue extractions or body fluids in volumes as small
as 1 .mu.l are directly applied to these surfaces, where proteins
with affinities to the bait surface will bind. Following a series
of washes to remove non-specifically or weakly bound proteins, the
bound proteins are laser desorbed and ionized for MS analysis.
Molecular weights of proteins ranging from small peptides to
proteins (1000 Dalton to 200 kD) are measured. These mass spectral
patterns are then used to differentiate one sample from another,
and identify lead candidate markers for further analysis. Candidate
marker have been identified by comparing the protein profiles of
conditioned versus conditioned stem cell culture medium. Once
candidate markers are identified, they are purified and
sequenced.
[0257] The fractionated serum samples were applied to different
chemically modified surface chips (cationic exchange, anionic
exchange, metal-affinity binding, hydrophobic and normal phase) and
profiled by SELDI, two-dimensional PAGE (2DE) and two-dimensional
liquid chromatography (2D/LC).
Two-Dimensional Liquid Chromatography (2D/LC)
[0258] The ProteomeLab PF 2D Protein Fractionation System is a
fully automated, two-dimensional fractionation system (in liquid
phase) that resolves and collects proteins by isoelectric point
(pI) in the first dimension and by hydrophobicity in the second
dimension. The system visualizes the complex pattern with a two
dimensional protein map that allows the direct comparison of
protein profiling between different samples. Since all components
are isolated and collected in liquid phase, it is ideal for
downstream protein identification using mass spectrometry and/or
protein extraction for antibody production.
[0259] The PF 2D system addresses many of the problems associated
with traditional proteomics research, such as detection of low
abundance proteins, run-to-run reproducibility, quantitation,
detection of membrane or hydrophobic proteins, detection of basic
proteins and detection of very low and very high molecular weight
proteins. Since the dynamic range of proteins in serum spans over
10 orders of magnitude, and the relatively few abundant proteins
make up over 95% of the total protein contents, this makes it very
difficult to detect low abundant proteins that are candidate
markers. In order to enrich and identify the less abundant
proteins, the serum samples were partitioned using IgY-R7 rodent
optimized partition column to separate the seven abundant proteins
(Albumin, IgG, Transferrin, Fibrinogen, IgM, .alpha.1-Antitrypsin,
Haptoglobin) from the less abundant ones.
[0260] The partitioned serum was applied to the PF-2D. The first
dimensional chromatofocusing was performed on an HPCF column with a
linear pH gradient generated using start buffer (pH 8.5) and eluent
buffer (pH 4.0). The proteins were separated based on the pi.
Fractions were collected and applied to a reverse-phase HPRP column
for a second dimensional separation. The 2D map generated from each
sample was then compared and differential peak patterns were
identified. The fraction was subsequently selected and subjected to
trypsin digestion. The digested samples were sequenced using LC/MS
for protein identification.
2-D Gel Electrophoresis
[0261] Two-dimensional electrophoresis has the ability to resolve
complex mixtures of thousands of proteins simultaneously in a
single gel. In the first dimension, proteins are separated by pI,
while in the second dimension, proteins are separated by MW.
Applications of 2D gel electrophoresis include proteome analysis,
cell differentiation, detection of disease markers, monitoring
response to treatment etc.
[0262] The IgY partitioned serum samples were applied to
immobilized pH gradient (IPG) strips with different pH gradients,
pH 3-10, pH 3-6 and pH 5-8. After the first dimensional run, the
IPG strip was laid on top of an 8-16% or 4-20% SDS-PAGE gradient
gel for second dimensional separation.
Results
[0263] A peak protein of approximately 4200 daltons was present in
the serum of CDr-RD and CDr-HSD, but not in the serum of CDs-RD or
CDs-HSD, as shown in FIG. 1. FIG. 2 is a MS/MS spectrum of the 4200
dalton fragment. This protein was sequenced and following extensive
database searches, was found to be a novel protein. The peptide was
designed "D3" and its sequence was found to be SGRPP MIVWF NRPFL
IAVSH THGQT ILFMA KVINP VGA (SEQ ID NO: 1). The D3 peptide is a
38-mer peptide sequence that corresponds to the first biomarker
discovered in the Cohen diabetic rat. Sequence alignment using the
BLAST algorithm available from the National Center for
Biotechnology Information (NCBI) was performed and the 38-amino
acid fragment was found to have sequence identity with at least ten
different amino acid sequences. Notably, BLAST alignment revealed
that the 38-amino acid D3 peptide contains conserved motifs
corresponding to: "FNRPFL" and "FMS/GKVT/VNP". FIG. 3A shows the
results of the BLAST alignment of amino acid sequences related to
the D3 peptide fragment, and FIG. 3B shows the results of a BLAST
alignment of nucleic acid sequences encoding the D3 peptide and the
peptides identified by protein BLAST. Degenerate primers were
designed to target the conserved motifs and comprise the following
sequences: Forward primer (targeting regions containing the amino
acid sequence "FNRPFL": 5'-TTC AAC MRR CCY TTY ST-3' (SEQ ID NO: 4)
and Reverse primer (targeting regions containing the sequence
"FMS/GKVT/VNP"): 5'-YVA CYT TKC YMA KRA AGA-3' (SEQ ID NO: 5);
wherein M=A or C; R=A or G; Y.dbd.C or T; S.dbd.C or G; K=G or T;
and V=A, C, or G. These degenerate primers were used in
reverse-transcription polymerase chain reactions (RT-PCR) to
amplify human SERPINA 3 in liver and pancreas. A 1.3 Kb fragment
was identified in human liver and pancreas.
[0264] Table 3 below represents additional identified candidate
markers identified by SELDI analysis.
TABLE-US-00003 Array Type CM10 (Anion exchange) Sample Fractioned
Serum F1 M/Z CDr-RD CDs-RD CDr-HSD CDs-HSD ~2156 + + - - ~2270 + +
- + ~3875 + - + - Sample Fractioned Serum F3 M/Z CDr-RD CDs-RD
CDr-HSD CDs-HSD ~3408 - + - + ~3422 + - + - ~3848 - + - + ~3861 + -
+ - Sample Fractioned Serum F4 M/Z CDr-RD CDs-RD CDr-HSD CDs-HSD
~4202 + - + - ~4423 + - + - Sample Fractioned Serum F5 M/Z CDr-RD
CDs-RD CDr-HSD CDs-HSD ~5377 ++ ++ ++ + ~5790 +/- +/- - + ~8813 +/-
+/- +/- + Sample Fractioned Serum F6 M/Z CDr-RD CDs-RD CDr-HSD
CDs-HSD ~4200 + - + - Sample Whole Serum M/Z CDr-RD CDs-RD CDr-HSD
CDs-HSD ~6631 - + - - ~7013 - - + + ~7027 + + - - ~7811 - + - -
Array Type Q10 Sample Fractioned Serum F1 M/Z CDr-RD CDs-RD CDr-HSD
CDs-HSD ~2627 + - + - ~2705 + - + - ~4290 + + ++ + ~5058 - - + -
~5220 + ++ + + ~5789 - - + - ~8818 + +/- ++ ++ Sample Fractioned
Serum F2 M/Z CDr-RD CDs-RD CDr-HSD CDs-HSD ~2359 + +/- - - ~2587 +
+ - +/- ~2879 + + - +/- ~2298 - + - - Sample Fractioned Serum F4
M/Z CDr-RD CDs-RD CDr-HSD CDs-HSD ~4200 + - + - ~2067 - - + + ~2092
- - + + ~2042 - - + + ~8810 - - + + ~8850 + + - - Sample Fractioned
Serum F5 M/Z CDr-RD CDs-RD CDr-HSD CDs-HSD ~3977 + - + - ~4200 + -
+ - ~2102 + - + - ~4030 + ++ + ++ Sample Fractioned Serum F6 M/Z
CDr-RD CDs-RD CDr-HSD CDs-HSD ~4200 + - + - ~17645 + - + - Sample
Whole Serum M/Z CDr-RD CDs-RD CDr-HSD CDs-HSD ~6632 - + - - ~3419 +
+ - - ~3435 + + - - ~4074 + + - - ~4090 + + - - ~4200 + - + - ~5152
+ + - - ~8915 + + - - Array Type H50 Sample Fractioned Serum F2 M/Z
CDr-RD CDs-RD CDr-HSD CDs-HSD ~5521 - + - - Sample Fractioned Serum
F5 M/Z CDr-RD CDs-RD CDr-HSD CDs-HSD ~34224 - - - + Array Type IMAC
Sample Whole Serum M/Z CDr-RD CDs-RD CDr-HSD CDs-HSD ~2714 + + - +
~4330 - + + +
[0265] The D3 peptide was used for the production of hyper-immune
serum in rabbits. FIGS. 4A and 4B provide bioinformatic and
sequence analysis of the D3 sequence. The D3 peptide was found to
be homologous to Rattus norvegicus Serpin 3M. FIG. 5 depicts
Western blots showing the reactivity of the D3 hyper-immune serum
with a .about.4 kD protein isolated from CDr-RD and CDr-HSD rat
serum fraction 6. Fractionated CD rat serum samples were run on a
10% SDS-PAGE gel, then transferred to PVDF membranes. A higher
molecular weight doublet (in the range of 49 and 62 kD) also
reacted with the hyper-immune sera, indicated that a parent protein
is expressed by all strains under treatment modalities RD or HSD,
however a derivative of smaller size (.about.4 kD) corresponding to
the D3 fragment is differentially expressed only in the CDr strain.
These results are consistent with the results obtained by SELDI
profiling. The concentration of the D3 fragment in CDr rat serum
was subsequently analyzed by SELDI. A series of synthetic D3
peptide standards (0.1, 0.033, 0.011, 0.0037, 0.0012 and 0 mg/ml)
and 10.times. diluted CDr-serum were spotted in duplicate on Q10
protein chip arrays. The peak intensity was plotted against the
concentration of D3 peptide standards. Based on the plot, the
linear range for concentration determination is from 0 to 0.01
mg/ml. Accordingly, the concentration of D3 in CDr-RD serum is
around 0.04 mg/ml, based on the peak intensity of the CDr-RD serum
sample.
[0266] Analysis of Serpina expression by Western blot analysis was
performed in Cohen rat liver extracts using anti D3 rabbit serum
(1:200) and secondary goat anti-rabbit IgG conjugated to HRP
(1:25,000 dilution). Controls containing liver extracts (10 .mu.g)
and secondary goat anti-rabbit IgG antibodies conjugated to HRP
(1:25,000 dilution), but no primary antibody were also analyzed. A
cluster of proteins (41, 45 and 47 kD) were visualized following
reaction of liver extracts with D3 hyper immune serum. The 41 and
45 kD proteins were expressed at approximately the same level while
the 47 kD protein is not detected in the diabetic rat--i.e.,
CDs-HSD (diabetic).
[0267] Table 4 contains a summary of biomarker data obtained from
CD rat serum samples.
TABLE-US-00004 TABLE 4 T2DBMARKER Data Summary Differential
profiling in MW Calculated Cohen Diabetic Rats Serum No. Protein
Gene Gi (KD) pI CDr-RD CDs-RD 1 C-terminal fragment of a Serpina
34867677 4.2 12.01 + - predicted protein, 3M similar to serine
protease inhibitor 2.4 2 unnamed protein product Spin 2a 57231 45
5.48 + - or Spin2a protein 56789860 46 5.48 3 Fetuin beta Fetub
17865327 42 6.71 + - or Fetub protein 47682636 44 7.47 - + 4
Apolipoprotein C-III Apoc 3 91990 11 4.65 + + precursor 5 Predicted
protein, Apoc 2 27676424 11 4.57 + + similar to predicted
Apolipoprotein C2 6 Aa2-066 None 33086518 61 4.39 + - or
alpha-2-HS- Ahsg 6978477 39 6.05 glycoprotein or alpha-2-HS-
60552688 39 6.05 glycoprotein 7 T-kininogen II precursor None
57526868 49 5.94 - + 8 alpha-1-macroglobulin Pzp 202857 168 6.46
TBD + or pregnancy-zone Pzp 21955142 + - protein 9 Serine/cysteine
Serpinc1 56789738 53 6.18 + - proteinase inhibitor, clade C, member
1 (predicted) 10 coagulation factor 2 F2 12621076 72 6.28 + - 11
inter-alpha-inhibitor H4 ITIH4 9506819 104 6.08 + - heavy chain
59808074 + - 12 vitamin D binding Gc 203927 55 5.65 + - protein
prepeptide 13 LMW T-kininogen I Map1 205085 49 6.29 + ++ precursor
or kininogen 56270334 or major acute phase 68791 alpha-1 protein
precursor 14 preapolipoprotein A-1 ApoA1 55747 30 5.52 + + or
apolipoprotein A-1 59808388 + + 15 predicted protein, Apoc2
109461385 11 4.57 TBD TBD similar to apolipoprotein C-II precursor
16 thrombin 207304 28 9.38 TBD TBD or prothrombin 56970 72 TBD TBD
precursor 17 Apolipoprotein E ApoE 37805241 36 5.23 + - or
Apolipoprotein E 55824759 36 5.53 + - or Apolipoprotein E 20301954
36 + - or ORF2 202959 38 + - + ++ 18 Liver regeneration- Tf
33187764 78 7.14 + + related protein LRRG03 19 Apolipoprotein A-IV
Apoa4 60552712 44 5.12 + - 20 LOC297568 protein 71051724 79 5.45 +
++ or Alpha-1-inhibitor 3 112893 165 + ++ precursor 21 hypothetical
protein 62718654 188 6.06 + ++ XP_579384 22 Histidine-rich Hrg
11066005 59 8.12 + ++ glycoprotein 23 unnamed protein product None
55562 167 5.68 +++ ++ or predicted: 62647940 167 +++ ++
hypothetical protein XP_579477 24 Complement component C9 2499467
63 5.51 +++ ++ C9 precursor 25 Apolipoprotein H ApoH 57528174 40
8.58 - + 26 B-factor, properdin Cfb 56268879 86 6.57 - + 27
Hemopexin Hpx 16758014 52 7.58 + ++ Differential profiling in Cohen
Diabetic Rats Serum Profiling Human No. Protein CDr-HSD CDs-HSD
technology Homologues 1 C-terminal fragment of a + - SELDI Serpina
3 predicted protein, similar to serine protease inhibitor 2.4 2
unnamed protein product - - PF-2D or Spin2a protein 3 Fetuin beta -
TBD PF-2D result Fetub_human or Fetub protein - + 2DE result 4
Apolipoprotein C-III + TBD PF-2D Apoc3_human precursor 5 Predicted
protein, + - PF-2D Apoc2_human similar to Apolipoprotein C2 6
Aa2-066 + + PF-2D Alpha-2-HS- glycoprotein or alpha-2-HS-
FetuA_Human glycoprotein or alpha-2-HS- glycoprotein 7 T-kininogen
II precursor - TBD PF-2D 8 alpha-1-macroglobulin TBD TBD PF-2D
result PZP_human and or pregnancy-zone - - 2DE result A2MG_human
protein 9 Serine/cysteine + - PF-2D proteinase inhibitor, clade C,
member 1 (predicted) 10 coagulation factor 2 TBD TBD PF-2D 11
inter-alpha-inhibitor H4 + TBD PF-2D ITIH4_human heavy chain + TBD
12 vitamin D binding TBD TBD PF-2D VTDB_human protein prepeptide 13
LMW T-kininogen I + ++++ PF-2D/2DE precursor or kininogen or major
acute phase alpha-1 protein precursor 14 preapolipoprotein A-1 + -
PF-2D ApoA1_human or apolipoprotein A-1 + - 15 predicted protein, +
- PF-2D similar to apolipoprotein C-II precursor 16 thrombin + TBD
PF-2D or prothrombin + TBD THRB_human precursor 17 Apolipoprotein E
- - 2DE or Apolipoprotein E - - or Apolipoprotein E - - or ORF2 - -
+ ++ 18 Liver regeneration- ++++ ++ 2DE related protein LRRG03 19
Apolipoprotein A-IV - - 2DE 20 LOC297568 protein + +++++ 2DE or
Alpha-1-inhibitor 3 + +++++ precursor 21 hypothetical protein + +++
2DE XP_579384 22 Histidine-rich + +++ 2DE glycoprotein 23 unnamed
protein product ++ + 2DE or predicted: ++ + 2DE hypothetical
protein XP_579477 24 Complement component ++ + PF/2DE C9 precursor
25 Apolipoprotein H + + 2DE 26 B-factor, properdin + + 2DE 27
Hemopexin + +++ 2DE Hemo_human
Example 2
Bi-Directional Immunological Contrasting and Generation of
Monoclonal Antibodies
[0268] From the pancreatic extract protein profiles obtained by
SDS-PAGE, obvious differences in the banding patterns were noted
between CDr-HSD and CDs-HSD samples. Bi-directional immunological
contrast was performed between these two samples. This technique
involves injecting two pancreatic extracts from the Cohen diabetic
rats to be contrasted separately into the footpads of an
experimental animal (e.g. a Balb/c mouse). Following uptake and
processing of the antigen at the site of injection by antigen
presenting cells (APCs), the activated APCs migrate to the local
lymph nodes (popliteal) to initiate an immune response. As these
lymph nodes are located in each leg, they are anatomically
separated from each other, which prevents mixing of
antigen-specific lymphocytes at this point. Later in the immune
response, these activated lymphocytes migrate from the local lymph
nodes to the spleen where they become mixed, and from where they
may circulate systemically.
[0269] Two weeks after footpad injection, the animals were boosted
by injecting each footpad with the same antigen as before. This
boost recalls antigen specific lymphocytes back to the site of
injection, again subsequently draining to the popliteal lymph
nodes. This technique uses the natural proliferation and cell
migration processes as a filtering mechanism to separate and enrich
specific lymphocytes in each lymph node, where they are
anatomically segregated to minimize mixing of cells that are
specific for antigen(s) expressed in only one of the extracts.
Three days after boosting, the popliteal lymph nodes were removed
and separated into pools derived from each side of the animals.
When boosting, it is imperative not to switch the antigenic
material, as this will cause specific lymphocytes to migrate to
both sets of popliteal lymph nodes and the anatomical segregation
of specific cells, and hence the advantage of the technique, will
be lost.
[0270] Fifteen female Balb/c mice ages 6-8 weeks were ordered from
Harlan. Each animal was injected with 25 .mu.g of CDr-HSD
pancreatic extract into the left hind footpad, and 25 .mu.g of
CDs-HSD pancreatic extract into the right hind footpad. Antigens
were prepared in 20% Ribi adjuvant in a final volume of 50 .mu.l as
follows:
TABLE-US-00005 TABLE 5 Right footpad Left footpad 375 mg of CDs-HSD
110 .mu.l -- 375 mg of CDr-HSD -- 62 .mu.l PBS 490 .mu.l 538 .mu.l
Ribi adjuvant 150 .mu.l 150 .mu.l
[0271] Ribi adjuvant was warmed to 37.degree. C. and reconstituted
with 1 ml of sterile PBS. The bottle was vortexed for at least 1
minute to fully reconstitute the material. The correct volume of
Ribi adjuvant was then added to the antigen preparation, and the
mixture was again vortexed for 1 minute. Any unused formulated
material was discarded, and any unused Ribi adjuvant was stored at
4.degree. C. and used to formulate booster injections. Animals were
primed on day 1 and boosted on day 14. Animals were euthanized on
day 17, when popliteal lymph nodes were excised post mortem and
returned to the lab for processing.
Generation of Hybridomas
[0272] Hybridoma cell lines were created essentially as described
by Kohler and Milstein (1975). Lymphocytes derived from immunized
animals were fused with a murine myeloma cell line (Sp2/0) by
incubation with polyethylene glycol (PEG). Following fusion, cells
were maintained in selective medium containing hypoxanthine,
aminopterin and thymidine (HAT medium) that facilitates only the
outgrowth of chimeric fused cells.
[0273] On the day before the fusion, the fusion partner
(Sp2/0.times.Ag14 cells in dividing stage with viability above 95%)
was split at 1.times.10.sup.5 viable cells/ml, 24 hours before the
fusion. On the day of the fusion, the mice were sacrificed and the
lymph nodes were excised and placed in a Petri dish containing
pre-warmed room temperature DMEM supplemented with 10% fetal bovine
serum (FBS). Using sterile microscope slides, the lymph nodes were
placed between the 2 frosty sides of the slides and crushed into a
single cell suspension. The cell suspension was then transferred to
a 15 ml tube and centrifuged for 1 minute at 1000 rpm. The
supernatant was removed by aspiration, and the cell pellet gently
resuspended in 12 ml of serum-free DMEM, after which they were
subjected to another round of centrifugation for 10 minutes at 1000
rpm. The process was repeated twice more to ensure that the serum
was completely removed. After washing, the cells were resuspended
in 5 ml of serum-free DMEM and counted under the microscope.
[0274] The fusion partner was collected by spinning in a centrifuge
for 10 minutes at 1000 rpm. The cells were washed three times in
serum-free DMEM, and finally resuspended in serum-free DMEM and
counted. The number of fusion partner cells were calculated based
on the number of lymph node cells. For every myeloma cell (fusion
partner), 2 lymph nodes cells is needed (ratio 1:2 of myeloma to
lymph node cells; e.g. for 10.times.10.sup.6 lymph node cells,
5.times.10.sup.6 fusion partner cells are needed). The appropriate
number of myeloma cells to the LN cells were added and the total
volume of cells was adjusted to 25 ml using serum free DMEM, and 25
ml of 3% dextran was then added to the cells. The mixture was spun
for 10 minutes at 1000 rpm, and the supernatant aspirated as much
as possible from the cell pellet. Once the lid was placed onto the
tube containing the cells, the bottom of the tube was gently tapped
the bottom of the tube to resuspend the cells and 1 ml of
pre-warmed 50% (v/v) PEG was added to the tube. The agglutinated
cells were allowed to sit for 1 minute, after which 20 ml of serum
free DMEM, followed by 25 ml of 20% FBS, DMEM with 25 mM Hepes was
added. The tube was inverted once to mix and then centrifuged for
10 minutes at 1000 rpm. The media was aspirated and the cells were
gently resuspended by tapping. HAT selection media was added such
that the cell suspension was either at 0.125.times.10.sup.6
cells/ml or 0.0625.times.10.sup.6 cells/ml. One hundred .mu.l of
cells per well were added to a 96-well flat bottom plate and
incubated at 37.degree. C. with CO.sub.2 at 8.5%. After 2 days, the
cells were fed with 100 .mu.l of fresh HAT selection media. Cells
were checked for colony growth after 7 days.
Hybridoma Screening
[0275] Once visible colonies were observed in the 96 well plates,
100 .mu.l of conditioned supernatant was harvested from each colony
for screening by ELISA. Supernatants were screened for the presence
of detectable levels of antigen-specific IgG against both CDr-HSD
and CDs-HSD extracts. Only colonies exhibiting a positive ELISA
reaction against one of the two extracts with at least a 2-fold
difference were selected for expansion and further
characterization.
[0276] Pancreas extract at a concentration of 25 .mu.g/ml to be
tested was diluted in carbonate bicarbonate buffer (1 capsule of
carbonate-bicarbonate was dissolved in 100 ml of deionized water).
Two extra wells for the positive control and two extra wells for
the negative control of a 96-well plate were reserved. The plate
was then covered using adhesive film and incubated at 4.degree. C.
overnight.
[0277] The plate was washed once with 200 .mu.l of PBS/Tween. The
well content was removed by flicking the plate into a sink, and
then gently tapping the plate against absorbent paper to remove
remaining liquid. Approximately 200 .mu.l of washing buffer
(PBS/Tween) was added and subsequently discarded as previously
described. The entire plate was then blocked for 1 hour at
37.degree. C. in 200 .mu.l of 5% powdered milk/PBS/Tween. The plate
was then washed 3 times using PBS/Tween as previously
described.
[0278] The fusion culture supernatant was diluted 1:1 in 0.5%
milk/PBS/Tween and each sample added to the wells (50 .mu.l; final
volume is 100 .mu.l per well) with 50 .mu.l of anti-actin Ab
(Sigma) at 20 .mu.g/ml to well containing 50 .mu.l of buffer. Fifty
111 of buffer was added to the negative control well. The plate was
covered and incubated overnight at 4.degree. C. The plate was
washed 3 times using PBS/Tween as previously described, and
anti-HRP anti-mouse IgG in 0.5% milk/PBS/Tween at 1:20000 (100
.mu.l) was added to each well. The plate was covered and incubated
at 37.degree. C. for two hours.
[0279] After incubation with secondary antibody, the plates were
washed 4 to 5 times as previously described. On the last wash, the
washing buffer was left on the plate for a couple of minutes before
discarding it. One hundred .mu.l of pre-warmed room temperature TMB
(VWR; stored in the dark) was added to each well while minimizing
the introduction of bubbles, until the color developed (20-30
minutes). The reaction was stopped by adding 50 .mu.l of 2M
sulfuric acid. The plate was read using a spectrophotometer at 450
nm.
[0280] Thirteen clones produced monoclonal antibodies (mAbs) that
met the experimental criteria outlined above, 9 against CDs-HSD and
4 against CDr-HSD. The ELISA data for these colonies is summarized
in Table 6 for monospecific CDr-HSD and CDs-HSD hybridomas.
Absolute absorbance values, and fold difference at OD 450 nm is
shown for each colony. To verify primary screening data, some
clones were retested during expansion to confirm the experimental
observations from the initial screen.
[0281] The composition of each mAb was defined by determining the
class of heavy and light chains, as well as the molecular weight,
of each component. Isotyping was performed using the Immunopure
monoclonal antibody isotyping kit I (Pierce) according to the
manufacturer's instructions. The molecular weight of heavy and
light chains was determined using the Experion automated
electrophoresis system from Bio-Rad. The Experion system
automatically performs the multiple steps of gel-based
electrophoresis: separation, staining, destaining, band detection,
imaging, and data analysis. The results of these analyses are shown
in Table 6, which shows the physical characterization of CDr-HSD
and CDs-HSD specific monoclonal antibodies. Identification of both
heavy and light chains was performed using the Immunopure
monoclonal antibody isotyping kit I (Pierce), and molecular weights
(in kD) were determined using the Experion automated
electrophoresis system (Bio-Rad).
TABLE-US-00006 TABLE 6 Light chain Heavy chain Clone ID Sub- Mol.
Sub- Mol. Whole IgG Accession No. type Wt. class Wt. Mol. Wt.
P1-5-F11 kappa -- IgG2b -- -- (Accession No.) P1-14-A2 Kappa/ --
IgG1 -- -- (Accession No.) lambda P1-17-E4 Kappa -- IgG1 -- --
(Accession No.) P1-18-C12 Kappa -- IgG2b -- -- (Accession No.)
P1-20-B7 Kappa -- IgG1 -- -- (Accession No.) P1-23-F7 Kappa --
IgG2b -- -- (Accession No.) P2-1-E8 Kappa -- IgG1 -- -- (Accession
No.) P2-10-E3 Kappa -- IgG2a -- -- (Accession No.) P2-14-C6 Kappa
-- IgG1 -- -- (Accession No.) P2-4-H5 Kappa -- IgG2b -- --
(Accession No.) P2-8-A3 Kappa -- IgG2b -- -- (Accession No.)
P2-10-B8 Kappa -- IgG2b -- -- (Accession No.) P2-13-A9 kappa --
IgG1 -- -- (Accession No.)
[0282] Purification of the monoclonal antibodies and
immunoprecipitation were conducted using standard protocols known
in the art.
[0283] Following precipitation, several bands were visible on the
gel after staining for total protein with Coomassie. A faint
doublet band was observed in the molecular weight range of 70 to 80
kD. The doublet was confirmed to be the bands of interest by
probing a Western Blot prepared from a similar gel with hybridoma
clones MAb-P2.19B8.KA8 or P2-4-H %-K-B4 (FIGS. 6 and 7). The
doublet bands were excised individually from the SDS-PAGE gel and
submitted for identification by mass spectrometry (FIG. 8). An
positive identification of the lower band as calnexin was made.
Calnexin is a molecular chaperone associated with the endoplasmic
reticulum.
[0284] Calnexin is a 90 kD integral protein of the endoplasmic
reticulum (ER). It consists of a large (50 kD) N-terminal
calcium-binding lumenal domain, a single transmembrane helix and a
short (90 residues), acidic cytoplasmic tail. Calnexin belongs to a
family of proteins known as "chaperones," which are characterized
by their main function of assisting protein folding and quality
control, ensuring that only properly folded and assembled proteins
proceed further along the secretory pathway. The function of
calnexin is to retain unfolded or unassembled N-linked
glycoproteins in the endoplasmic reticulum. Calnexin binds only
those N-glycoproteins that have GlcNAc2Man9Glc1 oligosaccharides.
Oligosaccharides with three sequential glucose residues are added
to asparagine residues of the nascent proteins in the ER. The
monoglucosylated oligosaccharides that are recognized by calnexin
result from the trimming of two glucose residues by the sequential
action of two glucosidases, I and II. Glucosidase II can also
remove the third and last glucose residue. If the glycoprotein is
not properly folded, an enzyme called UGGT will add the glucose
residue back onto the oligosaccharide thus regenerating the
glycoprotein ability to bind to calnexin. The glycoprotein chain
which for some reason has difficulty folding up properly thus
loiters in the ER, risking the encounter with MNS1
(.alpha.-mannosidase), which eventually sentences the
underperforming glycoprotein to degradation by removing its mannose
residue. ATP and Ca.sup.2+ are two of the cofactors involved in
substrate binding for calnexin.
Example 3
Microarray Analysis of Gene Expression in Tissues from Cohen Type 2
Diabetic Rats
[0285] The microarray data were analyzed through Phase I and Phase
II analyses. Phase I is based on the processed data from Gene
Logic. Phase II corresponds to data analysis using GeneSpring GX.
Additional criteria including statistics, signaling pathways and
clustering were used for the analyses.
[0286] The microarray results from Gene Logic (Phase I) that were
derived from comparisons of pancreatic total RNA of Cohen Type 2
Diabetes rats (CDs-HSD, CDr-HSD) were analyzed using MAS5.0
software from Affymetrix, Inc. The global gene expression analysis
showed that there were 1178 genes upregulated in CDr-HSD and 803
genes were downregulated in compared to CDs-HSD. Many of these
transcripts are involved in several signaling pathways related to
Type 2 Diabetes such as insulin signaling, beta-cell dysfunction
and lipid and glucose metabolisms. Also, several serpin family
members (serine proteinase inhibitors) are expressed differently in
the two models.
Table 7 provides a summary of the data derived from Gene Logic,
wherein changes greater than 3-fold were observed.
TABLE-US-00007 TABLE 7 Upregulated Downregulated genes genes CDR-HS
vs. CDR-HS vs. Signaling Pathways CDS-HS CDS-HS Insulin signaling
39 41 .beta. cell dysfunction (apoptosis, survival) 17 6
Inflammation and immune system 5 92 Mitochondrial dysfunction and
reactive 20 8 oxygen species Lipid and glucose metabolisms 17 13
proteinase and proteinase inhibitors 28 17 Amino acid, nucleic acid
transporters and 13 9 metabolisms Potassium channels 3 6 ER and
Golgi body related genes 8 8 Other unclassified genes 1028 603
Total 1178 803
[0287] Phase II data analysis was performed using GeneSpring GX,
which used normalized data (ratio=transcript signal/control signal)
to improve cross-chip comparison. GeneSpring GX allows for gene
lists to be filtered according to genes exhibiting a 2-fold or
3-fold change in the expression levels. GeneSpring GX also
comprises statistical algorithms, such as ANOVA, Post-Hoc Test, and
Cross-Gene Error Modeling, as well as gene clustering algorithms
like Gene Tree, K-mean clustering, and Self-Organizing Map (SOM)
clustering. GeneSpring GX also has the ability to integrate with
pathways that are published in the art, such as the Kyoto
Encyclopedia of Genes and Genomes ("KEGG pathways") and Gen Map
Annotator and Pathway Profiler (GenMAPP).
[0288] Microarray and quantitative PCR analyses were applied to
identify the transcriptome changes in pancreatic and epididymal fat
tissues of the two strains exposed to a regular diet (RD) or
diabetogenic/high sucrose diet (HSD). Both pancreatic tissues and
visceral fat tissue-epididymal fat tissue are deemed important
primary tissues to study gene transcripts that may play a crucial
role in the prediction, progression, and possibly prevention of the
disease.
[0289] Total RNA was extracted from pancreatic and epididymal fat
tissues from each of the strains (CDs, CDr) under regular diet (RD)
and diabetogenic diet (HSD). The transcriptome was then analyzed
using the Rat Expression Arrays (Affymetrix) set 230 which contains
oligonucleotide probes for over 30,000 transcripts. Three to five
rats from each groups (CDs-RD, CDs-HSD, CDr-RD and CDr-HSD) were
used for data analyses. The results were analyzed using GeneSpring
GX (Agilent, CA). Expression of several selected transcripts was
also confirmed by real-time PCR.
[0290] Transcriptome changes of pancreatic tissue were first
analyzed via microarray. For this experiment three animals from
each of the following groups CDr-HSD and CDs-HSD were analyzed. In
CDr-HSD and CDs-HSD rats, eighty-two (82) transcripts show a change
of three fold or higher when the two groups are compared (see
Tables 8 and 9); nineteen (19) transcripts are downregulated
(expression in CDr-HSD is decreased 3 fold or more; Table 9), and
sixty-three (63) transcripts were upregulated (expression in
CDr-HSD is increased 3 fold or more; Table 8). Fourteen of these
transcripts were selected and their changes in the expression
levels were confirmed by quantitative PCR. The quantitative PCR
analyses validated the changes of expression observed by micorarray
analyses.
TABLE-US-00008 TABLE 8 Upregulated transcripts expressed 3-fold in
CDr-HSD rats UniGene UniGene Name (rat) (human) Description and
Gene Ontology REG3G Rn.11222 Hs.447084 Regenerating islet-derived 3
gamma SDF2L1 Rn.1414 Hs.303116 Endoplasmic reticulum
stress-inducible gene REG3A Rn.9727 Hs.567312 Regenerating
islet-derived 3 alpha MAT1A Rn.10418 Hs.282670 Methionine
adenosyltransferase NUPR1 Rn.11182 Hs.513463 Nuclear protein 1
CHAC1 Rn.23367 Hs.155569 Cation transport regulator-like 1 SLC7A3
Rn.9804 Hs.175220 Solute carrier family 7, member 3 PRSS3 Rn.13006
Hs.128013 Protease serine 3 (mesotrypsin) BF415056 Rn.47821 n/a
Unknown cDNA PABPC4 Rn.199400 Hs.169900 Ploy A binding protein,
cytoplasmic 4 CYP2D6 Rn.91355 Hs.648256 Cytochrome P450, 2D6
AI044556 Rn.17900 n/a unknown PRSS4 Rn.10387 Hs.128013 Mesotrypsin
preproprotein GLS2 Rn.10202 Hs.212606 Glutaminase 2 (liver,
mitochondrial) NME2 Rn.927 Hs.463456 Nucleoside diphosphate
kinase-B P2RX1 Rn.91176 Hs.41735 Purinergic receptor P2X,
ligand-gated ion channel 1 PDK4 Rn.30070 Hs.8364 Pyruvate
dehydrogenase kinase, isoenzyme 4 AMY1A Rn.116361 Hs.484588 Amylase
1A, 1B and 2A and 2B are closely related CBS Rn.87853 Hs.533013
Cytathionine beta synthase MTE1 Rn.37524 Hs.446685 Acyl-CoA
thioesterase2 or mitochondrial acyl-CoA thioesterase SPINK1 Rn.9767
Hs.407856 Serine protease inhibitor, Kazal type 1, GATM Rn.17661
Hs.75335 Glycine amidinotransferase (L-arginine:glycine
amidinotransferase) TMED6 Rn.19837 Hs.130849 Transmembrane emp24
protein transport domain containing 6 TFF2 Rn.34367 Hs.2979 Trefoil
factor 2 (spasmolytic protein 1) HSD17B13 Rn.25104 Hs.284414
Hydroxysteriod (17-beta) dehydrogenase 13 GNMT Rn.11142 Hs.144914
Glycine N-methyltransferase LRRGT00012 Rn.11766 n/a unknown PAH
Rn.1652 Hs.652123 Phenylalanine hydroxylase SERPINI2 Rn.54500
Hs.445555 Serine proteinase inhibitor clade I, member 2 RGD1309615
Rn.167687 n/a Similar to hypothetical protein XP_580018 LRRC39
Rn.79735 Hs.44277 Leucine repeat containing 39 EPRS Rn.21240
Hs.497788 Glutamyl-prolyl-tRNA synthetase PCK2 Rn.35508 Hs.75812
Phosphoenolpyruvate carboxykinase 2 (mitrochondria) AA997640
Rn.12530 n/a unknown SERPINA10 Rn.10502 Hs.118620 Serine peptidase
inhibitor, clade A, member 10 SLC30A2 Rn.11135 Hs.143545 Solute
carrier family 30 (zinc transporter), member 2 CCKAR Rn.10184
Hs.129 Cholecystokinin A receptor BHLHB8 Rn.9897 Hs.511979 Basic
helix-loop-helix domain containing, class B, 8 ANPEP Rn.11132
Hs.1239 Alanyl aminopeptidase ASNS Rn.11172 Hs.489207 Asparagines
synthetase SLC7A5 Rn.32261 Hs.513797 Solute carrier family 7 member
5 PABPC4 Rn.2995 Hs.169900 Poly (A) binding protein, cytoplasmic
4(inducible) KLK1 Rn.11331 Hs.123107 Kallikrein 1 ERP27 Rn.16083
Hs.162143 Endoplasmic reticulum protein 27 KDa QSCN6 Rn.44920
Hs.518374 Quiescin 6 CLDN10 Rn.99994 Hs.534377 Claudin10 MARS
Rn.140163 Hs.632707 Methonine-tRNA synthetase EIF4B Rn.95954
Hs.292063 Eukaryotic translation initiation factor 4B RNASE4
Rn.1742 Hs.283749 Ribonuclease, Rnase A family 4 ST6GALNAC4
Rn.195322 Hs.3972 Alpha-2,6-sialytransferase ST6GALNAC 4 HERPUD1
Rn.4028 Hs.146393 Homocysteine-inducible, endoplasmic reticulum
stress- inducible, ubiquitin-like domain member 1 DBT Rn.198610
Hs.653216 Dihydrolipoamide branched chain transferase E2 FUT1
Rn.11382 Hs.69747 Fucosyltransferase 1 AL170755 Rn.22481 n/a
unknown VLDLR Rn.9975 Hs.370422 Very low density lipoprotein
receptor GNPNAT1 Rn.14702 Hs.478025 Glucosamine phosphate
N-acetyltransferase 1 DDAH1 Rn.7398 Hs.379858 Dimethylarginine
dimethylaminohydrolase 1 HSPA9 Rn.7535 Hs.184233 Heat shock 70 Kda
protein 9 PTGER3 Rn.10361 Hs.445000 Prostaglandin E receptor 3
AW523490 Rn.169405 n/a Unknown cNDA RAMP4 Rn.2119 Hs.518326
Ribosome associated membrane MTAC2D1 Rn.43919 Hs.510262 Membrane
targeting 9tandem) C2 domain containing 1 DNAJC3 Rn.162234
Hs.591209 DnaJ homolog, subfamily C, member 3
TABLE-US-00009 TABLE 9 Downregulated transcripts showing 3-fold
reduced in expression in CDr-HSD rats UniGene UniGene Name (rat)
(human) Description and Gene Ontology CCL21 Rn.39658 Hs.57907
chemokine (C-C motif) ligand 21b IGHG1 Rn.10956 Hs.510635 IGHG1 in
human: immunoglobulin heavy constant gamma 1 IGHM Rn.201760
Hs.510635 IGHM: immunoglobulin heavy constant mu Tnfrsf26 Rn.162508
n/a Tumor necrosis factor receptor superfamily, member 26
RGD1306939 Rn.95357 n/a Unknown CD32 Rn.33323 Hs.352642 Fc
receptor, IgG, low affinity IIb LCK Rn.22791 Hs.470627 Lymphocyte
protein tyrosine kinase SCG5 Rn.6173 Hs.156540 Secretogranin V
ARHGD1B Rn.15842 Hs.504877 Rho GDP dissociation inhibitor (GDI)
beta RAC2 Rn.2863 Hs.517601 RAS-related C3 botulinum toxin
substrate 2 CD45 Rn.90166 Hs.192039 Protein tyrosine phosphatase,
receptor type BAT3 Rn.40130 Hs.440900 HLA-B associated transcript 3
CD38 Rn.11414 Hs.479214 CD38 antigen CD132 Rn.14508 Hs.84
Interleukin 2 receptor, gamma ARHGAP30 Rn.131539 Hs.389374 Rho
GTPase activating protein 30 CD53 Rn.31988 Hs.443057 CD53 antigen
S100B Rn.8937 Hs.422181 S100 calcium binding protein B GIMAP4
Rn.198155 Hs.647101 GTPase, IMAP family member4 RGD1563461
Rn.199308 n/a Unknown
[0291] Given the changes observed in the pancreatic tissue and
their consistency by both methods microarray analyses and
quantitative PCR, changes in transcriptome levels in epidydimal fat
tissue for all four groups of Cohen Diabetic rats were also
analyzed. Comparisons among groups may lead to discovery of
biomarkers used for either predisposition, progression, and
resistance of Type 2 Diabetes. For example, CDr-RD versus
CDs-R.sup.D comparisons may indicate predisposition for Type 2
Diabetes, while CDs-RD versus CDs-HSD comparisons may serve as a
model for progression of the disease, and CDr-HSD versus CDs-HSD
comparisons may be used as a model for resistance against
development of Type 2 Diabetes.
[0292] Tissue samples from five animals from each of the
above-mentioned groups were analyzed and the results are summarized
herein. Two hundred (200) transcripts, eighty (80) known
transcripts and one hundred and twenty (120) unknown transcripts
were expressed only in CDs-HSD group, the group that develops Type
2 Diabetes. Twenty-five (25) transcripts with signal strengths
(arbitrary fluorescence units) significantly greater than the
background noise are listed in Table 10.
TABLE-US-00010 TABLE 10 Transcripts Expressed Only in CDs-HSD Rats
UniGene Name (rat) Description and Gene Ontology RGD1306952
Rn.64439 Similar to Ab2-225 Dmrt2 Rn.11448 Doublesex and mab-3
related transcription factor 2 (predicted) AA819893 Rn.148042
unknown cDNA Gpr176 Rn.44656 G protein-coupled receptor 176 Tmem45b
Rn.42073 Transmembrane protein 45b Nfkbil1 Rn.38632 Nuclear factor
of kappa light polypeptide gene enhancer in B-cells inhibitor-like
1 Dctn2 Rn.101923 Dynactin 2 Itpkc Rn.85907 Inositol
1,4,5-trisphosphate 3-kinase C BM389613 Rn.171826 unknown cDNA
Prodh2 Rn.4247 Proline dehydrogenase (oxidase) 2 BF288777 Rn.28947
unknown cDNA Abi3 Rn.95169 ABI gene family, member 3 Ring1
Rn.116589 Ring finger protein 1 Adrbk1 Rn.13010 Adrenergic receptor
kinase, beta 1 AW531966 Rn.8608 unknown cDNA RGD1560732 Rn.100399
Similar to LIM and senescent cell antigen-like domains 1
(predicted) Oxsr1 Rn.21097 Oxidative-stress responsive 1
(predicted) MGC114531 Rn.39247 unknown cDNA BF418465 Rn.123735
unknown cDNA LOC690911 Rn.25022 Similar to Msx2-interacting protein
(SPEN homolog) Pex6 Rn.10675 Peroxisomal biogenesis factor 6
RGD1311424 Rn.57800 Similar to hypothetical protein FLJ38348
(predicted) AI013238 Rn.135595 unknown cDNA BI288719 Rn.45106
unknown cDNA Evpl Rn.19832 Envoplakin (predicted)
[0293] The results of comparisons among the three groups are
presented in Table 11 below. Among the genes differentially
expressed for each of the models, there are several common
transcripts.
TABLE-US-00011 TABLE 11 Results of microarray analyses in
epididymal fat tissue. CDr-HSD CDs-HSD CDr-RD Comparisons vs.
CDs-HSD vs. CDs-RD vs. CDs-RD Type of model Resistance Progression
Predisposition >2 fold increase 140 79 288 >2 fold decrease
150 98 610 >3 fold increase 26 6 94 >3 fold decrease 27 22
203
[0294] Table 12 summarizes the results of common and unique
transcripts differentially expressed in the resistance and
progression models.
[0295] Table 12: Common and Unique transcripts differentially
expressed for each model
TABLE-US-00012 Common transcripts for both Unique transcripts
Comparisons Type of model models for each model CDr-HSD vs. CDs-HSD
Resistance 48 242 CDs-HSD vs. Progression 138 CDs-RD
[0296] The 48 common transcripts for these two models are listed in
Table 13. FIG. 9 is a graph depicting the fold changes in the 48
markers common to these two models.
TABLE-US-00013 TABLE 13 Common Transcripts Differentially Expressed
in Progression and Resistance Models UniGene UniGene Name (rat)
(human) Description and Gene Ontology SERPINE2 Rn.2271 Hs.38449
Serine proteinase inhibitor clade E member 2 C20orf160 Rn.6807
Hs.382157 C20orf160 predicted Cystein type endopeptidase Unknown
Rn.33396 n/a unknown LOC338328 Rn.7294 Hs.426410 High density
lipoprotein binding protein PTPRR Rn.6277 Hs.506076 Protein
tyrosine phosphatase receptor type R, LYPLA3 Rn.93631 Hs.632199
Lysophosphilipase 3 CYYR1 Rn.1528 Hs.37445 Cysteine/tyrosine-rich 1
Membrane-associated protein SOX17 Rn.7884 Hs.98367 SRY-box gene 17
LY6H Rn.40119 Hs.159590 Lymphocyte antigen 6 complex, locus H
SEMA3G Rn.32183 Hs.59729 Semaphorin 3G C1QTNF1 Rn.53880 Hs.201398
C1q and tumor necrosis factor related protein 1 ADCY4 Rn.1904
Hs.443428 Adenylate cyclase 4 RBP7 Rn.13092 Hs.422688 Retinol
binding protein 7, ADRB3 Rn.10100 Hs.2549 Adrenergic, beta-3-,
receptor NR1H3 Rn.11209 Hs.438863 Nuclear receptor subfamily, group
H, member 3 TMEFF1 Rn.162809 Hs.657066 Transmembrane protein with
EGF-like and two follistatin-like domains 1 TIMP-4 Rn.155651
Hs.591665 Tissue inhibitor of metalloproteinase 4 CYP4F8 Rn.10170
Hs.268554 Cytochrome P450, family 4, subfamily F, polypeptide 8
FOLR1 Rn.6912 Hs.73769 Folate receptor 1 SCD Rn.83595 Hs.558396
Stearoyl-CoA desaturase KIAA2022 Rn.62924 Hs.124128 DNA polymerase
activity GK Rn.44654 Hs.1466 Glycerol kinase OCLN Rn.31429
Hs.592605 Occludin SPINT2 Rn.3857 Hs.31439 Serine peptidase
inhibitor, Kunitz type, 2 RBM24 Rn.164640 Hs.519904 RNA binding
motif protein 24 SLC25A13 Rn.14686 Hs.489190 Solute carrier family
25, member 13 (citrin) TPMT Rn.112598 Hs.444319 Thiopurine
S-methyltransferase KRT18 Rn.103924 Hs.406013 Keratin 18 unknown
Rn.153497 n/a unknown C2orf40 Rn.16593 Hs.43125 Chromosome 2 open
reading frame 40 LOC440335 Rn.137175 Hs.390599 Hypothetical gene
supported by BC022385 BEXL1 Rn.9287 Hs.184736 Brain expressed
X-linked-like 1 CYB561 Rn.14673 Hs.355264 Cytochrome b-561 AMOT
Rn.149241 Hs.528051 Angiomotin SQLE Rn.33239 Hs.71465 Squalene
epoxidase ANKRD6 Rn.45844 Hs.656539 Ankyrin repeat domain 6 CCDC8
Rn.171055 Hs.97876 Coiled-coil domain containing 8 KRT8 Rn.11083
Hs.533782 Keratin 8 WWC1 Rn.101912 Hs.484047 WW and C2 domain
containing 1 PFKP Rn.2278 Hs.26010 Phosphofructokinase PEBP1
Rn.29745 Hs.433863 Phosphatidylethanolamine binding protein 1
SLC7A1 Rn.9439 Hs.14846 Solute carrier family 7 (cationic amino
acid transporter, y+ system), member 1 GSTM1 Rn.625 Hs.301961
Glutathione S-transferase M1 Glutathione metabolism CCL5 Rn.8019
Hs.514821 Chemokine (C-C motif) ligand 5 STEAP1 Rn.51773 Hs.61635
Six transmembrane epithelial antigen of the prostate 1 IAH1 Rn.8209
Hs.656852 Isoamyl acetate-hydrolyzing esterase 1 homolog (S.
cerevisiae) GNA14 Rn.35127 Hs.657795 Guanine nucleotide binding
protein (G protein), alpha 14 TMEM64 Rn.164935 Hs.567759
transmembrane protein 64
[0297] Unique transcripts that show a change in expression of 3
fold or higher are listed in Table 14. These transcripts are unique
in the sense that the changes of the expression level are observed
only within one of the models described and as such, they may serve
as markers to further study resistance against Type 2 Diabetes or
progression and predisposition for the disease.
TABLE-US-00014 TABLE 14 Unique Transcripts Found in Epididymal Fat
Tissue with Changes Greater than 3-Fold. (Appendix IV) UniGene
UniGene Name (rat) (human) Description and Gene Ontology SDF2L1
Rn.1414 Hs.303116 Stromal cell-derived factor 2-like 1 CCL11
Rn.10632 Hs.54460 Chemokine (C-C motif) ligand 11 CNN1 Rn.31788
Hs.465929 Calponin 1 ZCD2 Rn.24858 Hs.556638 Zinc finger,
CDGSH-type domain 2 CYR61 Rn.22129 Hs.8867 Cysteine-rich,
angiogenic inducer, 61 GGH Rn.10260 Hs.78619 Gamma-glutamyl
hydrolase TPM3 Rn.17580 Hs.645521 Tropomyosin 3 CSNK1A1 Rn.23810
Hs.654547 Casein kinase 1, alpha 1 PCDH7 Rn.25383 Hs.570785
Protocadherin 7 FHL2 Rn.3849 Hs.443687 Four and a half LIM domains
2 COL11A1 Rn.260 Hs.523446 Collagen, type XI, alpha 1 EMB Rn.16221
Hs.645309 Embigin homolog (mouse) ISG15 Rn.198318 Hs.458485 ISG15
ubiquitin-like modifier CRYAB Rn.98208 Hs.408767 Crystallin, alpha
B ACADSB Rn.44423 Hs.81934 Acyl-Coenzyme A dehydrogenase, Unknown
Rn.164743 n/a Unknown ABCA1 Rn.3724 Hs.429294 ATP-binding cassette,
sub-family A (ABC1), member 1 Unknown Rn.7699 n/a IMAGE clone:
BC086433 ACSM3 Rn.88644 Hs.653192 Acyl-CoA synthetase medium-chain
family member 3 CHD2 Rn.162437 Hs.220864 Chromodomain helicase DNA
binding protein 2 ACTA2 Rn.195319 Hs.500483 Actin, alpha 2, smooth
muscle, aorta RAMP3 Rn.48672 Hs.25691 Receptor (G protein-coupled)
activity modifying protein 3 DDEF1 Rn.63466 Hs.655552 Development
and differentiation enhancing factor 1 NIPSNAP3A Rn.8287 Hs.591897
Nipsnap homolog 3A (C. elegans) Unknown Rn.9546 n/a Unknown GPR64
Rn.57243 Hs.146978 G protein-coupled receptor 64 SGCB Rn.98258
Hs.438953 Sarcoglycan, beta Unknown Rn.146540 n/a Unknown Unknown
Rn.199679 n/a Unknown CALML3 Rn.105124 Hs.239600 Calmodulin-like 3
LOC645638 Rn.41321 Hs.463652 Similar to WDNM1-like protein RAB8B
Rn.10995 Hs.389733 RAB8B, a member RAS oncogene family Unknown
Rn.6638 n/a Unknown YTHDF2 Rn.21737 Hs.532286 YTH domain family,
member 2 SCEL Rn.34468 Hs.534699 Sciellin BNC1 Rn.26595 Hs.459153
Basonuclin 1 FGL2 Rn.64635 Hs.520989 Fibrinogen-like 2 UPK1B
Rn.9134 Hs.271580 Uroplakin 1B CTDSPL Rn.37030 Hs.475963 CTD
(carboxy-terminal domain, RNA polymerase II, polypeptide A) small
phosphatase- like PIK3R1 Rn.163585 Hs.132225
Phosphoinositide-3-kinase, regulatory subunit 1 (p85 alpha) POLA2
Rn.153998 Hs.201897 Polymerase (DNA directed), alpha 2 (70 kD
subunit) SPTBN1 Rn.93208 Hs.659362 Spectrin, beta, non-erythrocytic
1 RTEL1 Rn.98315 Hs.434878 Regulator of telomere elongation
helicase 1 MSLN Rn.18607 Hs.408488 Mesothelin ARVCF Rn.220
Hs.655877 Armadillo repeat gene deletes in velocardiofacial
syndrome ALB Rn.9174 Hs.418167 Albumin SLC6A4 Rn.1663 Hs.591192
Solute carrier family 6 (neurotransmitter transporter, serotonin),
member 4 SLC2A4 Rn.1314 Hs.380691 Solute carrier family 2
(facilitated glucose transporter), member 4 Unknown Rn.26537 n/a
Unknown Unknown Rn.44072 n/a Unknown Unknown Rn.199355 n/a Unknown
MRPL4 Rn.13113 Hs.279652 Mitochondrial ribosomal protein L4 GPR109A
Rn.79620 Hs.524812 G protein-coupled receptor 109A
[0298] Transcriptome/gene expression analyses were conducted on
pancreatic and epididymal fat tissue for the Cohen rat models.
Transcripts differentially expressed for both tissues have been
characterized as described above. For selected transcripts (14
transcripts for pancreatic tissue and 48 transcripts for epididymal
fat tissue), the microarray results have been confirmed by
quantitative PCR. FIG. 10 is a summary graph of the expression of
the selected markers measured in pancreatic tissue.
[0299] The 48 gene expression biomarkers common between models of
resistance and progression were mapped according to resistance
alone (FIG. 11A) or progression alone (FIG. 11B). FIG. 11C shows
the "merged" network, wherein the top biological functions
associated with the identified biomarkers include the following in
Table 15:
TABLE-US-00015 TABLE 15 Top Biological Functions Function P-value
confidence Biomarkers Hepatic system disease 5.4E-06 to 1.75E-02
SCD, SLC25A13, KRT8, KRT18, NR1H3, CCL5, GK, ADRB3 Cellular
assembly and 8.41E-06 to 4.88E-02 PEBP1, SCD, KRT8, organization
KRT18, NR1H3, CCL5, OCLN Hair and skin 8.41E-06 to 4.6E-02 KRT8,
KR18, CCL5 development and function Development and 8.41E-06 to
2.61E-02 SCD, SLC25A13, KRT8, function KRT18, NR1H3, CCL5
[0300] Table 16 lists the top canonical pathways associated with
the biomarkers identified in epididymal fat:
TABLE-US-00016 TABLE 16 Top Canonical Pathways Canonical Pathways
P-value confidence Biomarkers PXR/RXR Activation 1.92E+00 SCD,
GSTM1 (includes EG: 2944) LXR/RXR Activation 1.90E+00 SCD, NR1H3
Hepatic Cholestasis 1.34E+00 ADCY4, NR1H3 Synaptic Long Term
1.27E+00 ADCY4, GNA14 Depression CXCR4 Signaling 1.25E+00 ADCY4,
GNA14 RAR Activation 1.18E+00 RBP7, ADCY4 Biosynthesis of Steroids
1.17E_00 SQLE PPARO.+-./RXRO.+-. 1.17E+00 ADCY4, GK Activation
cAMP-mediated 1.16E+00 ADCY4, ADRB3 Signaling
[0301] FIG. 12A depicts a network combining most of the identified
biomarkers from serum in the Cohen rat models discussed herein.
FIG. 12B shows a simplified version of the network depicted in FIG.
12A. FIG. 12C is a bar graph depicting the top canonical pathways
implicated in the bioinformatics analyses discussed herein, while
FIG. 12D shows the top implicated biological functions.
Example 4
In Vivo Efficacy of D3 Peptide in the Streptozotocin (STZ) Model of
Type 1 Diabetes
[0302] To examine the possible role of D3 peptide in preventing the
onset of T2D in the CD rat model, the efficacy of synthetically
produced D3 peptide to ameliorate the severity of experimental type
2 Diabetes Mellitus in a multiple low dose streptozotocin
(STZ)-induced murine model was tested.
[0303] In a 36-day prophylactic study using STZ-induced male
C57BL/6 mice, treatment with 1 mg/kg of D3 peptide every 3 days
resulted in 100% survival of treated animals compared to 60%
survival in untreated controls. Peptide treatment also delayed the
onset of Diabetes (mean blood glucose levels.gtoreq.300 mg/dL) by
approximately 14 days, and reduced peak BG levels by 40%
(p<0.01) in treated animals. This naturally occurring peptide
may represent a safer alternative to small molecule kinase
inhibitors for the control of IDDM and associated
complications.
[0304] Streptozotocin (Streptozocin, STZ, Zanosar) is a naturally
occurring glucosamine-nitrosourea compound that is toxic to insulin
producing beta cells in the pancreas. This alkylating agent bears
enough similarity to the molecular structure of glucose to be
readily transported into beta cells by the highly abundant glucose
GLU2 glucose transporter protein. Once inside the cell STZ causes
damage to the DNA, resulting in a loss of cellular function.
Administration of this compound to experimental animals selectively
inhibits beta cell function resulting in a deregulation of glucose
metabolism and hyperglycemia, both characteristics of type 1 or
type 2 Diabetes.
[0305] Twenty five male C57BL/6 mice aged approximately 6 weeks
were randomized into 3 groups of 10, 10 and 5 animals. Beginning on
day -6, non-fasting blood glucose (BG) levels for all animals were
determined twice weekly using an Ascencia Contour blood glucose
reader (Beyer). Also beginning on day -6, one group of 10 animals
(Group 2) was administered 1 mg of D3 peptide in 200 .mu.l sterile
water intraperitoneally. Groups 1 and 3 were intraperitoneally
administered 200 .mu.l sterile water alone. Injections continued in
a similar fashion every 3 days for the duration of the experiment.
Beginning on day 0, Groups 2 and 3 also received 50 mg/kg of STZ in
200 .mu.l of sterile water via the intraperitoneal route for 5
consecutive days. Group 1 animals received a mock intraperitoneal
injection of 200 .mu.l of sterile water during the same period (see
Table 17). Animals were allowed access to food and water ad libitum
and this treatment schedule was followed until day 36.
TABLE-US-00017 TABLE 17 Experimental design for efficacy study of
D3 peptide in STZ induced diabetic C57BL/6 mice. Group No. of
Disease No. Animals BG Level Treatment Induction Description 1 5
Biweekly H.sub.2O H.sub.2O Normal Control 2 10 Biweekly D3 Peptide
STZ Treated Diabetic 3 10 Biweekly H.sub.2O STZ Untreated
Diabetic
[0306] In experiment 1, animals were fed normal mouse chow for the
duration of the experiment and a fresh vial of STZ was used. Blood
glucose measurements for each individual animal were recorded twice
weekly for the duration of the experiment. The blood glucose levels
in the non diabetic control animals remained stable at
approximately 150 mg/dl. However, immediately following the
administration of STZ to the two experimental groups, blood glucose
levels increased steadily over time and reached a maximum at
approximately day 15. No difference in blood glucose levels were
observed between D3 treated and untreated STZ-induced animals.
[0307] For the duration of the experiment, the mean blood glucose
level of normal control animals was approximately 150 mg/dl. For
the purposes of this study, a blood glucose reading in STZ-induced
animals more than 2-fold higher than normal (i.e. .gtoreq.300
mg/dl) was considered to indicate a diabetic animal. Using this
criterion, the incidence and time to onset of diabetes in the
treated and untreated STZ groups was determined. The results (FIG.
14) show that none of the normal control animals displayed any sign
of diabetes throughout the experiment. For both STZ groups, the
onset of Diabetes began as early as day 4 post STZ administration.
All of the untreated STZ animals were classed as diabetic by day
10. For the D3 peptide treated animals, the first signs of Diabetes
also manifested on day 4, although the time to establish Diabetes
in 100% of these animals slightly longer at day 15. The rate of
disease progression in treated animals (slope of the graph) was
essentially similar for both groups.
[0308] Once Diabetes is evident in these animals, several changes
in animal behavior were observed. Urination becomes more frequent
as animals try to excrete excess sugar, grooming is less evident
and fur becomes ruffled and pilated. As disease becomes more
severe, lethargy can ensue and animals can succumb to diabetic
complications rendering them moribund, or resulting in death. In
the untreated diabetic group, some initial deaths were recorded
following the initial onset of disease with only 80% of animals
surviving past day 4. Later in the experiment, further mortality
was observed for this group with only 60% of animals reaching the
end of the experiment on day 36 (see FIG. 15). In contrast, no
mortality was observed in D3 treated animals during the course of
the study. As expected, the survival rate was 100% in the normal
control group.
[0309] In experiment 2, animals were fed a high carbohydrate diet
(.about.50% sucrose) for the duration of the experiment. An older
stock vial of STZ was used. Blood glucose measurements for each
individual animal were recorded twice weekly for the duration of
the experiment. The data is summarized in FIG. 16. As expected, the
blood glucose levels in the non diabetic control animals remained
below 150 mg/dl. However, blood glucose levels for both STZ-induced
animals increased over time, compared to the control group, and
were significantly greater than normal from day 8 onwards
(p<0.05). For the groups receiving STZ, the mean blood glucose
levels in untreated diabetic animals were higher than those
observed in diabetic animals treated with D3 peptide every 3 days,
with significant differences (p<0.05) first observed on day 10.
This trend was maintained for the entire duration of the
experiment.
[0310] For the duration of the experiment, the mean blood glucose
level of normal control animals was approximately 150 mg/dl. For
the purposes of this study, a blood glucose reading in STZ-induced
animals more than 2-fold higher than normal (i.e. .gtoreq.300
mg/dl) was considered to indicate a diabetic animal. Using this
criterion, the incidence and time to onset of Diabetes in the
treated and untreated STZ groups was determined. The results showed
that none of the normal control animals showed any signed of
Diabetes throughout the experiment. For the untreated STZ group,
Diabetes onset began as early as day 8 post STZ administration, and
100% of animals were diabetic by day 18. For the D3 peptide treated
animals however, the first signs of diabetes did not manifest until
day 12, four days later than in untreated animals. The rate of
disease progression in treated animals (slope of the graph) was
much less pronounced in D3 treated animals. By day 36 only 70% of
animals in the D3 treated group were classified as diabetic. No
animals succumbed to diabetic complications or experienced any
toxic events related to peptide or STZ treatment during this
study.
[0311] In experiment 3, animals were fed a high sucrose diet ad
libitum upon arrival. D3 peptide was administered at 1 mg per
animal every 3 days in water via the intraperitoneal route
beginning on day -6. Diabetes was induced by the intraperitoneal
administration of 50 mg/kg of fresh STZ in 0.1 mM sodium citrate
buffer (pH 4.5) for 5 consecutive days, beginning on day 0. Blood
glucose was measured twice weekly using an Ascencia Countour BG
meter (Beyer, maximum reading is 600 mg/dL). On day 37, a glucose
tolerance test was administered by injecting 2 g/kg of glucose IP
and monitoring BG levels at 0, 15, 30, 60, 120 and 180 minutes post
injection. After GTT, animals were switched to regular mouse chow
and peptide treatment was halted. BG levels were followed for a
further 21 days. The experiment was terminated on day 69, at which
time serum samples were obtained from each animal and pancreata
were removed and formalin fixed for possible histological
analysis.
Example 5
Serpina D3 Peptide Inhibits Kinases In Vitro
[0312] Kinase assays were performed using the ProfilerPro kinase
panel kit available from NovaScreen and were validated using the
KinaseProfiler enzyme panel available from Millipore. To obtain
information of biological activity and selectivity of the D3
peptide, this lead biomarker was first tested at a concentration of
1 .mu.M for binding by NovaScreen to 59 receptors, including 25
neurotransmitter-related receptors, 4 steroid receptors, 3 ion
channels, 2 second messengers, 2 growth factors/hormones, 7
Brain/gut peptides, and 16 enzymes including 12 Kinases. The
preliminary results indicated that the D3 peptide showed specific
inhibition activities to 7 kinases that are involved in insulin
receptor signaling pathways. To confirm this finding, two
concentrations of D3 peptide (0.1 uM and 1 uM) were used to test
for binding in the same 12 kinases using the Millipore enzyme
panel. The data confirmed that the D3 peptides have specific
inhibition activity on 4 out of 7 kinases highlighted in Table 18
with IC.sub.50 values ranging from 0.3 .mu.M-1 .mu.M.
TABLE-US-00018 TABLE 18 Kinase Profiling Data Summary D3 at 0.1
.mu.M % D3 at 1 .mu.m % Kinase activity activity IC.sub.50 (.mu.M)
GSK3.beta. (h) 101 100 IKK.beta.(h) 103 71 IR (h) 96 103 MAPK1 (h)
86 92 MAPK2 (h) 96 96 P7OS6K (h) 73 22 0.373 PDK1 (h) 98 108 PKA(h)
115 105 PKB.beta. (h) 63 12 0.448 PKC.beta.II (h) 101 103 PKC.zeta.
(h) 95 42 1.045 SGK (h) 74 5 0.325
[0313] Two kinase screens were performed using the Invitrogen
SelectScreen.TM. biochemical kinase profiling service. The
Z'-LYTE.RTM. biochemical assay employs a fluorescence-based,
coupled-enzyme format and is based on the differential sensitivity
of phosphorylated and non-phosphorylated peptides to proteolytic
cleavage. The peptide substrate is labeled with two
fluorophores--one at each end--that make up a
FRET pair. In the primary reaction, the kinase transfers the
gamma-phosphate of ATP to a single tyrosine, serine or threonine
residue in a synthetic FRET-peptide. In the secondary reaction, a
site-specific protease recognizes and cleaves non-phosphorylated
FRET-peptides. Phosphorylation of FRET-peptides suppresses cleavage
by the Development Reagent. Cleavage disrupts FRET between the
donor (i.e., coumarin) and acceptor (i.e., fluorescein)
fluorophores on the FRET-peptide, whereas uncleaved, phosphorylated
FRET-peptides maintain FRET. A ratiometric method, which calculates
the ratio (the Emission Ratio) of donor emission to acceptor
emission after excitation of the donor fluorophore at 400 nm, is
used to quantitate reaction progress, as shown in the equation:
Emission Ratio = Coumarin Emission ( 445 nm ) Fluorescein Emission
( 520 nm ) ##EQU00001##
[0314] The assay yields very high Z'-factor values (>0.7) at a
low percent phosphorylation. Both cleaved and uncleaved
FRET-peptides contribute to the fluorescence signals and therefore
to the Emission Ratio. The extent of phosphorylation of the
FRET-peptide can be calculated from the Emission Ratio. The
Emission Ratio will remain low if the FRET-peptide is
phosphorylated (i.e., no kinase inhibition) and will be high if the
FRET-peptide is non-phosphorylated (i.e., kinase inhibition) Test
Compounds are screened in 1% DMSO (final) in the well. For 10 point
titrations, 3-fold serial dilutions are conducted from the starting
concentration selected by the present inventors. All Peptide/Kinase
Mixtures are diluted to a 2.times. working concentration in the
appropriate Kinase Buffer. All ATP Solutions are diluted to a
4.times. working concentration in Kinase Buffer (50 mM HEPES pH
7.5, 0.01% BRIJ-35, 10 mM MgCl.sub.2, 1 mM EGTA). ATP K.sub.m
apparent is previously determined using a Z'-LYTE.RTM.V assay. The
Development Reagent is diluted in Development Buffer: 10.times.
Novel PKC Lipid Mix: 2 mg/ml Phosphatidyl Serine, 0.2 mg/ml DAG in
20 mM HEPES, pH 7.4,
0.3% CHAPS. For 5 mL 10.times. Novel PKC Lipid Mix, ten mg of
phosphatidylserine and 1 mg diacylglycerol were added to a glass
tube. The lipid mixture was removed from the chloroform by
evaporation under a stream of nitrogen and dried. To the dried
lipid mixture, 5 mL of resuspension buffer, 10% CHAPS/500 mM HEPES,
pH 7.4, was added. The mixture was then heated gently to
50-60.degree. C. and vortexed in short intervals until the lipids
dissolved and the solution appeared clear. The mixture was divided
into single use volumes and stored at -20.degree. C.
[0315] To a bar-coded Corning, low volume NBS, black 384-well
plate, the following components were added: 2.5 .mu.L of 4.times.
Test Compound or 100 mL 100.times. plus 2.4 .mu.L kinase buffer, 5
.mu.L of 2.times. Peptide/Kinase Mixture, and 2.5 .mu.L of
4.times.ATP Solution. The plate was shaken for 30 seconds, then
incubated for 60 minutes at room temperature to allow the kinase
reaction to run. After incubation, 5 .mu.L of Development Reagent
Solution was added, then the plate was shaken for another
30-seconds and incubated at room temperature for another 60
minutes. Fluorescence was captured on a fluorescence plate reader
and the data was analyzed alongside the following controls, which
were made for each individual kinase and located on the same plate
as the kinase:
0% Phosphorylation Control (100% Inhibition Control), 100%
Phosphorylation Control, and 0% Inhibition Control. The maximum
Emission Ratio is established by the 0% Phosphorylation Control
(100% Inhibition Control), which contains no ATP and therefore
exhibits no kinase activity. This control yields 100% cleaved
peptide in the Development Reaction. The 100% Phosphorylation
Control, which consists of a synthetically phosphorylated peptide
of the same sequence as the peptide substrate, is designed to allow
for the calculation of percent phosphorylation. This control yields
a very low percentage of cleaved peptide in the Development
Reaction. The 0% Phosphorylation and 100% Phosphorylation Controls
allow one to calculate the percent Phosphorylation achieved in a
specific reaction well. Control wells do not include any kinase
inhibitors.
[0316] The minimum Emission Ratio in a screen is established by the
0% Inhibition Control, which contains active kinase. This control
is designed to produce a 10-50% phosphorylated peptide in the
Kinase Reaction. A known inhibitor control standard curve, 10 point
titration, is run for each individual kinase on the same plate as
the kinase to ensure the kinase is inhibited within an expected
IC.sub.50 range previously determined.
The following controls are prepared for each concentration of Test
Compound assayed: "Development Reaction Interference," which is
established by comparing the Test Compound Control wells that do
not contain ATP versus the 0% Phosphorylation Control (which does
not contain the Test Compound). The expected value for a
non-interfering compound should be 100% and any value outside of
90% to 110% is flagged; Test Compound Fluorescence Interference,
which is determined by comparing the Test Compound Control wells
that do not contain the Kinase/Peptide Mixture (zero peptide
control) versus the 0% Inhibition Control. The expected value for a
non-fluorescence compound should be 0%. Any value>20% is
flagged. Table 19 is a list of equations that were used for each
set of data points:
TABLE-US-00019 TABLE 19 Equations Used in Data Analysis Equation
Correction for Background Fluorescence FI.sub.Sample - FI.sub.TCFI
Ctl Emission Ration (using values corrected for background
fluorescence) Coumarin Emission ( 445 nm ) Fluorescein Emission (
520 nm ) ##EQU00002## % Phosphorylation (% Phos) { 1 - ( Emission
Ratio .times. F 100 % ) - C 100 % ( C 0 % - C 100 % ) + [ Emission
Ratio .times. ( F 100 % - F 0 % ) ] } * 100 ##EQU00003## %
Inhibition { 1 - % Phos Sample % Phos 0 % Inhibition Ctl } * 100
##EQU00004## Z' (using Emission Ratio values) 1 - 3 * Stdev 0 %
Phos Ctl + 3 * Stdev 0 % Inhibition Mean 0 % Phos Ctl - Mean 0 %
Inhibition ##EQU00005## Difference Between Data Points (single
point |% Inhibition.sub.Point 1 - % Inhibition.sub.Point 2| only)
Development Reaction Interference (DRI) (no ATP control) Emission
Ratio DRI Ctl Emission Ratio 0 % Phos Ctl ##EQU00006## Test
Compound Fluorescence Interference (TCFI) (check both Coumarin and
Fluorescein emissions) FI TCFI Ctl FI 0 % Inhibitor Ctl
##EQU00007## FI = Fluorescence Intensity C.sub.100% = Average
Coumarin emission signal of the 100% Phos. Control C.sub.0% =
Average Coumarin emission signal of the 0% Phos. Control F.sub.100%
= Average Fluorescein emission signal of the 100% Phos. Control
F.sub.0%=Average Fluorescein emission signal of the 0% Phos.
Control
[0317] The SelectScreen.TM. Kinase Profiling Service used XLfit
from IDBS as graphing software. The dose response curve is curve
fit to model number 205 (sigmoidal dose-response model). If the
bottom of the curve does not fit between -20% & 20% inhibition,
it is set to 0% inhibition. If the top of the curve does not fit
between 70% and 130% inhibition, it is set to 100% inhibition.
[0318] Table 20 below provides a summary of the results of the two
Invitrogen SelectScreen.TM. kinase profiling assays:
TABLE-US-00020 % Inhibition Annotation Kinase Tested mean Group
Family Subfamily NEK9 108.33 NEK NEK8 NIMA (never in mitosis gene
a)-related kinase 9 MAP2K2 (MEK2) 104.81 STE STE7 MEK1
Mitogen-activated protein kinase kinase 2 SGK2 101.80 AGC SGK
Serum/glucocorticoid regulated kinase 2 MAP3K9 (MLK1) 100.99 TKL
MLK MLK Mitogen-activated protein kinase kinase kinase 9 MINK1
100.96 Misshapen-like kinase 1 MST4 100.88 STE STE20 YSK PRKG2
(PKG2) 100.88 AGC PKG SGK (SGK1) 100.46 AGC SGK
Serum/glucocorticoid regulated kinase RPS6KB1 (p70S6K) 100 AGC RSK
p70 Ribosomal protein S6 kinase, 70 kDa, polypeptide 1 BRAF 100.40
TKL RAF RAF V-raf murine sarcoma viral oncogene homolog B1 MAP4K4
(HGK) 100 STE STE20 MSN PRKX 100.32 AGC PKA TAOK2 (TAO1) 100.15 STE
STE20 TAO TAO kinase 2 BRAF V599E 99.79 V-raf murine sarcoma viral
oncogene homolog B1 V599E mutation CAMK4 (CaMKIV) 99.58 CAMK CAMK1
Calcium/calmodulin-dependent protein kinase IV STK4 (MST1) 98.95
STE STE20 MST Serine/threonine kinase 4 STK24 (MST3) 98.63 STE
STE20 YSK Serine/threonine kinase 24 (STE20 homolog, yeast) MAP3K8
(COT) 98.58 STE STE-Unique Mitogen-activated protein kinase kinase
kinase 8 MAP4K2 (GCK) 98.41 STE STE20 KHS Mitogen-activated protein
kinase kinase kinase kinase 2 RPS6KA1 (RSK1) 98.30 AGC RSK RSK
Ribosomal protein S6 kinase, 90 kDa, polypeptide 1 CAMK1D (CaMKI
delta) 98.01 CAMK CAMK1 Calcium/calmodulin-dependent protein kinase
I delta NEK6 97.42 NEK NEK6 Other NIMA (never in mitosis gene
a)-related kinase 6 SGKL (SGK3) 97.00 AGC SGK Serum/glucocorticoid
regulated kinase family, member 3 RPS6KA4 (MSK2) 96.82 AGC RSK MSK
Ribosomal protein S6 kinase, 90 kDa, polypeptide 4 IRAK4 96 TKL
IRAK IRAK4 (interleukin-1 receptor- associated kinase 4) RAF1
(cRAF) Y340D Y341D 94.58 TKL RAF RAF c-Raf MAP2K1 (MEK1) 94 STE
STE7 MEK1 STK25 (YSK1) 93.14 STE STE20 YSK Serine/threonine kinase
25 (STE20 homolog, yeast) RPS6KA3 (RSK2) 93 AGC RSK RSK Ribosomal
protein S6 kinase, 90 kDa, polypeptide 3 MAPK9 (JNK2) 92.94 CMGC
MAPK JNK Mitogen-activated protein kinase 9 PRKD2 (PKD2) 92.53 CAMK
PKD Protein kinase D2 MAPK10 (JNK3) 90.65 CMGC MAPK JNK
Mitogen-activated protein kinase 10 STK3 (MST2) 90.37 STE STE20 MST
Serine/threonine kinase 3 (STE20 homolog, yeast) CAMK2B (CaMKII
beta) 90.26 PIM2 89.94 NEK2 89.57 PIM1 89 CAMK PIM Pim-1 oncogene
MAPK8 (JNK1) 89 CMGC MAPK JNK Mitogen-activated protein kinase 8
PRKCB1 (PKC beta I) 88 AGC PKC Alpha Protein kinase C, beta 1,
RPS6KA6 (RSK4) 87.62 PHKG2 87 CAMK PHK Phosphorylase kinase, gamma
2 AMPK A2/B1/G1 86.48 PRKCQ (PKC theta) 86.33 MAP4K5 (KHS1) 86.31
MELK 85.80 RPS6KA5 (MSK1) 85.05 NEK1 84 OTHER NEK NEK1 NIMA (never
in mitosis gene a)-related kinase 1 BRSK1 (SAD1) 83.46 MAPK14 (p38
alpha) 83 CMGC MAPK p38 Mitogen-activated protein kinase 14 CAMK2A
(CaMKII alpha) 82.58 PRKCG (PKC gamma) 81.02 PASK 80.82 PRKD1 (PKC
mu) 80.58 MERTK (cMER) 79.55 SRPK2 79.19 AMPK A1/B1/G1 79 CAMK
CAMKL AMPK AMP-activated protein kinase PRKCZ (PKC zeta) 74.39
CHEK1 (CHK1) 74 CAMK CAMKL CHK1 CHK1 checkpoint homolog NEK7 73.81
CAMK2D (CaMKII delta) 72.54 TYK2 70.50 ABL1 T315I 69.85 PRKCN
(PKD3) 69.58 AKT2 (PKB beta) 67.74 ADRBK2 (GRK3) 66.73 ROCK2 66.70
TEK (Tie2) 67 TK Tie Tyrosine kinase, endothelial CLK2 65.89 ABL1
E255K 65.63 MYLK2 (skMLCK) 64.57 MAPKAPK3 64.06 PKN1 (PRK1) 64.00
PDK1 63.95 CHEK2 (CHK2) 62.13 DAPK3 (ZIPK) 62.04 PRKCB2 (PKC beta
II) 61.65 DYRK4 61.23 GRK4 58.65 MAPKAPK2 58 CAMK MAPKAPK MAPKAPK
Mitogen-activated protein kinase-activated protein kinase 2 PRKCD
(PKC delta) 57.41 GRK6 57.06 ADRBK1 (GRK2) 56.06 MATK (HYL) 55.61
CSK 55.21 PHKG1 54.82 SYK 54 TKL Syk Spleen tyrosine kinase GRK5
50.83 ABL1 G250E 50.74 PLK1 51 Polo-like kinase 1 GRK7 49.86 PRKCI
(PKC iota) 48.93 PAK4 47 STE STE20 PAKB P21(CDKN1A)-activated
kinase 4 SRMS (Srm) 46.49 PRKCH (PKC eta) 46.46 CLK3 45.99 PRKCA
(PKC alpha) 45.09 IKBKB (IKK beta) IKK2 41 OTHER IKK
[0319] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the ambit of the following claims.
Sequence CWU 1
1
5138PRTRattus norvegicus 1Ser Gly Arg Pro Pro Met Ile Val Trp Phe
Asn Arg Pro Phe Leu Ile1 5 10 15Ala Val Ser His Thr His Gly Gln Thr
Ile Leu Phe Met Ala Lys Val20 25 30Ile Asn Pro Val Gly
Ala35285PRTRattus norvegicus 2Phe Ser Gln Gln Ala Asp Leu Ser Arg
Ile Thr Gly Ala Lys Asp Leu1 5 10 15Ser Val Ser Gln Val Val His Lys
Val Val Leu Asp Val Asn Glu Thr20 25 30Gly Thr Glu Ala Ala Ala Ala
Thr Gly Ala Asn Leu Val Pro Arg Ser35 40 45Gly Arg Pro Pro Met Ile
Val Trp Phe Asn Arg Pro Phe Leu Ile Ala50 55 60Val Ser His Thr His
Gly Gln Thr Ile Leu Phe Met Ala Lys Val Ile65 70 75 80Asn Pro Val
Gly Ala853419PRTRattus norvegicus 3Met Ala Phe Ile Ala Ala Leu Gly
Leu Leu Met Ala Gly Ile Cys Pro1 5 10 15Ala Val Leu Gly Phe Pro Asp
Gly Thr Leu Gly Asn Asp Thr Leu Leu20 25 30His Lys Asp Gln Asp Lys
Gly Thr Gln Leu Asp Ser Leu Thr Leu Glu35 40 45Ser Ile Asn Thr Asp
Phe Ala Phe Ser Leu Tyr Lys Met Leu Ala Leu50 55 60Lys Asn Pro Asp
Lys Asn Val Val Phe Ser Pro Leu Ser Ile Ser Ala65 70 75 80Ala Leu
Ala Ile Val Ser Leu Gly Ala Lys Gly Asn Thr Leu Glu Glu85 90 95Ile
Leu Glu Val Leu Arg Phe Asn Leu Thr Glu Ser Tyr Glu Thr Asp100 105
110Ile His Gln Gly Phe Gly His Leu Leu Gln Arg Leu Ser Gln Pro
Gly115 120 125Asp Gln Val Lys Ile Ile Thr Gly Asn Ala Leu Phe Ile
Asp Lys Asn130 135 140Leu Gln Val Leu Ala Glu Phe Gln Glu Lys Thr
Arg Ala Leu Tyr Gln145 150 155 160Val Glu Ala Phe Thr Ala Asp Phe
Gln Gln Pro Arg Val Thr Glu Lys165 170 175Leu Ile Asn Asp Tyr Val
Arg Asn Gln Thr Gln Gly Lys Ile Gln Glu180 185 190Leu Val Ser Gly
Leu Lys Glu Arg Thr Ser Met Val Leu Val Asn Tyr195 200 205Leu Leu
Phe Arg Gly Lys Trp Lys Val Pro Phe Asp Pro Asp Tyr Thr210 215
220Phe Glu Ser Glu Phe Tyr Val Asp Glu Lys Arg Ser Val Lys Val
Ser225 230 235 240Met Met Lys Ile Glu Glu Leu Thr Thr Pro Tyr Phe
Arg Asp Glu Glu245 250 255Leu Ser Cys Ser Val Leu Glu Leu Lys Tyr
Thr Gly Asn Ser Ser Ala260 265 270Leu Phe Ile Leu Pro Asp Lys Gly
Arg Met Gln Gln Val Glu Ala Ser275 280 285Leu Gln Pro Glu Thr Leu
Lys Lys Trp Lys Asp Ser Leu Arg Pro Arg290 295 300Lys Ile Asp Glu
Leu Tyr Leu Pro Arg Leu Ser Ile Ser Thr Asp Tyr305 310 315 320Ser
Leu Glu Glu Val Leu Pro Glu Leu Gly Ile Arg Asp Val Phe Ser325 330
335Gln Gln Ala Asp Leu Ser Arg Ile Thr Gly Ala Lys Asp Leu Ser
Val340 345 350Ser Gln Val Val His Lys Val Val Leu Asp Val Asn Glu
Thr Gly Thr355 360 365Glu Ala Ala Ala Ala Thr Gly Ala Asn Leu Val
Pro Arg Ser Gly Arg370 375 380Pro Pro Met Ile Val Trp Phe Asn Arg
Pro Phe Leu Ile Ala Val Ser385 390 395 400His Thr His Gly Gln Thr
Ile Leu Phe Met Ala Lys Val Ile Asn Pro405 410 415Val Gly
Ala417DNAArtificialSynthetic primer 4ttcaacmrrc cyttyst
17518DNAArtificialSynthetic primer 5yvacyttkcy makraaga 18
* * * * *