Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
G12L, A NOVEL GENE ASSOCIATED WITH THE THERMAL
RESPONSE
RELATED APPLICATIONS
This application claims priority to U.S. provisional application Serial No.
60/186,513,
filed March 2, 2000, which is incorporated herein by reference in its
entirety.
FIELD OF THE INVENTION
The invention generally relates to nucleic acids and polypeptides encoded
therefrom.
More specifically, the invention relates to nucleic acids encoding novel
polypeptides, as well as
vectors, host cells, antibodies, and recombinant methods for producing these
nucleic acids and
polypeptides.
BACKGROUND OF THE INVENTION
OBESITY
Obesity is the most prevalent metabolic disorder in the United States
affecting on the
order of 35°l0 of adults at an estimated cost of 300,000 lives and $70
billion in direct and indirect
costs. As an epidemic it is growing due to the increase in the number of
children who can be
considered overweight or obese. Obesity is defined as an excess of body fat,
frequently
resulting in a significant impairment of health. Obesity results when
adipocyte size or number
in a person's body increases to levels that may result in one or more of a
number of physical and
psychological disorders. A normal-sized person has between 30 and 35 billion
fat cells. When a
person gains weight, these fat cells increase in size at first and later in
number. Obesity is
influenced by genetic, metabolic, biochemical, psychological, and behavioral
factors. As such,
it is a complex disorder that must be addressed on several fronts to achieve
lasting positive
clinical outcome.
Obese individuals are prone to ailments including: type II diabetes mellitus
(NIDDM),
hypertension, coronary heart disease, hypercholesterolemia, osteoarthritis,
gallstones, cancers of
the reproductive organs, and sleep apnea. Sleep apnea is episodes of not
breathing during sleep
that correlates with higher incidence of strobe and heart attack, two other
factors contributing to
obesity-linked morbidity and mortality among the clinically obese.
There are several well established treatment modes ranging from non-
pharmaceutical to
pharmaceutical intervention. Non-pharmaceutical intervention includes diet,
exercise,
psychiatric treatment, and surgical treatments to reduce food consumption or
remove fat,
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
liposuction. Appetite suppressants and energy expenditurelnutrient-modifying
agents represent
the main focus of pharmacological intervention. Dexfenfluramine (REDUX~) and
sibutramine
(MERIDIA~) in the first class and beta3-adrenergic agonists and orlistat
(XENICAL~)
representing the latter.
Animal models have provided strong evidence that genetic make-up is
influential in the
determining the nature and extent of obesity. Though what is true in animals
may not be true for
humans, 40-80% of variation in body mass index (BMI, a measure of obesity
correlating weight
and height) can be attributed to genetic factors. While human obesity does not
generally follow
a Mendelian inheritance pattern, there are several rodent models that do so
(Spiegelman, Cell.
1996 Nov 1;87(3):377-89; Weigle, Bioessays. 1996 Nov;l8(11):867-74). As human
obesity is a
complex trait, it is therefore not surprising that single mutations in rodent
might not be
representative of causation in the majority of obese humans, though there are
examples of
humans with genetic lesions analogous to those found in rodents (Montague,
NatuYe. 1997 Jun
26;387(6636):903-8; Clement, Natuf°e. 1998 Mar 26;392(6674):398-401).
Interestingly, animal
models for complex phenotypes, such as hypertension and stroke, are also
obese. This suggests
that these animals may represent a more telling model for understanding the
complexities of
human obesity.
There are several rodent models of obesity that result in the inheritance of a
single
genetic lesion. Monogenetic obesity syndromes in mice that are well
characterized but rarely, if
ZO ever, observed in humans include: obese (ob), aberrant termination of the
translation of the
satiety factor leptin. Mutations of the leptin receptor results in the obese
diabetic mouse (db).
Agouti (Ay) is a coat color mutant that is obese. Normally only expressed in
the skin, in the
mutant animals it is ubiquitously expressed and may antagonize the binding of
melanocyte
stimulating hormone (MSH). MSH is derived from adrenocorticotropic hormone
(ACTH) a
ZS major pituitary hornone that results from the proteolytic processing of the
pro-hormone
proopiomelanocortin (POMC). The fat phenotype is the consequence of a mutation
in the
hypothalamic pro-hormone converting enzyme carboxypeptidase E. The least well
characterized obese mouse mutant is tub. tub encodes a cytosolic protein that
may influence the
processing of hypothalamic neuropeptide hormones such as neuropeptide Y (NPY,
an appetite
30 stimulating hormone) and POMC. Recently, a POMC knockout mouse was reported
that has a
phenotype analogous to several mouse models for obesity, particularly that of
Ay. The POMC
knockout has early onset obesity and has yellow hair color as well as adrenal
insufficiency due
to the apparent morphological absence of their adrenal gland. As there is no
detectable
corticosterone in these animals and corticosterones increase food intake, it
is surprising that they
2
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
are obese. The obese phenotype can be treated with alpha-MSH, a peptide
hormone derived
from POMC (Yaswen, Nat Med. 1999 Sep;S(9):1066-70).
Other animal models include fa/fa (fatty) rats, wluch bear many similarities
to the ob/ob
and db/db mice, discussed above. One difference is that, while fa/fa rats are
very sensitive to
cold, their capacity for non-shivering thermogenesis is normal. It is well
established that
thermogenesis and metabolism are closely coupled endocrinologically. Torpor, a
condition
analogous to hibernation and lethargy, seems to play a larger part in the
maintenance of obesity
in fa/fa rats than in the mice mutants. Further, several desert rodents, such
as the spiny mouse,
do not become obese in their natural habitats, but do become so when fed on
standard laboratory
feed.
BROWN ADIPOSE TISSUE
Brown Adipose Tissue (BAT), also known as multilocular adipose tissue, is so
called
because of the its color due to the large number of capillaries and
mitochondria in the cells
malting up this tissue. BAT is found primarily in the shoulder region and
flanks of human
embryo and newborn and then disappears in the first months of life. In
animals, particularly
hibernating animals and rodents, it is more abundant. BAT has features of an
endocrine organ in
that it is vascularized with capillaries and can receive direct sympathetic
innervation.
Sympathetic neurotransmission leads to the release of the catecholamines
noradrenaline and
adrenaline resulting in the activation of a hormone-sensitive lipase. This
results in the
hydrolysis of triglycerides that are converted to fatty acids and glycerol
leading to an increase in
oxygen consumption and heat production by uncoupling of the mitochondrial
proton gradient
from the formation of ATP via the activity of uncoupling proteins (IJCPs). BAT
stimulation by
catecholamines results in non-shivering thennogenesis.
Evidence of BAT as an endocrine organ comes from the work of Himms-Hagen done
in
the late 1960's. Experiments involving the removal of BAT from rats acclimated
to different
temperatures and the effects upon enhanced calorigenic response to
catecholamines lead to the
following observations i) removal of interscapular BAT (IBAT) from cold-
acclimated rats has
no immediate effect on the calorigenic response of rats to catecholamines. The
significance
being that BAT is not the organ directly responsive to this stimulus. ii) With
time (days) there is
a progressive loss of the enhanced catecholamines response by rats that have
had their IBAT
removed, suggesting that BAT is responsible for the long-term maintenance of
the
catecholamines-induced thennogenic response. Interestingly, the ability of
IBAT to maintain
the enhanced response correlated with the duration of exposure to cold. This
suggests that BAT
has short term and long term effects on acclimation. With long-term cold
acclimation there may
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
be a proliferation of BAT into regions other than that occupied by IBAT, thus
maintaining the
catecholamines response. Other work showing that transplantation of IBAT from
animals
acclimated to cold into those raised in the warm can confer a thermogenic
response under
condition that normally would not also supports the endocrine nature of BAT.
The role of this endocrine organ in the maintenance of body weight as well as
thermogenesis was demonstrated by the ablation of BAT in a transgenic mouse
model using a
BAT-specific promoter (UCP1) controlling the expression of diphtheria toxin
during the
development of this tissue. Animals were found to be unable to maintain core
body temperature
when exposed to the cold, were hyperphagic, leptin resistant, and obese. The
significance of this
latter observation is that in the absence of BAT the mice have increased
metabolic efficiency,
due to the loss of BAT thermogenesis and/or BAT derived hormones. That is to
say, in the
absence of BAT and UCP, there is a net accumulation of energy stored in the
form of fat.
Finally, in the case of one strain of mice with only a transient ablation of
BAT, the metabolic
defect is ameliorated with the reemergence of BAT [Lowell, Nature. 1993 Dec 23-
30;366(6457):740-2; Friedman, Nature. 1993 Dec 23-30;366(6457):720-1]. These
data taken
together support the contention that BAT is a unique thermogenic and endocrine
organ with a
pivotal role in the metabolic status of organisms in which it is observed.
It is well established that endocrine organs regulate metabolism and in doing
so must,
perforce, regulate gene expression. By understanding the mechanism by wluch
BAT provides
endocrine and thermogenic activity, insights into the regulation of metabolism
are gained.
Genes and proteins associated with BAT thermogenesis, proliferation, or
differentiation, will
provide novel tools to treat or diagnose metabolic or other disorders.
Furthermore, they may be
used in screening efforts to uncover pharmaceuticals or molecules to treat
metabolic and other
disease, and can be valuable markers in uses such as pharmacogenomics.
SUMMARY OF THE INVENTION
The present invention provides for an understanding of the mechanism by which
BAT
responds to environmental factors. Genes modulated in response to mouse
husbandry below the
thermal neutral zone of these animals represent important markers of metabolic
response, or lack
thereof, potential drug targets for metabolic disorders, and/or, in the case
of secreted/integral
membrane proteins, drugs themselves.
The invention is based in part upon the discovery of novel nucleic acid
sequences
encoding novel polypeptides. Nucleic acids encoding the polypeptides disclosed
in the
invention, and derivatives and fragments thereof, will hereinafter be
collectively designated as
"gl2L" nucleic acid or polypeptide sequences.
4
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
In one aspect, the invention provides an isolated gl2L nucleic acid molecule
encoding a
gl2L polypeptide that includes a nucleic acid sequence that has identity to
the nucleic acids
disclosed in SEQ ID NOS:1 or 3. In some embodiments, the gl2L nucleic acid
molecule can
hybridize under stringent conditions to a nucleic acid sequence complementary
to a nucleic acid
molecule that includes a protein-coding sequence of a gl2L nucleic acid
sequence. The
invention also includes an isolated nucleic acid that encodes a gl2L
polypeptide, or a fragment,
homolog, analog or derivative thereof. For example, the nucleic acid can
encode a polypeptide
at least 80% identical to a polypeptide comprising the amino acid sequences of
SEQ ID NOS:2
or 4. The nucleic acid can be, for example, a genomic DNA fragment or a cDNA
molecule that
includes the nucleic acid sequence of any of SEQ ID NOS:1 or 3.
Also included in the invention is an oligonucleotide, e.g., an oligonucleotide
which
includes at least 6 contiguous nucleotides of a gl2L nucleic acid (e.g., SEQ
117 NOS:1 or 3) or a
complement of said oligonucleotide.
Also included in the invention are substantially purified gl2L polypeptides
(SEQ ID
N0:2 or 4). In some embodiments, the gl2L polypeptides include an amino acid
sequence that
is substantially identical to the amino acid sequence of a human gl2L
polypeptide.
The invention also features antibodies that immunoselectively-bind to gl2L
polypeptides.
In another aspect, the invention includes pharmaceutical compositions that
include
therapeutically- or prophylactically-effective amounts of a therapeutic and a
pharmaceutically-
acceptable carrier. The therapeutic can be, e.g., a gl2L nucleic acid, a gl2L
polypeptide, or an
antibody specific for a gl2L polypeptide. In a further aspect, the invention
includes, in one or
more containers, a therapeutically- or prophylactically-effective amount of
this pharmaceutical
composition.
In a further aspect, the invention includes a method of producing a
polypeptide by
culturing a cell that includes a gl2L nucleic acid, under conditions allowing
for expression of
the gl2L polypeptide encoded by the DNA. If desired, the gl2L polypeptide can
then be
recovered.
W another aspect, the invention includes a method of detecting the presence of
a gl2L
polypeptide in a sample. In the method, a sample is contacted with a compound
that selectively
binds to the polypeptide under conditions allowing for formation of a complex
between the
polypeptide and the compound. The complex is detected, if present, thereby
identifying the
gl2L polypeptide within the sample.
The invention also includes methods to identify specific cell or tissue types
based on
their expression of a gl2L.
5
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
Also included in the invention is a method of detecting the presence of a gl2L
nucleic
acid molecule in a sample by contacting the sample with a gl2L nucleic acid
probe or primer,
and detecting whether the nucleic acid probe or primer bound to a gl2L nucleic
acid molecule in
the sample.
In a further aspect, the invention provides a method for modulating the
activity of a gl2L
polypeptide by contacting a cell sample that includes the gl2L polypeptide
with a compound
that binds to the gl2L polypeptide in an amount sufficient to modulate the
activity of said
polypeptide. The compound can be, e.g., a small molecule, such as a nucleic
acid, peptide,
polypeptide, peptidomimetic, carbohydrate, lipid or other organic (carbon
containing) or
inorganic molecule, as further described herein.
Also within the scope of the invention is the use of a Therapeutic in the
manufacture of a
medicament for treating or preventing disorders or syndromes including, e.g.,
metabolic
disorders and diseases, such as obesity, diabetes, and cachexia; cell
proliferative disorders and
diseases, such as hyperplasia, cancer, and restenosis; neurodegenerative
disorders and diseases,
such as Alzheimer's Disease, multiple sclerosis and Parkinson's Disorder;
immune disorders
and diseases, such as AmS, inflammation, and autoiimnune diseases; and
hematopoietic
disorders and diseases, such as SCm, cyclic neutropenia, and thrombocythemia.
In a preferred
embodiment, the Therapeutic is used in the manufacture of a medicament for
treating obesity
and obesity-related disorders. Obesity-related disorders include, but are not
limited to, type II
diabetes mellitus (NIDDM), hypertension, coronary heart disease,
hypercholesterolemia,
osteoarthritis, gallstones, cancers of the reproductive organs, and sleep
apnea. The Therapeutic
can be, e.g., a gl2L nucleic acid, a gl2L polypeptide, or a gl2L-specific
antibody, or
biologically-active derivatives or fragments thereof.
The invention further includes a method for screening for a modulator of
disorders or
syndromes including, e.g., metabolic disorders and diseases, such as obesity,
diabetes, and
cachexia; cell proliferative disorders and diseases, such as hyperplasia,
cancer, and restenosis;
neurodegenerative disorders and diseases, such as Alzheimer's Disease and
Parkinson's
Disorder; immune disorders and diseases, such as AmS, inflarmnation, and
autoimmune
diseases; and hematopoietic disorders and diseases, such as SCm, cyclic
neutropenia, and
thrombocythemia. In a preferred embodiment, a method screens for a modulator
of obesity and
obesity-related disorders. The method includes contacting a test compound with
a gl2L
polypeptide and determining if the test compound binds to the gl2L
polypeptide. Binding of the
test compound to the gl2L polypeptide indicates the test compound is a
modulator of activity, or
of latency or predisposition to the aforementioned disorders or syndromes.
6
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
Also within the scope of the invention is a method for screening for a
modulator of
activity, or of latency or predisposition to an disorders or syndromes
including, e.g., metabolic
disorders and diseases, such as obesity, diabetes, and cachexia; cell
proliferative disorders and
diseases, such as hyperplasia, cancer, and restenosis; neurodegenerative
disorders and diseases,
such as Alzheimer's Disease and Parkinson's Disorder; immune disorders and
diseases, such as
AmS, inflammation, and autoimmune diseases; and hematopoietic disorders and
diseases, such
as SC)D, cyclic neutropenia, and thrombocythemia, by administering a test
compound to a test
animal at increased risk for the aforementioned disorders or syndromes. In a
preferred
embodiment, the test animal has an increased rislc of obesity and obesity-
related disorders. The
test animal expresses a recombinant polypeptide encoded by a gl2L nucleic
acid. Expression or
activity of gl2L polypeptide is then measured in the test animal, as is
expression or activity of
the protein in a control animal which recombinantly-expresses gl2L polypeptide
and is not at
increased risk for~the disorder or syndrome. Next, the expression of gl2L
polypeptide in both
the test animal and the control animal is compared. A change in the activity
of gl2L
polypeptide in the test animal relative to the control animal indicates the
test compound is a
modulator of latency of the disorder or syndrome.
In yet another aspect, the invention includes a method for determining the
presence of or
predisposition to a disease associated with altered levels of a gl2L
polypeptide, a gl2L nucleic
acid, or both, in a subject (e.g., a human subject). The method includes
measuring the amount of
the gl2L polypeptide in a test sample from the subject and comparing the
amount of the
polypeptide in the test sample to the amount of the gl2L polypeptide present
in a control
sample. An alteration in the level of the gl2L polypeptide in the test sample
as compared to the
control sample indicates the presence of or predisposition to a disease in the
subject. Preferably,
the predisposition includes, e.g., metabolic disorders and diseases, such as
obesity, diabetes, and
cachexia; cell proliferative disorders and diseases, such as hyperplasia,
cancer, and restenosis;
neurodegenerative disorders and diseases, such as Alzheimer's Disease and
Parkinson's
Disorder; immune disorders and diseases, such as AmS, inflammation, and
autoimmune
diseases; and hematopoietic disorders and diseases, such as SCm, cyclic
neutropenia, and
thrombocythemia. In more preferred embodiments, the new polypeptides of the
invention can
be used in a method to screen for the presence of or predisposition to obesity
and obesity-related
disorders. Also, the expression levels of the new polypeptides of the
invention can be used in a
method to screen for the presence of or predisposition to various cancers,
e.g., breast cancer.
In a further aspect, the invention includes a method of treating or preventing
a
pathological condition associated with a disorder in a mammal by administering
to the subject a
gl2L polypeptide, a gl2L nucleic acid, or a gl2L-specific antibody to a
subject (e.g., a human
7
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
subject), in an amount sufficient to alleviate or prevent the pathological
condition. In preferred
embodiments, the disorder, includes, e.g., metabolic disorders and diseases,
such as obesity,
diabetes, and cachexia; cell proliferative disorders and diseases, such as
hyperplasia, cancer, and
restenosis; neurodegenerative disorders and diseases, such as Alzheimer's
Disease and
Parkinson's Disorder; immune disorders and diseases, such as AmS,
inflammation, and
autoimmune diseases; and hematopoietic disorders and diseases, such as SCm,
cyclic
neutropenia, and thrombocythemia. Iii more preferred embodiments,
administration is to a
subject suspected of suffering from obesity or obesity-related disorders.
In yet another aspect, the invention can be used in a method to identity the
cellular
components that interact with the gl2L nucleic acids and polypeptides,
including cellular
receptors and downstream effectors, by any one of a number of techniques
commonly employed
in the art. These include but are not limited to the two-hybrid system,
affinity purification, co-
precipitation with antibodies or other specific-interacting molecules.
Although methods and materials similar or equivalent to those described herein
can be
used in the practice or testing of the present invention, suitable methods and
materials are
described below. All publications, patent applications, patents, and other
references mentioned
herein are incorporated by reference in their entirety. In the case of
conflict, the present
specification, including definitions, will control. In addition, the
materials, methods, and
examples are illustrative only and not intended to be limiting.
BRIEF DESCRIPTION OF THE DRAWING
FIG.1. Multiple sequence analysis shows that the Spot 14's and the g12's are
distinct,
but related families of proteins. The boxed area is the location of the "Spot
Box" and the
underlined axea indicates the "acid box" for the two gene families. mspotl4 =
mouse Spot 14
(GenBanlc X95279; SEQ m NO:S), rnspotl4 = rat Spot 14 (GenBank NM 003251; SEQ
m
N0:6), hsspotl4 = human Spot 14 (GenBanlc Y08409; SEQ m N0:7), hsdrg121ike =
human
gl2L (SEQ m N0:2), mmdrg121ilce = mouse gl2L (SEQ m N0:4), and drgl2 =
zebrafish g12
(GenBank U27121; SEQ m N0:8).
FIG. 2. TaqMan time course of mouse gl2L modulation.
FIG. 3. Tissue distribution of mouse gl2L at 48hrs of acclimation at 4C, 22C,
and 33C
by TaqMan analysis.
FIG. 4. Relative protein homology of Spot 14's, zebrafish g12, and human and
mouse
gl2L's.
FIG. 5. Relative cDNA homology of Spot 14's, zebrafish g12, and human and
mouse
gl2L's.
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
FIG. 6. Radiation Hybrid map of mouse gl2L. Mouse gl2L is located near the
centromere of chromosome X. Radiation hybrid mapping vectors were submitted to
Whitehead
Institute/MIT Center for Genome Research's radiation hybrid map of the mouse
genome with a
lod score cut off of 14. The data format was the Whitehead Order using 93
hybrids.
(http://www.~enome.wi.mit.edu/c~i-bin/mouse rh/rlnnap-auto/rhma~per.cgii.)
i0q0-90.5 is the
band identifier for mouse gl2L. Cybb is cytochrome b 245 (GenBank U43384) and
Xkh
(GenBank AF155511) is the mouse homologue of the XK gene. Scale is 71.5 cR per
cM.
DETAILED DESCRIPTION
By identifying genes expressed in Brown Adipose Tissue (BAT) under conditions
that
affect the metabolic activity and proliferative status of BAT, the inventors
have isolated novel
nucleic acids and polypeptides with substantial homology to known nucleic
acids and proteins.
One such homologous protein is Spot 14.
Spotl4 in human, mouse, and rat, are nuclearly localized proteins of
approximately
~l8kD that are highly conserved between mouse, rat, and human. The human gene
is 81%
identical at the amino acid level to the rat Spotl4 gene and the mouse homolog
is 94% identical
to the rat gene product (Grillasca, FEBSLett. 1997 Jan 13;401(1):38-42). Rat
Spotl4 has been
relatively well studied and has been shown to have its expression modulated
under a number of
conditions relevant to metabolic regulation in WAT (White Adipose Tissue), BAT
(insulin,
retinoic acid, cAMP), and liver. It is up-regulated in liver by insulin,
carbohydrates, and thyroid
hormone (TH). This latter observed modulation lead to its identification as a
TH-responsive
gene in liver. Whereas insulin, TH, and carbohydrates up-regulate the
expression of Spot 14,
dietary fats and polyunsaturated fatty acids result in its down-regulation
(Clarke, JNut~. 1990
Feb;120(2):225-31; Jump, P~oc Natl Acad Sci ZI SA. 1993 Sep 15;90(18):8454-8).
Spot 14,
given its expression pattern and modulation in response to metabolism
influencing compounds,
likely is playing an important role in lipogenesis and/or the
development/proliferation of
lipogenic tissues under normal and disease conditions (Grillasca, FEBS Lett.
1997 Jan
13;401(1):38-42).
Spot 14 amplification is associated with cancer. Enhanced long-chain fatty
acid
synthesis may occur in breast cancer, where it is necessary for tmnor growth
and predicts a poor
prognosis. The Spot 14 protein functions to activate genes encoding the
enzymes of fatty acid
synthesis. Amplification of chromosome region l 1q13, where the human Spotl4
gene resides,
also predicts a poor prognosis in breast tumors. Human Spot 14 gene is
localized between
markers D11S906 and D11S937, at the telomeric end of the amplified region at l
1q13, and
found that it was amplified and expressed in breast cancer-derived cell lines.
Other findings
9
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
supported a role for the protein as a determinant of tumor lipid metabolism.
Expression of Spot
14 provided a pathophysiologic link between 2 prognostic indicators in breast
cancer: enhanced
lipogenesis and l 1q13 amplification (Moncur, Cytogeyaet Cell Gehet.
1997;78(2):131-2;
Cumungham, Tlzyroid. 1998 Sep;8(9):815-25; Moncur, P~oc Natl Acad Sci USA.
1998 Jun
9;95(12):6989-94).
Spot 14 proteins are believed to by the mammalian homologs of a small
Zebrafish
(Dahio ~erio) gastrulation-specific protein, g12 (Grillasca, FEBSLett. 1997
Jan 13;401(1):38-
42). Spot 14 has a high degree of homology to g12. The Spot protein and
closely related
proteins may, as a larger family of g12-like proteins, play a role in
differentiation during the
development and/or proliferation of lipogenic (and other) tissues under normal
and diseased
conditions. The putative family members have several features in common: low
molecular
weight, a "spot box", and acidic pI's that are probably due to the presence of
highly acidic
regions of the carboxyterminus of 10-20 amino acids with high aspartic acid
and glutamic acid
content (Grillasca, FEBSLett. 1997 Jan 13;401(1):38-42).
The invention is based, in part, upon the discovery of a novel mouse gene, as
well as its
human homolog, encoding novel polypeptides that are more similar to g12 than
the Spot 14
proteins from mouse, rat, and human. These novel genes and polypeptides are
collectively
designated herein as "gl2-like" (gl2L).
The novel gl2L nucleic acids of the invention include the nucleic acids whose
sequences
are provided in Tables 1A and 2A, or a fragnnent thereof. The invention also
includes a mutant
or variant gl2L nucleic acid, any of whose bases may be changed from the
corresponding base
shown in Tables 1A and 2A while still encoding a protein that maintains the
activities and
physiological functions of the gl2L protein fragment, or a fragment of such a
nucleic acid. The
invention further includes nucleic acids whose sequences are complementary to
those just
described, including complementary nucleic acid fragments. The invention
additionally
includes nucleic acids or nucleic acid fragments, or complements thereto,
whose structures
include chemical modifications. Such modifications include, by way of
nonlimiting example,
modified bases, and nucleic acids whose sugar phosphate backbones are modified
or derivatized.
These modifications are carried out at least in part to enhance the chemical
stability of the
modified nucleic acid, such that they may be used, for example, as antisense
binding nucleic
acids in therapeutic applications in a subject. In the mutant or variant
nucleic acids, and their
complements, up to 20% or more of the bases may be so changed.
The novel gl2L proteins of the invention include the protein fragments whose
sequences
are provided in Tables 1B and 2B. The invention also includes a gl2L mutant or
variant protein,
any of whose residues may be changed from the corresponding residue shown in
Tables 1B and
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
2B while still encoding a protein that maintains its native activities and
physiological functions,
or a functional fragment thereof. I11 the mutant or variant gl2L protein, up
to 20% or more of
the residues may be so changed. The invention further encompasses antibodies
and antibody
fragments, such as Fab or (Fab)a, that bind irmnunospecifically to any of the
gl2L proteins of the
invention.
Human gl2L
Table 1A describes the nucleic acid sequence of a nucleotide fragment encoding
a human
gl2L protein. Putative ATG start codon is labeled in bold underline type.
Putative stop codon
is in bold italic underline type. Sequence is presented 45 characters per
line.
11
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
Table 1A. Human gl2L nucleotide fragment (SEQ m NO:1).
1 GCGCGCCAGGGGTGGCCCTGAGCGCCGGCGACACCTTTCCTGGAC
46 TATAAATTGAGCACCTGGGATGGGTAGGGGGCCAACGCAGTCACC
91 GCCGTCCGCAGTCACAGTCCAGCCACTGACCGCAGCAGCGCCCTT
136 GCGTAGCAGCCGCTTGCAGCGAGAACACTGAATTGCCAACGAGCA
181 GGAGAGTCTCAAGGCGCAAGAGGAGGCCAGGGCTCGACCCACAGA
226 GCACCCTCAGCCATCGCGAGTTTCCGGGCGCCAAAGCCAGGAGAA
271 GCCGCCCATCCCGCAGGGCCGGTCTGCCAGCGAGACGAGAGTTGG
316 CGAGGGCGGAGGAGTGCCGGGAATCCCGCCACACCGGCTATAGCC
361 AGGCCCCCAGCGCGGGCCTTGGAGAGCGCGTGAAGGCGGGCATCC
406 CCTTGACCCGGCCGACCATCCCCGTGCCCCTGCGTCCCTGCGCTC
451 CAACGTCCGCGCGGCCACCATGATGCAAATCTGCGACACCTACAA
496 CCAGAAGCACTCGCTCTTTAACGCCATGAATCGCTTCATTGGCGC
541 CGTGAACAACATGGACCAGACGGTGATGGTGCCCAGCTTGCTGCG
586 CGACGTGCCCCTGGCTGACCCCGGGTTAGACAACGAGGTCAGCGT
631 GGAGGTAGGCGGCAGTGGCAGCTGCCTGGAGGAGCGCACGACCCC
676 GGCCCCAAGCCCGGGCAGCGCCAATGGAAGCTTTTTCGCGCCCTC
721 CCGGGACATGTACAGCCACTACGTGCTGCTCAAGTCCATCCGCAA
766 CGATATTGAGTGGGGAGTCCTGCACCAGCCGCCTCCACCGGCTGG
811 GAGCGAGGAGGGCAGTGCCTGGAAGTCCAAGGACATCCTGGTGGA
856 CCTGGGCCACTTGGAGGGTGCGGACGCCGGCGAAGAAGACCTGGA
901 ACAGCAGTTCCACTACCACCTGCGCGGGCTGCACACTGTGCTCTC
946 GAAACTCACGCGCAAAGCCAACATCCTCACTAACAGATACAAGCA
991 GGAGATCGGCTTCGGCAATTGGGGCCACTGAGGCGTGGCGCCCGT
1036 GGCTGCCCAGCACCTTCTTCGACCCATCTCACCCTCTCTCATTCC
1081 TCAAAGCTTTTTTTTTTTCCCTGGCTGGGGGGCGGAAAGGGCAAA
1126 CTG
12
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
A polypeptide encoded by SEQ ID NO:1 is presented using the one-letter code in
Table
1B.
Table 1S. Human gl2L polypeptide sequence (SEQ m N0:2).
1 MMQICDTYNQKHSLFNAMNRFIGAVNNMDQTVMVPSLLRDVPLADPGLDNEVSVEVGGSGSCLEERTTPA
71 PSPGSANGSFFAPSRDMYSHYVLLKSIRNDIEWGVLHQPPPPAGSEEGSAWKSKDILVDLGHLEGADAGE
141 EDLEQQFHYHLRGLHTVLSKLTRKANILTNRYKQEIGFGNWGH
Mouse ~12L
Table 2A describes the nucleic acid sequence of a nucleotide fragment encoding
a mouse
gl2L protein. Putative ATG start codon is labeled in bold underline type.
Putative stop codon
is in bold italic underline type. Putative polyadenylation site is in italics
underline type.
Sequence is presented 45 characters per line.
Table 2A. Mouse gl2L nucleotide fragment (SEQ m NO:3).
1 CGGACGCGTGGGGGAGGTAGGAGGAGGAGACATCAGGGGTGGTCC
46 TGGGCGCCTGGGACACCTTTCCCGGACTATAAATTGAGCACCTGG
91 AATGGGCAGGGGGCCGGAGCAACCACAGTCGCCCTTACTCACAGT
136 CCGATCAGTGACCGCAGCAGCGCCCTTGGGCAGCCACCGTCCGCA
181 ACGCAAGCACTGAGAACCAGGGGATTTCGCAGTGCAAGAGAA.A.A.A
226 GGCTAGACCCAGCCACCCACCGTCAATCCTGAGCCAAAGATAAGA
271 GCAGCCGGGCCTCACGAAGGGCTGAGCTGAGAAAGAAGCAAGTTA
316 GAGAGGGCGGAGAAGGATCTGGGAATCCCGTCACACCGGCTTCAA
361 GCAGGCTCCCGGCATCAGCCTCTGAGAGCGCTTGAAGGCGGCATC
406 GCCAGCGGTCTATCTCCGTGTACCAGCGTCCCTGTGTTTCCGCGC
451 CCGCTCGGCCACCATGATGCAAATCTGCGACACATATAACCAGAA
496 GCACTCGCTCTTTAACGCCATGAATCGCTTCATTGGCGCGGTGAA
541 CAACATGGACCAGACGGTGATGGTGCCCAGTCTGCTGCGCGACGT
586 ACCCCTGTCCGAGCCGGAGATAGACGAGGTCAGCGTGGAGGTAGG
13
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
631 CGGCAGTGGCGGCTGCCTGGAGGAGCGCACGACCCCGGCCCCAAG
676 CCCGGGCAGCGCCAATGAAAGCTTTTTCGCGCCCTCCCGGGACAT
721 GTACAGCCACTACGTGCTGCTCAAGTCCATCCGCAATGATATCGA
766 GTGGGGAGTCCTGCACCAGCCTTCGTCTCCGCCGGCCGGGAGCGA
811 GGAGAGCACCTGGAAGCCCAAGGACATCCTGGTGGGCCTGAGTCA
856 CTTGGAGAGCGCGGATGCGGGCGAGGAAGATCTGGAGCAGCAGTT
901 CCACTACCACCTGCGCGGGCTGCACACCGTGCTCTCCAAACTCAC
946 CCGAA.A.AGCCAACATCCTCACCAATAGATACAAGCAGGAGATCGG
991 CTTCAGTAATTGGGGCCACTGAGGCGGGGGCTGTCCCCGCTGCCC
1036 AGCACCCTCTCTCGGGTCGGCTCTACCACCCCTCTCTTTCCTCCA
1081 AGCTATTTTCTTCCTGGTTGTGGGGCGCGAAGGGCACACTGTAAA
1126 GTTGGGCTGTGTACTTGGTGGGGTTAGTGTGGAGAAGAGGGCCTC
1171 ATCGCGAGAGCAGAGGAAAGTAGTCGCCAGAGAGGGGGGTTCAAA.
1216 GACCCCCGGAGGGGGCCTACTCTGTGTTGGTGGGAATGGAACTGG
1261 GCCGATGTCCTTCATTCAGCCTGTGCCTTTCTTGGGGTTTCTTTT
1306 CTGTTTTTCTTTCCGGAAGAGAAGGGCCTGAGAA.AGGGCCATGCC
1351 AGGGCACAGTGCTGGGTTGCCACACATGGGAGGGCAGCTTCTAGC
1396 CGGGTGCTTGGGGGAGGCGGGGCTCAGCCTCCTGCTGCCCTGCCT
1441 TGAGCTGCCAGAGGAGGCCTTGGCGTTGCTAGGATTGCGTCAGTT
1486 TTCCTGTTTGCACTATTTCTTTTTGTAACAGTGACCCTGTCTTAA
1531 GTCTTTCAGATCTCTTTGCTTTGAAACTTCGTCGATTCCATTGTG
1576 ATAAGCGCACAAACAGCACTGTTGGTAACCGGTACTACTTTATTA
1621 ATGATTTTCTGTTACACTGTACAGTAGTCCTGTGGCACCCTATCC
14
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
1666 CTTTCACGCCACCCCTCCCCCGCCCGTGTGTGTAAACTGGCGATG
1711 TGCCAGCTAGGATGAAGCTTGCCACTCGGCTAGCGAAA.ATAATTA
1756 ACATTATTATGAGAA.AGTGGATTTATCTAAAGTGGAACCAGCTGA
1801 CATTATATCTGTATCGTATGGAGAATGATGAAGGGCTCCACTGTT
1846 GTTATATGTCTTGTTTATTTAA.AACTTTTTTTAATCCAGATGTAG
1891 ACTATATTCTAA.A.AAATAA.A.AACGCAGATGTGTTAAC
A polypeptide encoded by SEQ ID N0:3 is presented using the one-letter code in
Table
2B.
Table 2B. Mouse gl2L polypeptide sequence (SEQ ID N0:4).
1 MMQICDTYNQKHSLFNAMNRFIGAVNNMDQTVMVPSLLRDVPLSEPEIDEVSVEVGGSGGCLEERTTPAP
71 SPGSANESFFAPSRDMYSHYVLLKSIRNDIEWGVLHQPSSPPAGSEESTWKPKDILVGLSHLESADAGEE
141 DLEQQFHYHLRGLHTVLSKLTRKANILTNRYKQEIGFSNWGH
An alignment of the gl2L polypeptides with zebrafish g12 and the mouse, rat,
and
human Spot 14 polypeptides is shown in FIG. 1.
The gl2L nucleic acids and proteins of the invention are useful in potential
therapeutic
applications implicated in metabolic disorders and diseases, such as obesity,
diabetes, and
cachexia; cell proliferative disorders and diseases, such as hyperplasia,
cancer, and restenosis;
neurodegenerative disorders and diseases, such as Alzheimer's Disease and
Parkinson's
Disorder; irx~mune disorders and diseases, such as AIDS, inflammation, and
autoimmune
diseases; and hematopoietic disorders and diseases, such as SCID, cyclic
neutropenia, and
thrombocythemia. In preferred embodiments, the nucleic acids and proteins of
the invention are
used in therapeutic applications for obesity-related disorders, other
metabolic diseases,
proliferative disorders, and other diseases. Obesity related disorders
include, but are not limited
to, type II diabetes mellitus (N7DDM), hypertension, coronary heart disease,
hypercholesterolemia, osteoaxthritis, gallstones, cancers of the reproductive
organs, and sleep
apnea. For example, a cDNA encoding human gl2L may be useful in gene therapy,
and the
human gl2L protein may be useful when administered to a subject in need
thereof. The novel
nucleic acid encoding human gl2L protein of the invention, or fragments
thereof, may further be
useful in diagnostic applications, wherein the presence or amount of the
nucleic acid or the
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
protein are to be assessed, diseases related to single nucleotide
polymorphisms, or other
mutations. These materials are further useful in the generation of antibodies
that bind
immunospecifically to the novel substances of the invention for use in
therapeutic or diagnostic
methods.
gl2L Nucleic Acids and Polypeptides
One aspect of the invention pertains to isolated nucleic acid molecules that
encode gl2L
polypeptides or biologically-active portions thereof. Also included in the
invention are nucleic
acid fragments sufficient for use as hybridization probes to identify gl2L-
encoding nucleic acids
(e.g., gl2L mRNAs) and fragments for use as PCR primers for the amplification
and/or mutation
of gl2L nucleic acid molecules. As used herein, the term "nucleic acid
molecule" is intended to
include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA),
analogs
of the DNA or RNA generated using nucleotide analogs, and derivatives,
fragments and
homologs thereof. The nucleic acid molecule may be single-stranded or double-
stranded, but
preferably is comprised double-stranded DNA.
A gl2L nucleic acid can encode a mature gl2L polypeptide. As used herein, a
"mature"
form of a polypeptide or protein disclosed in the present invention is the
product of a naturally
occurring polypeptide or precursor form or proprotein. The naturally occurring
polypeptide,
precursor or proprotein includes, by way of nonlimiting example, the full
length gene product,
encoded by the corresponding gene. Alternatively, it may be defined as the
polypeptide,
ZO precursor or proprotein encoded by an open reading frame described herein.
The product
"mature" form arises, again by way of nonlimiting example, as a result of one
or more naturally
occurring processing steps as they may take place within the cell, or host
cell, in which the gene
product arises. Examples of such processing steps leading to a "mature" form
of a polypeptide
or protein include the cleavage of the N-terminal methionine residue encoded
by the initiation
codon of an open reading frame, or the proteolytic cleavage of a signal
peptide or leader
sequence. Thus a mature form arising from a precursor polypeptide or protein
that has residues
1 to N, where residue 1 is the N-terminal methionine, would have residues 2
through N
remaining after removal of the N-terminal methionine. Alternatively, a mature
form arising
from a precursor polypeptide or protein having residues 1 to N, in which an N-
terminal signal
sequence from residue 1 to residue M is cleaved, would have the residues from
residue M+1 to
residue N remaining. Further as used herein, a "mature" form of a polypeptide
or protein may
arise from a step of post-translational modification other than a proteolytic
cleavage event. Such
additional processes include, by way of non-limiting example, glycosylation,
myristoylation or
16
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
phosphorylation. In general, a mature polypeptide or protein may result from
the operation of
only one of these processes, or a combination of any of them.
The term "probes", as utilized herein, refers to nucleic acid sequences of
variable length,
preferably between at least about 10 nucleotides (nt), 100 nt, or as many as
approximately, e.g.,
6,000 nt, depending upon the specific use. Probes are used in the detection of
identical, similar,
or complementary nucleic acid sequences. Longer length probes are generally
obtained from a
natural or recombinant source, are highly specific, and much slower to
hybridize than shorter-
length oligomer probes. Probes may be single- or double-stranded and designed
to have
specificity in PCR, membrane-based hybridization technologies, or ELISA-like
technologies.
The term "isolated" nucleic acid molecule, as utilized herein, is one which is
separated
from other nucleic acid molecules which are present in the natural source of
the nucleic acid.
Preferably, an "isolated" nucleic acid is free of sequences that naturally
flank the nucleic acid
(i.e., sequences located at the 5'- and 3'-termini of the nucleic acid) in the
genomic DNA of the
organism from which the nucleic acid is derived. For example, in various
embodiments, the
isolated gl2L nucleic acid molecules can contain less than about 5 kb, 4 kb, 3
lcb, 2 kb, 1 lcb, 0.5
lcb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid
molecule in genomic
DNA of the cell/tissue from which the nucleic acid is derived (e.g., brain,
heart, liver, spleen,
etc.). Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule,
can be
substantially free of other cellular material or culture medium when produced
by recombinant
techniques, or of chemical precursors or other chemicals when chemically
synthesized.
A nucleic acid molecule of the invention, e.g., a nucleic acid molecule having
the
nucleotide sequence of SEQ ID NOS:1 or 3, or a complement of this
aforementioned nucleotide
sequence, can be isolated using standard molecular biology techniques and the
sequence
information provided herein. Using all or a portion of the nucleic acid
sequence of SEQ ID
NOS:1 or 3 as a hybridization probe, gl2L molecules can be isolated using
standard
hybridization and cloning techniques (e.g., as described in Sambrook, et al.,
(eds.), MOLECULAR
CLONING: A LABORATORY MANUAL 2"d Ed., Cold Spring Harbor Laboratory Press,
Cold Spring
Harbor, NY, 1959; and Ausubeh, et al., (eds.), CURRENT PROTOCOLS IN MOLECULAR
BIOLOGY,
John Wiley & Sons, New Yorhc, NY, 1993.)
A nucleic acid of the invention can be amplified using cDNA, mRNA or
alternatively,
genomic DNA, as a template and appropriate ohigonucleotide primers according
to standaxd
PCR amplification techniques. The nucleic acid so amplified can be cloned into
an appropriate
vector and characterized by DNA sequence analysis. Furthermore,
oligonucleotides
corresponding to gl2L nucleotide sequences can be prepared by standard
synthetic techniques,
e.g., using an automated DNA synthesizer.
17
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
As used herein, the term "oligonucleotide" refers to a series of linked
nucleotide
residues, which oligonucleotide has a sufficient number of nucleotide bases to
be used in a PCR
reaction. A short oligonucleotide sequence may be based on, or designed from,
a genomic or
cDNA sequence and is used to amplify, confirm, or reveal the presence of an
identical, similar
or complementary DNA or RNA in a particular cell or tissue. Oligonucleotides
comprise
portions of a nucleic acid sequence having about 10 nt, 50 nt, or 100 nt in
length, preferably
about 15 nt to 30 nt in length. In one embodiment of the invention, an
oligonucleotide
comprising a nucleic acid molecule less than 100 nt in length would further
comprise at least 6
contiguous nucleotides of SEQ ID NOS:1 or 3, or a complement thereof.
Oligonucleotides may
be chemically synthesized and may also be used as probes.
In another embodiment, an isolated nucleic acid molecule of the invention
comprises a
nucleic acid molecule that is a complement of the nucleotide sequence shown in
SEQ m NOS:1
or 3, or a portion of this nucleotide sequence (e.g., a fragment that can be
used as a probe or
primer or a fragment encoding a biologically-active portion of an gl2,L
polypeptide). A nucleic
acid molecule that is complementary to the nucleotide sequence shown in SEQ ID
NOS:1 or 3,
is one that is sufficiently complementary to the nucleotide sequence shown in
SEQ m NOS:1 or
3, that it can hydrogen bond with little or no mismatches to the nucleotide
sequence shown in
SEQ ID NOS:1 or 3, thereby forming a stable duplex.
As used herein, the term "complementary" refers to Watson-Crick or Hoogsteen
base
pairing between nucleotides units of a nucleic acid molecule, and the term
"binding" means the
physical or chemical interaction between two polypeptides or compounds or
associated
polypeptides or compounds or combinations thereof. Binding includes ionic, non-
ionic, van der
Waals, hydrophobic interactions, and the lilce. A physical interaction can be
either direct or
indirect. Indirect interactions may be through or due to the effects of
another polypeptide or
~5 compound. Direct binding refers to interactions that do not take place
through, or due to, the
effect of another polypeptide or-compound, but instead are without other
substantial chemical
intermediates.
Fragments provided herein are defined as sequences of at least 6 (contiguous)
nucleic
acids or at least 4 (contiguous) amino acids, a length sufficient to allow for
specific
hybridization in the case of nucleic acids or for specific recognition of an
epitope in the case of
amino acids, respectively, and are at most some portion less than a full
length sequence.
Fragments may be derived from any contiguous portion of a nucleic acid or
amino acid sequence
of choice. Derivatives are nucleic acid sequences or amino acid sequences
formed from the
native compounds either directly or by modification or partial substitution.
Analogs are nucleic
acid sequences or amino acid sequences that have a structure similar to, but
not identical to, the
18
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
native compound but differs from it in respect to certain components or side
chains. Analogs
may be synthetic or from a different evolutionary origin and may have a
similar or opposite
metabolic activity compared to wild type. Homologs are nucleic acid sequences
or amino acid
sequences of a particular gene that are derived from different species.
Derivatives and analogs may be full length or other than full length, if the
derivative or
analog contains a modified nucleic acid or amino acid, as described below.
Derivatives or
analogs of the nucleic acids or proteins of the invention include, but are not
limited to,
molecules comprising regions that are substantially homologous to the nucleic
acids or proteins
of the invention, in various embodiments, by at least about 70%, 80%, or 95%
identity (with a
preferred identity of 80-95%) over a nucleic acid or amino acid sequence of
identical size or
when compared to an aligned sequence in which the alignment is done by a
computer homology
program known in the art, or whose encoding nucleic acid is capable of
hybridizing to the
complement of a sequence encoding the aforementioned proteins under stringent,
moderately
stringent, or low stringent conditions. See e.g. Ausubel, et al., CURRENT
PROTOCOLS IN
1 S MQLECULAR BIOLOGY, John Wiley & Sons, New Yorlc, NY, 1993, and below.
A "homologous nucleic acid sequence" or "homologous amino acid sequence," or
variations thereof, refer to sequences characterized by a homology at the
nucleotide level or
amino acid level as discussed above. Homologous nucleotide sequences encode
those sequences
coding for isoforms of gl2L polypeptides. Isoforms can be expressed in
different tissues of the
same organism as a result of, for example, alternative splicing of RNA.
Alternatively, isoforms
can be encoded by different genes. In the invention, homologous nucleotide
sequences include
nucleotide sequences encoding for a gl2L polypeptide of species other than
humans, including,
but not limited to: vertebrates, and thus can include, e.g., frog, mouse, rat,
rabbit, dog, cat cow,
horse, and other organisms. Homologous nucleotide sequences also include, but
are not limited
to, naturally occurring allelic variations and mutations of the nucleotide
sequences set forth
herein. A homologous nucleotide sequence does not, however, include the exact
nucleotide
sequence encoding human gl2L protein. Homologous nucleic acid sequences
include those
nucleic acid sequences that encode conservative amino acid substitutions (see
below) in SEQ ID
NOS:2 or 4, as well as a polypeptide possessing gl2L biological activity.
Various biological
activities of the gl2L proteins are described below.
A gl2L polypeptide is encoded by the open reading frame ("ORF") of a gl2L
nucleic
acid. An ORF corresponds to a nucleotide sequence that could potentially be
translated into a
polypeptide. A stretch of nucleic acids comprising an ORF is uninterrupted by
a stop codon.
An ORF that represents the coding sequence for a full protein begins with an
ATG "start" codon
and terminates with one of the three "stop" codons, namely, TAA, TAG, or TGA.
For the
19
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
purposes of this invention, an ORF may be any part of a coding sequence, with
or without a start
codon, a stop codon, or both. For an ORF to be considered as a good candidate
for coding for a
boyaa fide cellular protein, a minimum size requirement is often set, e.g., a
stretch of DNA that
would encode a protein of 50 amino acids or more.
The nucleotide sequences determined from the cloning of the human gl2L genes
allows
for the generation of probes and primers designed for use in identifying
and/or cloning gl2L
homologues in other cell types, e.g. from other tissues, as well as gl2L
homologues from other
vertebrates. The probe/primer typically comprises substantially purified
oligonucleotide. The
oligonucleotide typically comprises a region of nucleotide sequence that
hybridizes under
stringent conditions to at least about 12, 25, 50, 100, 150, 200, 250, 300,
350 or 400 consecutive
sense strand nucleotide sequence of SEQ m NOS:l or 3; or an anti-sense strand
nucleotide
sequence of SEQ m NOS:1 or 3; or of a naturally occurring mutant of SEQ m
NOS:I or 3.
Probes based on the human gl2L nucleotide sequences can be used to detect
transcripts
or genomic sequences encoding the same or homologous proteins. In various
embodiments, the
probe further comprises a label group attached thereto, e.g. the label group
can be a radioisotope,
a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be
used as a part
of a diagnostic test kit for identifying cells or tissues which mis-express a
gl2L protein, such as
by measuring a level of a gl2L-encoding nucleic acid in a sample of cells from
a subject e.g.,
detecting gl2L ml2NA levels or determining whether a genomic gl2L gene has
been mutated or
deleted.
"A polypeptide having a biologically-active portion of a gl2L polypeptide"
refers to
polypeptides exhibiting activity similar, but not necessarily identical to, an
activity of a
polypeptide of the invention, including mature forms, as measured in a
particular biological
assay, with or without dose dependency. A nucleic acid fragment encoding a
"biologically-
active portion of gl2L " can be prepared by isolating a portion of SEQ m NOS:1
or 3, that
encodes a polypeptide having a gl2L biological activity (the biological
activities of the gl2L
proteins are described below), expressing the encoded portion of gl2L protein
(e.g., by
recombinaait expression iya vitro) and assessing the activity of the encoded
portion of gl2L.
gl2L Nucleic Acid and Polypeptide Variants
The invention further encompasses nucleic acid molecules that differ from the
nucleotide
sequences shown in SEQ m NOS:1 or 3, due to degeneracy of the genetic code and
thus encode
the same gl2L proteins as that encoded by the nucleotide sequences shown in
SEQ m NOS:1 or
3. In another embodiment, an isolated nucleic acid molecule of the invention
has a nucleotide
sequence encoding a protein having an amino acid sequence shown in SEQ m NOS:2
or 4.
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
In addition to the human gl2L nucleotide sequences shown in SEQ m N0:1, it
will be
appreciated by those skilled in the art that DNA sequence polymorphisms that
lead to changes in
the amino acid sequences of the gl2L polypeptides may exist witlun a
population (e.g., the
human population). Such genetic polymorphism in the gl2L genes may exist among
individuals
within a population due to natural allelic variation. As used herein, the
terms "gene" and
"recombinant gene" refer to nucleic acid molecules comprising an open reading
frame (ORF)
encoding a gl2L protein, preferably a vertebrate gl2L protein. Such natural
allelic variations
can typically result in 1-5% variance in the nucleotide sequence of the gl2L
genes. Any and all
such nucleotide variations and resulting amino acid polymorphisms in the gl2L
polypeptides,
which are the result of natural allelic variation and that do not alter the
functional activity of the
gl2L polypeptides, are intended to be within the scope of the invention.
Moreover, nucleic acid molecules encoding gl2L proteins from other species,
and thus
that have a nucleotide sequence that differs from the human sequence of SEQ m
NOS:1, axe
intended to be within the scope of the invention. Nucleic acid molecules
corresponding to
natural allelic variants and homologues of the gl2L cDNAs of the invention can
be isolated
based on their homology to the human gl2L nucleic acids disclosed herein using
the human
cDNAs, or a portion thereof, as a hybridization probe according to standard
hybridization
techniques under stringent hybridization conditions.
Accordingly, in another embodiment, an isolated nucleic acid molecule of the
invention
is at least 6 nucleotides in length and hybridizes wider stringent conditions
to the nucleic acid
molecule comprising the nucleotide sequence of SEQ m NOS:l or 3. In another
embodiment,
the nucleic acid is at least 10, 25, 50, 100, 250, 500, 750, 1000, 1500, or
2000 or more
nucleotides in length. In yet another embodiment, an isolated nucleic acid
molecule of the
invention hybridizes to the coding region. As used herein, the term
"hybridizes under stringent
conditions" is intended to describe conditions for hybridization and washing
under which
nucleotide sequences at least 60% homologous to each other typically remain
hybridized to each
other.
Homologs (i.e., nucleic acids encoding gl2L proteins derived from species
other tha~l
human) or other related sequences (e.g., paralogs) can be obtained by low,
moderate or high
stringency hybridization with all or a portion of the particular human
sequence as a probe using
methods well known in the art for nucleic acid hybridization and cloning.
As used herein, the phrase "stringent hybridization conditions" refers to
conditions under
which a probe, primer or oligonucleotide will hybridize to its target
sequence, but to no other
sequences. Stringent conditions are sequence-dependent and will be different
in different
circumstances. Longer sequences hybridize specifically at higher temperatures
than shorter
21
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
sequences. Generally, stringent conditions are selected to be about 5°C
lower than the thermal
melting point (Tm) for the specific sequence at a defined ionic strength and
pH. The Tm is the
temperature (under defined ionic strength, pH and nucleic acid concentration)
at which 50% of
the probes complementary to the target sequence hybridize to the target
sequence at equilibrium.
Since the target sequences are generally present at excess, at Tm, 50% of the
probes are
occupied at equilibrium. Typically, stringent conditions will be those in
which the salt
concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0
M sodium ion (or
other salts) at pH 7.0 to 8.3 and the temperature is at least about
30°C for short probes, primers
or oligonucleotides (e.g., 10 nt to 50 nt) and at least about 60°C for
longer probes, primers and
oligonucleotides. Stringent conditions may also be achieved with the addition
of destabilizing
agents, such as formamide.
Stringent conditions are known to those skilled in the art and can be found in
Ausubel, et
al., (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, N.Y.
(1989),
6.3.1-6.3.6. Preferably, the conditions are such that sequences at least about
65%, 70%, 75%,
85%, 90%, 95%, 98%, or 99% homologous to each other typically remain
hybridized to each
other. A non-limiting example of stringent hybridization conditions are
hybridization in a high
salt buffer comprising 6X SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP,
0.02%
Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm DNA at 65°C,
followed by one or
more washes in 0.2X SSC, 0.01% BSA at 50°C. An isolated nucleic acid
molecule of the
invention that hybridizes under stringent conditions to the sequences of SEQ m
NOS:1 or 3,
corresponds to a naturally-occurring nucleic acid molecule. As used herein, a
"naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule
having a
nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
In a second embodiment, a nucleic acid sequence that is hybridizable to the
nucleic acid
molecule comprising the nucleotide sequence of SEQ m NOS:1 or 3, or fragments,
analogs or
derivatives thereof, under conditions of moderate stringency is provided. A
non-limiting
example of moderate stringency hybridization conditions are hybridization in
6X SSC, SX
Denhardt's solution, 0.5% SDS and 100 mg/ml denatured salmon sperm DNA at
55°C, followed
by one or more washes in 1X SSC, 0.1 % SDS at 37°C. Other conditions of
moderate stringency
that may be used are well-lcnown within the art. See, e.g., Ausubel ,et al.
(eds.), 1993, CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, NY, and Kriegler, 1990;
GENE
TRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY.
In a third embodiment, a nucleic acid that is hybridizable to the nucleic acid
molecule
comprising the nucleotide sequences of SEQ ID NOS:l or 3 or fragments, analogs
or derivatives
thereof, under conditions of low stringency, is provided. A non-limiting
example of low
22
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
stringency hybridization conditions are hybridization in 35% formamide, SX
SSC, 50 mM
Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 mg/ml
denatured
salmon sperm DNA, 10% (wt/vol) dextran sulfate at 40°C, followed by one
or more washes in
2X SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS at 50°C. Other
conditions of
low stringency that may be used are well known in the art (e.g., as employed
for cross-species
hybridizations). See, e.g., Ausubel, et al. (eds.), 1993, CURRENT PROTOCOLS IN
MOLECULAR
BIOLOGY, John Wiley & Sons, NY, and Kriegler, 1990, GENE TRANSFER Arrn
EXPRESSION, A
LABORATORY MANUAL, Stockton Press, NY; Shilo and Weinberg, 1981. Proc Natl
Acad Sci
USA 78: 6789-6792.
CojzseYVative Mutations
In addition to naturally-occurnng allelic variants of gl2L sequences that may
exist in the
population, the skilled artisan will further appreciate that changes can be
introduced by mutation
into the nucleotide sequences of SEQ )D NO NOS:1 or 3, thereby leading to
changes in the
amino acid sequences of the encoded gl2L proteins, without altering the
functional ability of
said gl2L proteins. For example, nucleotide substitutions leading to amino
acid substitutions at
"non-essential" amino acid residues can be made in the sequence of SEQ ID
NOS:2 or 4. A
"non-essential" amino acid residue is a residue that can be altered from the
wild-type sequences
of the gl2L proteins without altering their biological activity, whereas an
"essential" amino acid
residue is required for such biological activity. For example, amino acid
residues that are
conserved among the gl2L proteins of the invention are predicted to be
particularly non-
amenable to alteration. Amino acids for which conservative substitutions can
be made are well-
known within the art.
Another aspect of the invention pertains to nucleic acid molecules encoding
gl2L
proteins that contain changes in amino acid residues that are not essential
for activity. Such
gl2L proteins differ in amino acid sequence from SEQ JD NOS:2 or 4, yet retain
biological
activity. hl one embodiment, the isolated nucleic acid molecule comprises a
nucleotide
sequence encoding a protein, wherein the protein comprises an amino acid
sequence at least
about 45% homologous to the amino acid sequences of SEQ >D NOS:2 or 4.
Preferably, the
protein encoded by the nucleic acid molecule is at least about 60% homologous
to SEQ 1D
NOS:2 or 4; more preferably at least about 70% homologous to SEQ )D NOS:2 or
4; still more
preferably at least about 80% homologous to SEQ m NOS:2 or 4; even more
preferably at least
about 90% homologous to SEQ m NOS:2 or 4; and most preferably at least about
95%
homologous to SEQ )D NOS:2 or 4.
An isolated nucleic acid molecule encoding a gl2L protein homologous to the
protein of
SEQ m NOS:2 or 4, can be created by introducing one or more nucleotide
substitutions,
23
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
additions or deletions into the nucleotide sequence of SEQ m NOS:1 or 3, such
that one or more
amino acid substitutions, additions or deletions are introduced into the
encoded protein.
Mutations can be introduced into SEQ m NOS:2 or 4, by standard techniques,
such as
site-directed mutagenesis and PCR-mediated mutagenesis. Preferably,
conservative amino acid
substitutions are made at one or more predicted, non-essential amino acid
residues. A
"conservative amino acid substitution" is one in which the amino acid residue
is replaced with
an amino acid residue having a similar side chain. Families of amino acid
residues having
similar side chains have been defined within the art. These families include
amino acids with
basic side chains (e.g., lysine, arginine, histidine), acidic side chains
(e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine,
serine, threonine,
tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine,
isoleucine, proline,
phenylalanine, methionine, tryptophan), beta-branched side chains (e.g.,
threonine, valine,
isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine,
tryptophan, histidine). Thus,
a predicted non-essential amino acid residue in the gl2L protein is replaced
with another amino
acid residue from the same side chain family. Alternatively, in another
embodiment, mutations
can be introduced randomly along all or part of a gl2L coding sequence, such
as by saturation
mutagenesis, and the resultant mutants can be screened for gl2L biological
activity to identify
mutants that retain activity. Following mutagenesis of SEQ ID NOS:2 or 4, the
encoded protein
can be expressed by any recombinant technology known in the art and the
activity of the protein
can be determined.
The relatedness of amino acid families may also be determined based on side
chain
interactions. Substituted amino acids may be fully conserved "strong" residues
or fully
conserved "weak" residues. The "strong" group of conserved amino acid residues
may be any
one of the following groups: STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW,
wherein the single letter amino acid codes are grouped by those amino acids
that may be
substituted for each other. Likewise, the "weak" group of conserved residues
may be any one of
the following: CSA, ATV, SAG, STNK, STPA, SGND, SNDEQK, NDEQHK, NEQHRK,
VLIM, HFY, wherein the letters within each group represent the single letter
amino acid code.
In one embodiment, a mutant gl2L protein can be assayed for the ability to the
ability to
affect adipose tissue differentiation or development. In yet another
embodiment, a mutant gl2L
protein can be assayed for the ability to regulate a more general biological
function (e.g., the
metabolism of an animal.).
24.
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
Antisense Nucleic Acids
Another aspect of the invention pertains to isolated antisense nucleic acid
molecules that
are hybridizable to or complementary to the nucleic acid molecule comprising
the nucleotide
sequence of SEQ ID NOS:1 or 3, or fragments, analogs or derivatives thereof.
An "antisense"
nucleic acid comprises a nucleotide sequence that is complementary to a
"sense" nucleic acid
encoding a protein (e.g., complementary to the coding strand of a double-
stranded cDNA
molecule or complementary to an ml2NA sequence). In specific aspects,
axitisense nucleic acid
molecules are provided that comprise a sequence complementary to at least
about 10, 25, 50,
100, 250 or 500 nucleotides or an entire gl2L coding strand, or to only a
portion thereof.
Nucleic acid molecules encoding fragments, homologs, derivatives and analogs
of an gl2L
protein of SEQ 1D NOS:2 or 4; or antisense nucleic acids complementary to an
gl2L nucleic
acid sequence of SEQ m NOS:1 or 3, are additionally provided.
In one embodiment, an antisense nucleic acid molecule is antisense to a
"coding region"
of the coding strand of a nucleotide sequence encoding a gl2L protein. The
teen "coding
region" refers to the region of the nucleotide sequence comprising codons that
are translated into
amino acid residues. In another embodiment, the antisense nucleic acid
molecule is antisense to
a "noncoding region" of the coding strand of a nucleotide sequence encoding
the gl2L protein.
The term "noncoding region" refers to 5' and 3' sequences that flank the
coding region that are
not translated into amino acids (i. e., also referred to as 5' and 3'
untranslated regions).
Given the coding strand sequences encoding the gl2L protein disclosed herein,
antisense
nucleic acids of the invention can be designed according to the rules of
Watson and Crick or
Hoogsteen base pairing. The antisense nucleic acid molecule can be
complementary to the
entire coding region of gl2L mRNA, but more preferably is an oligonucleotide
that is antisense
to only a portion of the coding or noncoding region of gl2L mRNA. For example,
the antisense
oligonucleotide can be complementary to the region surrounding the translation
start site of
gl2L mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15,
20, 25, 30, 35,
40, 45 or 50 nucleotides in length. An antisense nucleic acid of the invention
can be constructed
using chemical synthesis or enzymatic ligation reactions using procedures
known in the art. For
example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be
chemically
synthesized using naturally-occurnng nucleotides or variously modified
nucleotides designed to
increase the biological stability of the molecules or to increase the physical
stability of the
duplex formed between the antisense and sense nucleic acids (e.g.,
phosphorothioate derivatives
and acridine substituted nucleotides can be used).
Examples of modified nucleotides that can be used to generate the antisense
nucleic acid
include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,
hypoxanthine, xanthine, 4-
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-
thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine,
inosine, N6-
isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-
methyladenine,
2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-
methylguaune, 5-
methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-
mannosylqueosine, 5'-
methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-
isopentenyladenine, uracil-5-
oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-tluocytosine, 5-
methyl-2-thiouracil,
2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid
methylester, uracil-5-oxyacetic
acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil,
(acp3)w, and 2,6-
diaminopurine. Alternatively, the antisense nucleic acid can be produced
biologically using an
expression vector into which a nucleic acid has been subcloned in an antisense
orientation (i. e.,
RNA transcribed from the inserted nucleic acid will be of an antisense
orientation to a target
nucleic acid of interest, described further in the following subsection).
The antisense nucleic acid molecules of the invention are typically
administered to a
subject or generated ifz situ such that they hybridize with or bind to
cellular mRNA and/or
genomic DNA encoding a gl2L protein to thereby inhibit expression of the
protein (e.g., by
inhibiting transcription and/or translation). The hybridization can be by
conventional nucleotide
complementarity to form a stable duplex, or, for example, in the case of an
antisense nucleic
acid molecule that binds to DNA duplexes, through specific interactions in the
major groove of
the double helix. An example of a route of administration of antisense nucleic
acid molecules of
the invention includes direct injection at a tissue site. Alternatively,
antisense nucleic acid
molecules can be modified to target selected cells and then administered
systemically. For
example, for systemic administration, antisense molecules can be modified such
that they
specifically bind to receptors or antigens expressed on a selected cell
surface (e.g., by linking the
antisense nucleic acid molecules to peptides or antibodies that bind to cell
surface receptors or
antigens). The antisense nucleic acid molecules can also be delivered to cells
using the vectors
described herein. To achieve sufficient nucleic acid molecules, vector
constructs in which the
antisense nucleic acid molecule is placed under the control of a strong pol II
or pol BI promoter
are preferred.
In yet another embodiment, the antisense nucleic acid molecule of the
invention is an oc-
anomeric nucleic acid molecule. An oc-anomeric nucleic acid molecule forms
specific double-
stranded hybrids with complementary RNA in which, contrary to the usual (3-
units, the strands
run parallel to each other. See, e.g., Gaultier, et al., 1987. Nucl. Acids
Res. 15: 6625-6641. The
antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide
(see, e.g., moue,
26
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
et al. 1987. Nucl. Acids Res. 15: 6131-6148) or a chimeric RNA-DNA analogue
(see, e.g.,
moue, et al., 1987. FEBSLett. Z15: 327-330.
Ribozymes and PNA Moieties
Nucleic acid modifications include, by way of non-limiting example, modified
bases,
and nucleic acids whose sugar phosphate backbones are modified or derivatized.
These
modifications are carried out at least in part to enhance the chemical
stability of the modified
nucleic acid, such that they may be used, for example, as antisense binding
nucleic acids in
therapeutic applications in a subject.
In one embodiment, an antisense nucleic acid of the invention is a ribozyme.
Ribozymes
are catalytic RNA molecules with ribonuclease activity that are capable of
cleaving a single-
stranded nucleic acid, such as an mRNA, to which they have a complementary
region. Thus,
ribozymes (e.g., hammerhead ribozymes as described in Haselhoff and Gerlach
1988. Nature
334: 585-591) can be used to catalytically cleave gl2L mRNA transcripts to
thereby inhibit
translation of gl2L mRNA. A ribozyme having specificity for an gl2L-encoding
nucleic acid
can be designed based upon the nucleotide sequence of an gl2L cDNA disclosed
herein (i.e.,
SEQ ID NOS:1 or 3). For example, a derivative of a TetYahyrraena L-19 IVS RNA
can be
constructed in which the nucleotide sequence of the active site is
complementary to the
nucleotide sequence to be cleaved in a gl2L-encoding mRNA. See, e.g., U.S.
Patent 4,987,071
to Cech, et al. and U.S. Patent 5,116,742 to Cech, et al. gl2L mRNA can also
be used to select
a catalytic RNA having a specific ribonuclease activity from a pool of RNA
molecules. See, e.g.,
Bartel et al., (1993) Science 261:1411-1418.
Alternatively, gl2L gene expression can be inhibited by targeting nucleotide
sequences
complementary to the regulatory region of the gl2L nucleic acid (e.g., the
gl2L promoter and/or
enhancers) to form triple helical structures that prevent transcription of the
gl2L gene in target
cells. See, e.g., Helene, 1991. Aratican.cer Drug Des. 6: 569-84; Helene, et
al. 1992. Ann. N Y.
Acad. Sci. 660: 27-36; Maher, 1992. Bioassays 14: 807-15.
In various embodiments, the gl2L nucleic acids can be modified at the base
moiety,
sugar moiety or phosphate backbone to improve, e.g., the stability,
hybridization, or solubility of
the molecule. For example, the deoxyribose phosphate baclcbone of the nucleic
acids can be
modified to generate peptide nucleic acids. See, e.g., Hyrup, et al., 1996.
Bioor~g Med Cherry 4:
5-23. As used herein, the terms "peptide nucleic acids" or "PNAs" refer to
nucleic acid mimics
(e.g., DNA mimics) in that the deoxyribose phosphate backbone is replaced by a
pseudopeptide
backbone and only the four natural nucleobases are retained. The neutral
backbone of PNAs has
been shown to allow for specific hybridization to DNA and RNA under conditions
of low ionic
strength. The synthesis of PNA oligomers can be performed using standard solid
phase peptide
27
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
synthesis protocols as described in Hyrup, et al., 1996. supra; Perry-O'Keefe,
et al., 1996. Pf~oc.
Natl. Acad. Sci. USA 93: 14670-14675.
PNAs of gl2L can be used in therapeutic and diagnostic applications. For
example,
PNAs can be used as antisense or antigene agents for sequence-specific
modulation of gene
expression by, e.g., inducing transcription or translation arrest or
inlubiting replication. PNAs of
gl2L can also be used, for example, in the analysis of single base pair
mutations in a gene (e.g.,
PNA directed PCR clamping; as artificial restriction enzymes when used in
combination with
other enzymes, e.g., S1 nucleases (see, Hyrup, et al., 1996.sup~a); or as
probes or primers for
DNA sequence and hybridization (see, Hyrup, et al., 1996, supra; Perry-
O'Keefe, et al., 1996.
supYa).
In another embodiment, PNAs of gl2L can be modified, e.g., to enhance their
stability or
cellular uptalce, by attaching lipophilic or other helper groups to PNA, by
the formation of PNA-
DNA chimeras, or by the use of liposomes or other techniques of drug delivery
known in the art.
For example, PNA-DNA chimeras of gl2L can be generated that may combine the
advantageous properties of PNA and DNA. Such chimeras allow DNA recognition
enzymes
(e.g., RNase H and DNA polymerases) to interact with the DNA portion while the
PNA portion
would provide high binding affinity and specificity. PNA-DNA chimeras can be
linked using
linkers of appropriate lengths selected in terms of base stacking, number of
bonds between the
nucleobases, and orientation (see, Hyrup, etal., 1996. supra). The synthesis
of PNA-DNA
chimeras can be performed as described in Hyrup, et al., 1996. supra and Finn,
et al., 1996. Nucl
Acids Res 24: 3357-3363. For example, a DNA chain can be synthesized on a
solid support
. using standard phosphoramidite coupling chemistry, and modified nucleoside
analogs, e.g., 5'-
(4-methoxytrityl)amino-5'-deoxy-thynidine phosphoramidite, can be used between
the PNA and
the 5' end of DNA. See, e.g., Mag, et al., 1989. Nucl Acid Res 17: 5973-5988.
PNA monomers
are then coupled in a stepwise manner to produce a chimeric molecule with a 5'
PNA segment
and a 3' DNA segment. See, e.g., Finn, et al., 1996. supra. Alternatively,
chimeric molecules
can be synthesized with a 5' DNA segment and a 3' PNA segment. See, e.g.,
Petersen, et al.,
1975. Bioo~g. Med. Cltern. Lett. 5: 1119-11124.
In other embodiments, the oligonucleotide may include other appended groups
such as
peptides (e.g., for targeting host cell receptors in vivo), or agents
facilitating transport across the
cell membrane (see, e.g., Letsinger, et al., 1989. PPOC. Natl. Acad. Sci.
U.S.A. 86: 6553-6556;
Lemaitre, et al., 1987. Proc. Natl. Acad. Sci. 84: 648-652; PCT Publication
No. W088/09810)
or the blood-brain barrier (see, e.g., PCT Publication No. WO 89/10134). In
addition,
oligonucleotides can be modified with hybridization triggered cleavage agents
(see, e.g., I~rol, et
al., 1988. BioTechniques 6:958-976) or intercalating agents (see, e.g., Zon,
1988. Phat°fra. Res. 5:
28
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
539-549). To this end, the oligonucleotide may be conjugated to another
molecule, e.g., a
peptide, a hybridization triggered cross-linking agent, a transport agent, a
hybridization-
triggered cleavage agent, and the like.
gl2L Polypeptides
A polypeptide according to the invention includes a polypeptide including the
amino
acid sequence of gl2L polypeptides whose sequences are provided in SEQ ID
NOS:2 or 4. The
invention also includes a mutant or variant protein any of whose residues may
be changed from
the corresponding residues shown in SEQ m NOS:2 or 4, while still encoding a
protein that
maintains its gl2L activities and physiological functions, or a functional
fragment thereof.
In general, an gl2L variant that preserves gl2L-lilce fiulction includes any
variant in
which residues at a particular position in the sequence have been substituted
by other amino
acids, and further include the possibility of inserting an additional residue
or residues between
two residues of the parent protein as well as the possibility of deleting one
or more residues from
the parent sequence. Any amino acid substitution, insertion, or deletion is
encompassed by the
invention. In favorable circumstances, the substitution is a conservative
substitution as defined
above.
One aspect of the invention pertains to isolated gl2L proteins, and
biologically-active
portions thereof, or derivatives, fragments, analogs or homologs thereof. Also
provided are
polypeptide fragments suitable for use as irmnunogens to raise anti-gl2L
antibodies. In one
embodiment, native gl2L proteins can be isolated from cells or tissue sources
by an appropriate
purification scheme using standard protein purification teclnuques. In another
embodiment,
gl2L proteins are produced by recombinant DNA techniques. Alternative to
recombinant
expression, a gl2L protein or polypeptide can be synthesized chemically using
standard peptide
synthesis techniques.
An "isolated" or "purified" polypeptide or protein or biologically-active
portion thereof is
substantially free of cellular material or other contaminating proteins from
the cell or tissue
source from which the gl2L protein is derived, or substantially free from
chemical precursors or
other chemicals when chemically synthesized. The language "substantially free
of cellular
material" includes preparations of gl2L proteins in which the protein is
separated from cellular
components of the cells from which it is isolated or recombinantly-produced.
In one
embodiment, the language "substantially free of cellular material" includes
preparations of gl2L
proteins having less than about 30% (by dry weight) of non-gl2L proteins (also
referred to
herein as a "contaminating protein"), more preferably less than about 20% of
non-gl2L proteins,
still more preferably less than about 10% of non-gl2L proteins, and most
preferably less than
29
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
about 5% of non-gl2L proteins. When the gl2L protein or biologically-active
portion thereof is
recombinantly-produced, it is also preferably substantially free of culture
medium, i. e., culture
medium represents less than about 20%, more preferably less than about 10%,
and most
preferably less than about 5% of the volume of the gl2L protein preparation.
The language "substantially free of chemical precursors or other chemicals"
includes
preparations of gl2L proteins in which the protein is separated from chemical
precursors or
other chemicals that are involved in the synthesis of the protein. In one
embodiment, the
language "substantially free of chemical precursors or other chemicals"
includes preparations of
gl2L proteins having less than about 30% (by dry weight) of chemical
precursors or non-gl2L
chemicals, more preferably less than about 20% chemical precursors or non-gl2L
chemicals,
still more preferably less than about 10% chemical precursors or non-gl2L
chemicals, and most
preferably less than about 5% chemical precursors or non-gl2L chemicals.
Biologically-active portions of gl2L proteins include peptides comprising
amino acid
sequences sufficiently homologous to or derived from the amino acid sequences
of the gl2L
proteins (e.g., the amino acid sequence shown in SEQ m NOS:2 or 4) that
include fewer amino
acids than the full-length gl2L proteins, and exhibit at least one activity of
a gl2L protein.
Typically, biologically-active portions comprise a domain or motif with at
least one activity of
the gl2L protein. A biologically-active portion of a gl2L protein can be a
polypeptide which is,
for example, 10, 25, 50, 100 or more amino acid residues in length.
Moreover, other biologically-active portions, in which other regions of the
protein are
deleted, can be prepared by recombinant techniques and evaluated for one or
more of the
functional activities of a native gl2L protein.
In an embodiment, the gl2L protein has an amino acid sequence shown in SEQ m
NOS:2 or 4. In other embodiments, the gl2L protein is substantially homologous
to SEQ ID
NOS:2 or 4, and retains the functional activity of the protein of SEQ m NOS:2
or 4, yet differs
in amino acid sequence due to natural allelic variation or mutagenesis, as
described in detail,
below. Accordingly, in another embodiment, the gl2L protein is a protein that
comprises an
amino acid sequence at least about 45% homologous to the amino acid sequence
of SEQ ID
NOS:2 or 4, and retains the functional activity of the gl2L proteins of SEQ ID
NOS:2 or 4.
Detef°mining Homology Between Two o~ MoYe Sequences
To determine the percent homology of two amino acid sequences or of two
nucleic acids,
the sequences are aligned for optimal comparison purposes (e.g., gaps can be
introduced in the
sequence of a first amino acid or nucleic acid sequence for optimal alignment
with a second
amino or nucleic acid sequence). The amino acid residues or nucleotides at
corresponding
amino acid positions or nucleotide positions are then compared. When a
position in the first
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
sequence is occupied by the same amino acid residue or nucleotide as the
coiTesponding position
in the second sequence, then the molecules are homologous at that position
(i.e., as used herein
amino acid or nucleic acid "homology" is equivalent to amino acid or nucleic
acid "identity").
The nucleic acid sequence homology may be determined as the degree of identity
between two sequences. The homology may be determined using computer programs
known in
the art, such as GAP software provided in the GCG program package. See,
Needleman and
Wunsch, 1970. JMoI Biol 48: 443-453. Using GCG GAP software with the following
settings
for nucleic acid sequence comparison: GAP creation penalty of 5.0 and GAP
extension penalty
of 0.3, the coding region of the analogous nucleic acid sequences referred to
above exhibits a
degree of identity preferably of at least 70%, 75%, 80%, 85%, 90%, 95%, 98%,
or 99%, with the
CDS (encoding) part of the DNA sequence shown in SEQ ID NOS:l or 3.
The term "sequence identity" refers to the degree to which two polynucleotide
or
polypeptide sequences are identical on a residue-by-residue basis over a
particular region of
comparison. The term "percentage of sequence identity" is calculated by
comparing two
optimally aligned sequences over that region of comparison, determining the
number of
positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I,
in the case of nucleic
acids) occurs in both sequences to yield the number of matched positions,
dividing the number
of matched positions by the total number of positions in the region of
comparison (i.e., the
window size), and multiplying the result by 100 to yield the percentage of
sequence identity.
The term "substantial identity" as used herein denotes a characteristic of a
polynucleotide
sequence, wherein the polynucleotide comprises a sequence that has at least 80
percent sequence
identity, preferably at least 85 percent identity and often 90 to 95 percent
sequence identity,
more usually at least 99 percent sequence identity as compaxed to a reference
sequence over a
comparison region.
Ch.ifyteric a>zd Fusion Pt~oteitzs
The invention also provides gl2L chimeric or ftision proteins. As used herein,
a gl2L
"chimeric protein" or "fusion protein" comprises a gl2L polypeptide
operatively-linked to a
non-gl2L polypeptide. An "gl2L polypeptide" refers to a polypeptide having an
amino acid
sequence corresponding to an gl2L protein (SEQ ID NOS:2 or 4), whereas a "non-
gl2L
polypeptide" refers to a polypeptide having an amino acid sequence
corresponding to a protein
that is not substantially homologous to the gl2L protein, e.g., a protein that
is different from the
gl2L protein and that is derived from the same or a different organism. Within
a gl2L fusion
protein the gl2L polypeptide can correspond to all or a portion of a gl2L
protein. In one
embodiment, a gl2L fusion protein comprises at least one biologically-active
portion of an gl2L
protein. In another embodiment, a gl2L fusion protein comprises at least two
biologically-
31
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
active portions of a gl2L protein. In yet another embodiment, a gl2L fusion
protein comprises
at least three biologically-active portions of a gl2L protein. Within the
fusion protein, the term
"operatively-linked" is intended to indicate that the gl2L polypeptide and the
non-gl2L
pohypeptide are fused in-frame with one another. The non-gl2L polypeptide can
be fused to the
N-terminus or C-terminus of the gl2L polypeptide.
In one embodiment, the fusion protein is a GST-gl2L fusion protein in which
the gl2L
sequences are fused to the C-terminus of the GST (glutathione S-transferase)
sequences. Such
fusion proteins can facilitate the purification of recombinant gl2L
polypeptides.
In another embodiment, the fusion protein is a gl2L protein containing a
heterohogous
signal sequence at its N-terminus. In certain host cells (e.g., mammalian host
cells), expression
and/or secretion of gl2L can be increased through use of a heterologous signal
sequence.
In yet another embodiment, the fusion protein is a gl2L-immunoglobulin fusion
protein
in which the gl2L sequences are fused to sequences derived from a member of
the
immunoglobulin protein family. The gl2L-immunoglobulin fusion proteins of the
invention can
be incorporated into pharmaceutical compositions and administered to a subject
to inhibit an
interaction between a gl2L ligand and a gl2L protein, to thereby suppress gl2L-
mediated signal
transduction in vivo. The gl2L-immunoglobulin fusion proteins can be used to
affect the
bioavailab'ility of a gl2L cognate ligand. Inhibition of the gl2L ligand/ gl2L
interaction may be
useful therapeutically for both the treatment of proliferative and
differentiative disorders, as well
as modulating (e.g. promoting or inhibiting) cell survival. Moreover, the gl2L-
immunoglobulin
fusion proteins of the invention can be used as immunogens to produce anti-
gl2L antibodies in a
subject, to purify gl2L higands, and in screening assays to identify molecules
that inhibit the
interaction of gl2L with a gl2L ligand.
A gl2L chimeric or fusion protein of the invention can be produced by standard
recombinant DNA techniques. For example, DNA fragments coding for the
different
polypeptide sequences are ligated together in-frame in accordance with
conventional techniques,
e.g., by employing blunt-ended or stagger-ended termini for higation,
restriction enzyme
digestion to provide for appropriate termini, filling-in of cohesive ends as
appropriate, alkaline
phosphatase treatment to avoid undesirable joiiung, and enzymatic ligation. In
another
embodiment, the fusion gene can be synthesized by conventional techniques
including
automated DNA synthesizers. Alternatively, PCR amplification of gene fragments
can be
carried out using anchor primers that give rise to complementary overhangs
between two
consecutive gene fragments that can subsequently be annealed and reamplified
to generate a
chimeric gene sequence (see, e.g., Ausubel, et al. (eds.) CURRENT PROTOCOLS IN
MOLECULAR
BIOLOGY, John Wiley & Sons, 1992). Moreover, many expression vectors are
commercially
32
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
available that already encode a fusion moiety (e.g., a GST polypeptide). A
gl2L-encoding
nucleic acid can be cloned into such an expression vector such that the fusion
moiety is linked
in-frame to the gl2L protein.
gl2L Agoyzists ahd Ahtagohists
It is useful to test for compounds having the property of increasing or
decreasing gl2L
activity. This increase in activity may come about in a variety of ways, for
example: (1) by
increasing or decreasing the copies of the gene in the cell (amplifiers and
deamplifiers); (2) by
increasing or decreasing transcription of the gl2L gene (transcription up-
regulators and down-
regulators); (3) by increasing or decreasing the translation of gl2L mRNA into
protein
(translation up-regulators and down-regulators); or (4) by increasing or
decreasing the activity
of gl2L itself (agonists and antagonists).
Compounds that are amplifiers and deamplifiers may be identified by contacting
cells or
organisms with the compound, and then measuring the amount of DNA present that
encodes
gl2L (Ausubel, Brent et al. 1987). Compounds that are transcription up-
regulators and down-
regulators may be identified by contacting cells or organisms with the
compound, and then
measuring the amount of mRNA produced that encodes gl2L (Ausubel, Brent et al.
1987).
Compounds that are translation up-regulators and down-regulators may be
identified by
contacting cells or organisms with the compound, and then measuring the amount
of gl2L
polypeptide produced (Ausubel, Brent et al. 1987). Compounds that are agonists
or antagonists
may be identified by contacting cells or organisms with the compound, and then
measuring
gl2L activity; this may also be carried out in. vitro.
The invention also pertains to variants of the gl2L proteins that function as
either gl2L
agonists (i.e., mimetics) or as gl2L antagonists. Variants of the gl2L protein
can be generated
by mutagenesis (e.g., discrete point mutation or truncation of the gl2L
protein). An agonist of
the gl2L protein can retain substantially the same, or a subset of, the
biological activities of the
naturally occurring form of the gl2L protein. An antagonist of the gl2L
protein can inhibit one
or more of the activities of the naturally occurring form of the gl2L protein
by, for example,
competitively binding to a downstream or upstream member of a cellular
signaling cascade that
includes the gl2L protein. Thus, specific biological effects can be elicited
by treatment with a
variant of limited function. In one embodiment, treatment of a subject with a
variant having a
subset of the biological activities of the naturally occurring form of the
protein has fewer side
effects in a subject relative to treatment with the naturally occurring form
of the gl2L proteins.
Variants of the gl2L proteins that function as either gl2L agonists (i.e.,
mimetics) or as
gl2L antagonists can be identified by screening combinatorial libraries of
mutants (e.g.,
truncation mutants) of the gl2L proteins for gl2L protein agonist or
antagonist activity. In one
33
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
embodiment, a variegated library of gl2L variants is generated by
combinatorial mutagenesis at
the nucleic acid level and is encoded by a variegated gene library. A
variegated library of gl2L
variants can be produced by, for example, enzymatically ligating a mixture of
synthetic
oligonucleotides into gene sequences such that a degenerate set of potential
gl2L sequences is
expressible as individual polypeptides, or alternatively, as a set of larger
fusion proteins (e.g., for
phage display) containing the set of gl2L sequences therein. There are a
variety of methods that
can be used to produce libraries of potential gl2L variants from a degenerate
oligonucleotide
sequence. Chemical synthesis of a degenerate gene sequence can be performed in
an automatic
DNA synthesizer, and the synthetic gene then ligated into an appropriate
expression vector. Use
of a degenerate set of genes allows for the provision, in one mixture, of all
of the sequences
encoding the desired set of potential gl2L sequences. Methods for synthesizing
degenerate
oligonucleotides are well-known within the art. See, e.g., Narang, 1983.
Tetrahedron 39: 3;
Italcura, et al., 1984. Anrau. Rev. Biochefrz. 53: 323; Italcura, et al.,
1984. Science 198: 1056; Ike,
et al., 1983. Nucl. Acids Res. 11: 477.
Pol~peptide Libraries
In addition, libraries of fragments of the gl2L protein coding sequences can
be used to
generate a variegated population of gl2L fragments for screening and
subsequent selection of
variants of a gl2L protein. In one embodiment, a library of coding sequence
fragments can be
generated by treating a double stranded PCR fragment of a gl2L coding sequence
with a
nuclease under conditions wherein nicking occurs only about once per molecule,
denaturing the
double stranded DNA, renaturing the DNA to form double-stranded DNA that can
include
sense/antisense pairs from different nicked products, removing single stranded
portions from
reformed duplexes by treatment with S 1 nuclease, and ligating the resulting
fragment library into
an expression vector. By this method, expression libraries can be derived
which encodes
N-terminal and internal fragments of various sizes of the gl2L proteins.
Various techniques are known in the art for screening gene products of
combinatorial
libraries made by point mutations or truncation, and for screening cDNA
libraries for gene
products having a selected property. Such techniques are adaptable for rapid
screening of the
gene libraries generated by the combinatorial mutagenesis of gl2L proteins.
The most widely
used techniques, which are amenable to high throughput analysis, for screening
large gene
libraries typically include cloning the gene library into replicable
expression vectors,
transforming appropriate cells with the resulting library of vectors, and
expressing the
combinatorial genes under conditions in which detection of a desired activity
facilitates isolation
of the vector encoding the gene whose product was detected. Recursive ensemble
mutagenesis
(REM), a new technique that enhances the frequency of functional mutants in
the libraries, can
34
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
be used in combination with the screening assays to identify gl2L variants.
See, e.g., Arkin and
Yourvan, 1992. Proc. Natl. Acad. Sci. ZISA 89: 7811-7815; Delgrave, et al.,
1993. Protein
E~rgineerihg 6:327-331.
Anti-gl2L Antibodies
The invention encompasses antibodies and antibody fragments, such as Fab or
(Fab)z, that
bind immunospecifically to any of the gl2L polypeptides of said invention.
The term "antibody" as used herein refers to immmoglobulin molecules and
innnunologically active portions of innnunoglobulin (Ig) molecules, i.e.,
molecules that contain
an antigen-binding site that specifically binds (immunoreacts with) an
antigen. Such antibodies
include, but are not limited to, polyclonal, monoclonal, chimeric, single
chain, Fab, Fab° and F(ab~)z
fragments, and an Fab expression library. In general, antibody molecules
obtained from humans
relates to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from
one another by the
nature of the heavy chain present in the molecule. Certain classes have
subclasses as well, such
as IgGI, IgGz, and others. Furthermore, in humans, the light chain may be a
lcappa chain or a
lambda chain. Reference herein to antibodies includes a reference to all such
classes, subclasses
and types of human antibody species.
An isolated protein of the invention intended to serve as an antigen, or a
portion or
fragment thereof, can be used as an immunogen to generate antibodies that
immunospecifically
bind the antigen, using standard techniques for polyclonal and monoclonal
antibody preparation.
The full-length protein can be used or, alternatively, the invention provides
antigenic peptide
fragments of the antigen for use as immunogens. An antigenic peptide fragment
comprises at
least 6 amino acid residues of the amino acid sequence of the full length
protein, such as an
amino acid sequence shown in SEQ ID NOs:2 or 4, and encompasses an epitope
thereof such
that an antibody raised against the peptide forms a specific immune complex
with the full length
protein or with any fragment that contains the epitope. Preferably, the
antigenic peptide
comprises at least 10 amino acid residues, or at least 15 amino acid residues,
or at least 20 amino
acid residues, or at least 30 amino acid residues. Preferred epitopes
encompassed by the
antigenic peptide are regions of the protein that are located on its surface;
commonly these are
hydrophilic regions.
In certain embodiments of the invention, at least one epitope encompassed by
the
antigenic peptide is a region of gl2L that is located on the surface of the
protein, e.g., a
hydrophilic region. A hydrophobicity analysis of the gl2L protein sequence
will indicate which
regions of a gl2L polypeptide are particularly hydrophilic and, therefore, are
likely to encode
surface residues useful for targeting antibody production. As a means for
targeting antibody
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
production, hydropathy plots showing regions of hydrophilicity and
hydrophobicity may be
generated by any method well known in the art, including, for example, the
Kyte Doolittle or the
Hopp Woods methods, either with or without Fourier transformation. See, e.g.,
Hopp and
Woods, 1981, P3~oc. Nat. Acad. Sci. USA 78: 3824-3828; Kyte and Doolittle
1982, J. Mol. Biol.
157: 105-142, each incorporated herein by reference in their entirety.
Antibodies that are
specific for one or more domains within an antigenic protein, or derivatives,
fragments, analogs
or homologs thereof, are also provided herein.
A protein of the invention, or a derivative, fragment, analog, homolog or
ortholog
thereof, may be utilized as an immunogen in the generation of antibodies that
irmnunospecifically bind these protein components.
Various procedures known within the art may be used for the production of
polyclonal or
monoclonal antibodies directed against a protein of the invention, or against
derivatives,
fragments, analogs homologs or orthologs thereof (see, for example,
Antibodies: A Laboratory
Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold
Spring
Harbor, NY, incorporated herein by reference). Some of these antibodies are
discussed below.
1. Polyclonal Antibodies
For the production of polyclonal antibodies, various suitable host animals
(e.g., rabbit,
goat, mouse or other mammal) may be irmnunized by one or more inj ections with
the native
protein, a synthetic variant thereof, or a derivative of the foregoing. An
appropriate
immunogenic preparation can contain, fox example, the naturally occurnng
imxnunogenic
protein, a chemically synthesized polypeptide representing the immunogenic
protein, or a
recombinantly expressed immunogenic protein. Furthermore, the protein may be
conjugated to
a second protein known to be immunogenic in the mammal being immunized.
Examples of
such immunogenic proteins include but are not limited to keyhole limpet
hemocyanin, serum
albumin, bovine thyroglobulin, and soybean trypsin inhibitor. The preparation
can further
include an adjuvant. Various adjuvants used to increase the immunological
response include,
but are not limited to, Freund's (complete and incomplete), mineral gels
(e.g., aluminum
hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols,
polyanions, peptides,
oil emulsions, dinitrophenol, etc.), adjuvants usable in humans such as
Bacille Calmette-Guerin
and Corynebacterium parvum, or similar immunostimulatory agents. Additional
examples of
adjuvants that can be employed include MPL-TDM adjuvant (monophosphoryl Lipid
A,
synthetic trehalose dicorynomycolate).
The polyclonal antibody molecules directed against the immunogenic protein can
be
isolated from the mammal (e.g., from the blood) and further purified by well
lmown techniques,
such as affinity chromatography using protein A or protein G, which provide
primarily the IgG
36
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
fraction of immune serum. Subsequently, or alternatively, the specific antigen
that is the target
of the immunoglobulin sought, or an epitope thereof, may be immobilized on a
column to purify
the immune specific antibody by immunoaffinity chromatography. Purification of
immunoglobulins is discussed, for example, by D. Wilkinson (The Scientist,
published by The
Scientist, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).
2. Monoclonal Antibodies
The term "monoclonal antibody" (MAb) or "monoclonal antibody composition", as
used
herein, refers to a population of antibody molecules that contain only one
molecular species of
antibody molecule consisting of a unique light chain gene product and a unique
heavy chain
gene product. In particular, the complementarity determining regions (CDRs) of
the monoclonal
antibody are identical in all the molecules of the population. MAbs thus
contain an antigen-
binding site capable of inmnunoreacting with a particular epitope of the
antigen characterized by
a unique binding affinity for it.
Monoclonal antibodies can be prepared using hybridoma methods, such as those
described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma
method, a mouse,
hamster, or other appropriate host animal, is typically immunized with an
immunizing agent to
elicit lymphocytes that produce or are capable of producing antibodies that
will specifically bind
to the immunizing agent. Alternatively, the lymphocytes can be irmnunized in
vitro.
The immunizing agent will typically include the protein antigen, a fragment
thereof or a
fusion protein thereof. Generally, either peripheral blood lymphocytes are
used if cells of
human origin are desired, or spleen cells or lymph node cells are used if non-
human mammalian
sources are desired. The lymphocytes are then fused with an immortalized cell
line using a
suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell
[Goding,
Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-
103].
Jmmortalized cell lines are usually transformed mammalian cells, particularly
myeloma cells of
rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are
employed. The
hybridoma cells can be cultured in a suitable culture medium that preferably
contains one or
more substances that inhibit the growth or survival of the unfitsed,
immortalized cells. For
example, if the parental cells laclc the enzyme hypoxanthine guanine
phosphoribosyl transferase
(HGPRT or HPRT), the culture medium for the hybridomas typically will include
hypoxanthine,
aminopterin, and thymidine ("HAT medium"), which substances prevent the growth
of HGPRT-
deficient cells.
Preferred immortalized cell lines are those that fuse efficiently, support
stable high level
expression of antibody by the selected antibody-producing cells, and are
sensitive to a medium
such as HAT medium. More preferred immortalized cell lines are marine myeloma
lines, which
37
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
can be obtained, for instance, from the Salk Institute Cell Distribution
Center, San Diego,
California and the American Type Culture Collection, Manassas, Virginia. Human
myeloma
and mouse-human heteromyeloma cell lines also have been described for the
production of
human monoclonal antibodies [Kozbor, J. Irnrnunol., 133:3001 (1984); Brodeur
et al.,
Monoclonal Antibody Production Techniques and Applications, Marcel Dekker,
Inc., New
York, (1987) pp. 51-63].
The culture medium in which the hybridoma cells are cultured can then be
assayed for
the presence of monoclonal antibodies directed against the antigen.
Preferably, the binding
specificity of monoclonal antibodies produced by the hybridoma cells is
determined by
immunoprecipitation or by an iyZ vitro binding assay, such as radioimmunoassay
(RIA) or
enzyme-linlced immunoabsorbent assay (ELISA). Such techniques and assays are
known in the
art. The binding affinity of the monoclonal antibody can, for example, be
determined by the
Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980). It
is an objective,
especially important in therapeutic applications of monoclonal antibodies, to
identify antibodies
having a high degree of specificity and a high binding affinity for the target
antigen.
After the desired hybridoma cells are identified, the clones can be subcloned
by limiting
dilution procedures and grown by standard methods (coding, 1986). Suitable
culture media for
this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-
1640
medimn. Alternatively, the hybridoma cells can be grown in. vivo as ascites in
a mammal.
The monoclonal antibodies secreted by the subclones cal be isolated or
purified from the
culture medium or ascites fluid by conventional immunoglobulin purification
procedures such
as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis,
dialysis, or affinity chromatography.
The monoclonal antibodies 'can also be made by recombinant DNA methods, such
as
those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal
antibodies of the
invention can be readily isolated and sequenced using conventional procedures
(e.g., by using
oligonucleotide probes that are capable of binding specifically to genes
encoding the heavy and
light chains of marine antibodies). The hybridoma cells of the invention serve
as a preferred
source of such DNA. Once isolated, the DNA can be placed into expression
vectors, which are
then transfected into host cells such as simian COS cells, Chinese hamster
ovary (CHO) cells, or
myeloma cells that do not otherwise produce immunoglobulin protein, to obtain
the synthesis of
monoclonal antibodies in the recombinant host cells. The DNA also can be
modified, for
example, by substituting the coding sequence for human heavy and light chain
constant domains
in place of the homologous marine sequences (U.S. Patent No. 4,816,567;
Morrison, Nature
368, 812-13 (1994)) or by covalently joining to the immunoglobulin coding
sequence all or part
38
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
of the coding sequence for a non-immunoglobulin polypeptide. Such a non-
immunoglobulin
polypeptide can be substituted for the constant domains of an antibody of the
invention, or can
be substituted for the variable domains of one antigen-combining site of an
antibody of the
invention to create a chimeric bivalent antibody.
3. Humanized Antibodies
The antibodies directed against the protein antigens of the invention can
further comprise
humanzed antibodies or human antibodies. These antibodies are suitable for
admiustration to
humans without engendering an immune response by the human against the
administered
immunoglobulin. Humaiuzed forms of antibodies are chimeric immunoglobulins,
immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or
other antigen-
binding subsequences of antibodies) that are principally comprised of the
sequence of a hmnan
immunoglobulin, and contain minimal sequence derived from a non-human
immunoglobulin.
Humanization can be performed following the method of Winter and co-workers
(Jones et al.,
Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988);
Verhoeyen et al.,
Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences
for the
corresponding sequences of a human antibody. (See also U.S. Patent No.
5,225,539.) In some
instances, Fv framework residues of the human immunoglobulin axe replaced by
corresponding
non-hmnan residues. Humanized antibodies can also comprise residues that are
found neither in
the recipient antibody nor in the imported CDR or framework sequences. In
general, the
humanized antibody will comprise substantially all of at least one, and
typically two, variable
domains, in which all or substantially all of the CDR regions correspond to
those of a non-
human immunoglobulin and all or substantially all of the framework regions are
those of a
human immunoglobulin consensus sequence. The humanized antibody optimally also
will
comprise at least a portion of an immunoglobulin constant region (Fc),
typically that of a human
immunoglobulin (Jones et al., 1986; Riechmann et al., 1988; and Presta, Curr.
Op. Struct. Biol.,
2:593-596 (1992)).
4. Human Antibodies
Fully human antibodies essentially relate to antibody molecules in which the
entire
sequence of both the light chain and the heavy chain, including the CDRs,
arise from human
genes. Such antibodies are termed "human antibodies", or "fully human
antibodies" herein.
Human monoclonal antibodies can be prepared by the trioma technique; the human
B-cell
hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72) and the
EBV. hybridoma
technique to produce human monoclonal antibodies (see Cole, et al., 1985 In:
MONOCLONAL
ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human
monoclonal
antibodies may be utilized in the practice of the present invention and may be
produced by using
39
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80: 2026-
2030) or by
transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al.,
1985 In:
MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, lizc., pp: 77-96).
In addition, human antibodies can also be produced using additional
techniques,
including phage display libraries (Hoogenboom and Winter, J. Mol. Biol.,
227:381 (1991);
Marks et al., J. Mol. Biol., 222:581 (1991)). Similarly, human antibodies can
be made by
introducing human immunoglobulin loci into transgenic animals, e.g., mice in
which the
endogenous irmnunoglobulin genes have been partially or completely
inactivated. Upon
challenge, human antibody production is observed, which closely resembles that
seen in humans
in all respects, including gene rearrangement, assembly, and antibody
repertoire. Tlus approach
is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806;
5,569,825; 5,625,126;
5,633,425; 5,661,016, and in Marlcs et al. (Bio/Technolo~y 10, 779-783
(I992)); Lonberg et aI.
(Nature 368 856-859 (1994)); Morrison ( Nature 368, 812-13 (1994)); Fishwild
et al,( Nature
BiotechnoloQV 14, 845-51 (1996)); Neuberger (Nature Biotechnolo~y 14, 826
(1996)); and
Lonberg and Huszar (Intern. Rev. T_m_m__unol. 13 65-93 (1995)).
Human antibodies may additionally be produced using transgenic non-human
aumals
that are modified so as to produce fully human antibodies rather than the
animal's endogenous
antibodies in response to challenge by an antigen. (See PCT publication
W094/02602). The
endogenous genes encoding the heavy and light immunoglobulin chains in the
nonhuman host
have been incapacitated, and active loci encoding human heavy and light chain
irnmunoglobulins are inserted into the host's genome. The human genes are
incorporated, for
example, using yeast artificial chromosomes containing the requisite human DNA
segments. An
animal which provides all the desired modifications is then obtained as
progeny by
crossbreeding intermediate transgenic animals containing fewer than the full
complement of the
modifications. The preferred embodiment of such a nonhuman animal is a mouse,
and is termed
the XenomouseTM as disclosed in PCT publications WO 96/33735 and WO 96/34096.
This
animal produces B cells that secrete fully human immunoglobulins. The
antibodies can be
obtained directly from the animal after immunization with an immunogen of
interest, as, for
example, a preparation of a polyclonal antibody, or alternatively from
immortalized B cells
derived from the animal, such as hybridomas producing monoclonal antibodies.
Additionally,
the genes encoding the immunoglobulins with human variable regions can be
recovered and
expressed to obtain the antibodies directly, or can be further modified to
obtain analogs of
antibodies such as, for example, single chain Fv molecules.
An example of a method of producing a non-human host, exemplified as a mouse,
~ lacking expression of an endogenous immunoglobulin heavy chain is disclosed
in U.S. Patent
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
No. 5,939,598. It can be obtained by a method including deleting the J segment
genes from at
least one endogenous heavy chain locus in an embryonic stem cell to prevent
rearrangement of
the locus and to prevent formation of a transcript of a rearranged
immunoglobulin heavy chain
locus, the deletion being effected by a targeting vector containing a gene
encoding a selectable
marker; and producing from the embryoiuc stem cell a transgenic mouse whose
somatic and
germ cells contain the gene encoding the selectable marker.
A method for producing an antibody of interest, such as a human antibody, is
disclosed
in U.S. Patent No. 5,916,771. It includes introducing an expression vector
that contains a
nucleotide sequence encoding a heavy chain into one mammalian host cell in
culture,
introducing an expression vector containing a nucleotide sequence encoding a
light chain into
another mammalian host cell, and fusing the two cells to form a hybrid cell.
The hybrid cell
expresses an antibody containing the heavy chain and the light chain.
W a fiu-ther improvement on this procedure, a method for identifying a
clinically
relevant epitope on an ixmnunogen, and a correlative method for selecting an
antibody that binds
immunospecifically to the relevant epitope with high affinity, are disclosed
in PCT publication
WO 99/53049.
5. Fab Fragments and Single Chain Antibodies
According to the invention, techniques can be adapted for the production of
single-chain
antibodies specific to an antigenic protein of the invention (see e.g., U.S.
Patent No. 4,946,778).
In addition, methods can be adapted for the construction of Fab expression
libraries (see e.g.,
Huse, et al., 1989 Science 246: 1275-1281) to allow rapid and effective
identification of
monoclonal Fab fragments with the desired specificity for a protein or
derivatives, fragments,
analogs or homologs thereof. Antibody fragments that contain the idiotypes to
a protein antigen
may be produced by techniques known in the art including, but not limited to:
(i) an F(ab~72
fragment produced by pepsin digestion of an antibody molecule; (ii) an Fab
fragment generated
by reducing the disulfide bridges of an F~ab')2 fragment; (iii) an Fab
fragment generated by the
treatment of the antibody molecule with papain arid a reducing agent and (iv)
F,, fragments.
6. Bispecific Antibodies
Bispecific antibodies are monoclonal, preferably human or humanized,
antibodies that
have binding specificities for at least two different antigens. In the present
case, one of the
binding specificities is for an antigenic protein of the invention. The second
binding target is
any other antigen, and advantageously is a cell-surface protein or receptor or
receptor subunit.
Methods for making bispecific antibodies are known in the art. Traditionally,
the
recombinant production of bispecific antibodies is based on the co-expression
of two
41
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have
different
specificities (Milstein and Cuello, Nature, 305:537-539 (1983)). Because of
the random
assortment of immunoglobulin heavy and light chains, these hybridomas
(quadromas) produce a
potential mixture of ten different antibody molecules, of which only one has
the correct
bispecific structure. The purification of the correct molecule is usually
accomplished by affinity
chromatography steps. Similar procedures are disclosed in WO 93/08829,
published 13 May
1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).
Antibody variable domains with the desired binding specificities (antibody-
antigen
combining sites) can be fused to immunoglobulin constant domain sequences. The
fusion
preferably is with an irmnunoglobulin heavy-chain constant domain, comprising
at least part of
the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain
constant region
(CH1) containing the site necessary for light-chain binding present in at
least one of the fusions.
DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the
immunoglobulin
light chain, are inserted into separate expression vectors, and are co-
transfected into a suitable
host organism. For further details of generating bispecific antibodies see,
for example, Suresh et
al., Methods in Enzymology, 121:210 (1986).
According to another approach described in WO 96/2701 l, the interface between
a pair
of antibody molecules can be engineered to maximize the percentage of
heterodimers that are
recovered from recombinant cell culture. The preferred interface comprises at
least a part of the
CH3 region of an antibody constant domain. In this method, one or more small
amino acid side
chains from the interface of the first antibody molecule are replaced with
larger side chains (e.g.
tyrosine or tryptophan). Compensatory "cavities" of identical or similar size
to the large side
chains) are created on the interface of the second antibody molecule by
replacing large amino
acid side chains with smaller ones (e.g. alanine or threonine). This provides
a mechanism for
increasing the yield of the heterodimer over other unwanted end-products such
as homodimers.
Bispecific antibodies can be prepared as full length antibodies or antibody
fragments
(e.g. F(ab')2 bispecific antibodies). Techniques for generating bispecific
antibodies from
antibody fragments have been described in the literature. For example,
bispecific antibodies can
be prepared using chemical linkage. Brennan et al., Science 229:81 (1985)
describe a procedure
wherein intact antibodies are proteolytically cleaved to generate F(ab')Z
fragments. These
fragments are reduced in the presence of the dithiol complexing agent sodium
arsenite to
stabilize vicinal dithiols and prevent intermolecular disulfide formation. The
Fab' fragments
generated are then converted to thionitrobenzoate (TNB) derivatives. One of
the Fab'-TNB
derivatives is then reconverted to the Fab'-thiol by reduction with
mercaptoethylamine and is
mixed with an equimolar amount of the other Fab'-TNB derivative to form the
bispecific
42
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
antibody. The bispecific antibodies produced can be used as agents for the
selective
immobilization of enzymes.
Additionally, Fab' fragments can be directly recovered from E. coli and
chemically
coupled to form bispecific antibodies. Shahaby et al., J. Exp. Med. 175:217-
225 (1992) describe
the production of a fully humanized bispecific antibody F(ab')2 molecule. Each
Fab' fragment
was separately secreted from E. coli and subjected to directed chemical
coupling in vitro to form
the bispecific antibody. The bispecific antibody thus formed was able to bind
to cells
overexpressing the ErbB2 receptor and normal human T cells, as well as trigger
the hytic activity
of human cytotoxic lymphocytes against human breast tumor targets.
Various techniques for malting and isolating bispecific antibody fragments
directly from
recombinant cell culture have also been described. For example, bispecific
antibodies have been
produced using leucine zippers. I~ostelny et al., J. Immunol. 148(5):1547-1553
(1992). The
leucine zipper peptides from the Fos and Jun proteins were linked to the Fab'
portions of two
different antibodies by gene fusion. The antibody homodimers were reduced at
the hinge region
to form monomers and then re-oxidized to form the antibody heterodimers. This
method can
also be utilized for the production of antibody homodimers. The "diabody"
technology
described by Hollinger et a1., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)
has provided an
alternative mechanism for making bispecific antibody fragments. The fragments
comprise a
heavy-chain variable domain (VH) connected to a light-chain variable domain
(VL) by a hinlcer
which is too short to allow pairing between the two domains on the same chain.
Accordingly,
the VH and VL domains of one fragment are forced to pair with the
complementary VL and VH
domains of another fragment, thereby forming two antigen-binding sites.
Another strategy for
making bispecific antibody fragments by the use of single-chain Fv (sFv)
dimers has also been
reported. See, Gruber et al., J. Immunol. 152:5368 (1994).
Antibodies with more than two valencies are contemplated. For example,
trispecific
antibodies can be prepared. Tutt et al., J. Immunoh. 147:60 (1991).
Exemplary bispecific antibodies can bind to two different epitopes, at least
one of which
originates in the protein antigen of the invention. Alternatively, an anti-
antigenic ann of an
immunoghobulin molecule can be combined with an arm which binds to a
triggering molecule
on a leukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, or
B7), or Fc receptors
for IgG (FcyR), such as FcyRI (CD64), FcyRII (CD32) and FcyRIII (CD16) so as
to focus
cellular defense mechanisms to the cell expressing the particular antigen.
Bispecific antibodies
can also be used to direct cytotoxic agents to cells that express a particular
antigen. These
antibodies possess an antigen-binding arm and an arm that binds a cytotoxic
agent or a
43
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific
antibody
of interest binds the protein antigen described herein and further binds
tissue factor (TF).
7. Heteroconiu~ate Antibodies
Heteroconjugate antibodies are also within the scope of the present invention.
Heteroconjugate antibodies are composed of two covalerltly joined antibodies.
Such antibodies
have, for example, been proposed to target immune system cells to iulwanted
cells (U.S. Patent
No. 4,676,980), and for treatment of HIV infection (WO 91/00360; WO 92/200373;
EP 03089).
It is contemplated that the antibodies can be prepared ih vitro using known
methods in synthetic
protein chemistry, including those involving crosslinking agents. For example,
immunotoxins
can be constructed using a disulfide exchange reaction or by forming a
thioether bond.
Examples of suitable reagents for this purpose include iminothiolate and
methyl-4-
mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No.
4,676,980.
8. Effector Function Engineering
It can be desirable to modify the antibody of the invention with respect to
effector
function, so as to enhance, e.g., the effectiveness of the antibody in
treating cancer. For
example, cysteine residues) can be introduced into the Fc region, thereby
allowing interchain
disulfide bond formation in this region. The homodimeric antibody thus
generated can have
improved internalization capability and/or increased complement-mediated cell
lcilling and
antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp
Med., 176: 1191-
' 1195 (1992) and Shopes, J. Itnmunol., 148: 2918-2922 (1992). Homodimeric
antibodies with
enhanced anti-tumor activity can also be prepared using heterobifunctional
cross-linkers as
described in Wolff et. al. Cancer Research, 53: 2560-2565 (1993).
Alternatively, an antibody can
be engineered that has dual Fc regions and can thereby have enhanced
complement lysis and
ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230
(1989).
9. Immunoco~ugates
The invention also pertains to immunoconjugates comprising an antibody
conjugated to a
cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an
enzymatically active toxin of
bacterial, fungal, plant, or animal origin, or fragments thereof), or a
radioactive isotope (i.e., a
radioconjugate).
Chemotherapeutic agents useful in the generation of such immunoconjugates have
been
described above. Enzymatically active toxins and fragments thereof that can be
used include
diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin
A chain (from
Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-
sarcin,
Aleurites fordii proteins, dianthin proteins, Phytolaca anaericana proteins
(PAPI, PAPA, and
PAP-S), momordica charantia inhibitor, curcin, croon, sapaonaria officinalis
inhibitor, gelonin,
44
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A
variety of
radionuclides are available for the production of radioconjugated antibodies.
Examples include
zizBi~ isih isy~ soy and ls6Re.
Conjugates of the antibody and cytotoxic agent are made using a variety of
bifunctional
protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate
(SPDP),
iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl
adipimidate HCL),
active esters (such as disuccinimidyl suberate), aldehydes (such as
glutareldehyde), bis-azido
compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as
bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-
diisocyanate),
and bis-active fluorine compounds (such as 1,5-difluoro-2,4-diW trobenzene).
For example, a
ricin immunotoxin can be prepared as described in Vitetta et al., Science,
238: 1098 (1987).
Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene
triaminepentaacetic acid (MX-
DTPA) is an exemplary chelating agent for conjugation of radionucleotide to
the antibody. See
W094/11026.
In another embodiment, the antibody can be conjugated to a "receptor" (such as
streptavidin) for utilization in tumor pretargeting wherein the antibody-
receptor conjugate is
administered to the patient, followed by removal of unbound conjugate from the
circulation
using a clearing agent and then administration of a "ligand" (e.g., avidin)
that is in turn
conjugated to a cytotoxic agent.
10. Iminunoliposomes
The antibodies disclosed herein can also be formulated as immunoliposomes.
Liposomes containing the antibody are prepared by methods known in the art,
such as described
in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al.,
Proc. Natl Acad.
Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.
Liposomes with
enhanced circulation time are disclosed in U.S. Patent No. 5,013,556.
Particularly useful liposomes can be generated by the reverse-phase
evaporation method
with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-
derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of
defined pore
size to yield liposomes with the desired diameter. Fab' fragments of the
antibody of the present
invention can be conjugated to the liposomes as described in Martin et al., J.
Biol. Chem., 257:
286-288 (1982) via a disulfide-interchange reaction. A chemotherapeutic agent
(such as
Doxorubicin) is optionally contained within the liposome. See Gabizon et al.,
J. National
Cancer Inst., 81(19): 1484 (1989).
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
11. Dia~ostic Applications of Antibodies Directed Against the Proteins of the
Invention
Antibodies directed against a protein of the invention may be used in methods
known
within the art relating to the localization and/or quantitation of the protein
(e.g., for use in
measuring levels of the protein within appropriate physiological samples, for
use in diagnostic
methods, for use in imaging the protein, and the like). In a given embodiment,
antibodies
against the proteins, or derivatives, fragments, analogs or homologs thereof,
that contain the
antigen-binding domain, are utilized as pharmacologically-active compounds
(see below).
An antibody specific for a protein of the invention can be used to isolate the
protein by
standard techniques, such as immunoaffinity chromatography or
immunoprecipitation. Such an
antibody can facilitate the purification of the natural protein antigen from
cells and of
recombinantly produced antigen expressed in host cells. Moreover, such an
antibody can be
used to detect the antigenic protein (e.g., in a cellular lysate or cell
supernatant) in order to
evaluate the abundance and pattern of expression of the antigenic protein.
Antibodies directed
against the protein can be used diagnostically to monitor protein levels in
tissue as part of a
clinical testing procedure, e.g., to, for example, determine the efficacy of a
given treatment
regimen. Detection can be facilitated by coupling (i.e., physically linking)
the antibody to a
detectable substance. Examples of detectable substances include various
enzymes, prosthetic
groups, fluorescent materials, luminescent materials, bioluminescent
materials, and radioactive
materials. Examples of suitable enzymes include horseradish peroxidase,
alkaline phosphatase,
(3-galactosidase, or acetylcholinesterase; examples of suitable prosthetic
group complexes
include streptavidin/biotin and avidin/biotin; examples of suitable
fluorescent materials include
umbelliferone, fluorescein, fluorescein isotluocyanate, rhodamine,
dichlorotriazinylasnine
fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent
material includes
luminol; examples of bioluminescent materials include luciferase, luciferin,
and aequorin, and
examples of suitable radioactive material include lash isih 3sS or 3H.
12. Antibody Therapeutics
Antibodies of the invention, including polyclonal, monoclonal, humanized and
fully
human antibodies, may used as therapeutic agents. Such agents will generally
be employed to
treat or prevent a disease or pathology in a subject. An antibody preparation,
preferably one
having high specificity and high affinity for its target antigen, is
admiiustered to the subject and
will generally have an effect due to its binding with~the target. Such an
effect may be one of two
lcinds, depending on the specific nature of the interaction between the given
antibody molecule
and the target antigen in question. In the first instance, administration of
the antibody may
abrogate or inhibit the binding of the target with an endogenous ligand to
which it naturally
binds. In this case, the antibody binds to the target and masks a binding site
of the naturally
46
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
occurring ligand, wherein the ligand serves as an effector molecule. Thus the
receptor mediates
a signal transduction pathway for which ligand is responsible.
Alternatively, the effect may be one in which the antibody elicits a
physiological result
by virtue of binding to an effector binding site on the target molecule. In
this case the target, a
receptor having an endogenous ligand that may be absent or defective in the
disease or
pathology, binds the antibody as a surrogate effector ligand, initiating a
receptor-based signal
transduction event by the receptor.
A therapeutically effective amount of an antibody of the invention relates
generally to
the amount needed to achieve a therapeutic objective. As noted above, this may
be a binding
interaction between the antibody and its target antigen that, in certain
cases, interferes with the
functioning of the target, and in other cases, promotes a physiological
response. The amount
required to be administered will furthermore depend on the binding affinity of
the antibody for
its specific antigen, and will also depend on the rate at ,which an
administered antibody is
depleted from the free volume other subject to which it is administered.
Common ranges for
therapeutically effective dosing of an antibody or antibody fragment of the
invention may be, by
way of nonlimiting example, from about 0.1 mg/kg body weight to about 50 mg/kg
body weight.
Common dosing frequencies may range, for example, from twice daily to once a
week.
13. Pharmaceutical Compositions of Antibodies
Antibodies specifically binding a protein of the invention, as well as other
molecules
identified by the screening assays disclosed herein, can be administered for
the treatment of
various disorders in the form of pharmaceutical compositions. Principles and
considerations
involved in preparing such compositions, as well as guidance in the choice of
components are
provided, for example, in Remington: The Science And Practice Of Pharmacy 19th
ed. (Alfonso
R. Gennaro, et al., editors) Mack Pub. Co., Easton, Pa.: 1995; Drug Absorption
Enhancement
Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers,
Langhorne,
Pa., 1994; and Peptide And Protein Drug Delivery (Advances hl Parenteral
Sciences, Vol. 4),
1991, M. Dekker, New York.
If the antigenic protein is intracellular and whole antibodies are used as
inhibitors,
internalizing antibodies are preferred. However, liposomes can also be used to
deliver the
antibody, or an antibody fragment, into cells. Where antibody fragments are
used, the smallest
inhibitory fragment that specifically binds to the binding domain of the
target protein is
preferred. For example, based upon the variable-region sequences of an
antibody, peptide
molecules can be designed that retain the ability to bind the target protein
sequence. Such
peptides can be synthesized chemically and/or produced by recombinant DNA
technology. See,
e.g., Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993). The
formulation herein
47
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
can also contain more than one active compound as necessary for the particular
indication being
treated, preferably those with complementary activities that do not adversely
affect each other.
Alternatively, or in addition, the composition can comprise an agent that
enhances its function,
such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or
growth-inhibitory
agent. Such molecules are suitably present in combination in amounts that are
effective for the
purpose intended.
The active ingredients can also be entrapped in microcapsules prepared, for
example, by
coacervation techniques or by interfacial pol5nnerization, for example,
hydroxymethylcellulose
or gelatin-microcapsules and poly-(methylinethacrylate) microcapsules,
respectively, in
colloidal drug delivery systems (for example, liposomes, albumin microspheres,
microemulsions, nano-particles, and nanocapsules) or in macroemulsions.
The formulations to be used for in vivo admiustration are highly preferred to
be sterile.
This is readily accomplished by filtration through sterile filtration
membranes.
Sustained-release preparations can be prepared. Suitable examples of sustained-
release
preparations include semipermeable matrices of solid hydrophobic polymers
containing the
antibody, which matrices are in the form of shaped articles, e.g., films, or
inicrocapsules.
Examples of sustained-release matrices include polyesters, hydrogels (for
example, poly(2-
hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat.
No. 3,773,919),
copolymers of L-glutamic acid and y ethyl-L-glutamate, non-degradable ethylene-
vinyl acetate,
degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT TM
(injectable
microspheres composed of lactic acid-glycolic acid copolymer and leuprolide
acetate), and poly-
D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and
lactic acid-
glycolic acid enable release of molecules for over 100 days, certain hydrogels
release proteins
for shorter time periods.
gl2L Recombinant Expression Vectors and Host Cells
Another aspect of the invention pertains to vectors, preferably expression
vectors,
containing a nucleic acid encoding a gl2L protein, or derivatives, fragments,
analogs or
homologs thereof. As used herein, the term "vector" refers to a nucleic acid
molecule capable of
transporting another nucleic acid to which it has been linked. One type of
vector is a "plasmid",
which refers to a circular double stranded DNA loop into which additional DNA
segments can
be ligated. Another type of vector is a viral vector, wherein additional DNA
segments can be
ligated into the viral genome. Certain vectors are capable of autonomous
replication in a host
cell into which they are introduced (e.g., bacterial vectors having a
bacterial origin of replication
and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian
vectors) axe
48
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
integrated into the genome of a host cell upon introduction into the host
cell, and thereby are
replicated along with the host genome. Moreover, certain vectors are capable
of directing the
expression of genes to which they are operatively-linked. Such vectors are
referred to herein as
"expression vectors". W general, expression vectors of utility in recombinant
DNA techniques
are often in the form of plasmids. In the present specification, "plasmid" and
"vector" can be
used interchangeably as the plasmid is the most commonly used form of vector.
However, the
invention is intended to include such other forms of expression vectors, such
as viral vectors
(e.g., replication defective retroviruses, adenoviruses and adeno-associated
viruses), which serve
equivalent functions.
The recombinant expression vectors of the invention comprise a nucleic acid of
the
invention in a form suitable for expression of the nucleic acid in a host
cell, which means that
the recombinant expression vectors include one or more regulatory sequences,
selected on the
basis of the host cells to be used for expression, that is operatively-linked
to the nucleic acid
sequence to be expressed. Within a recombinant expression vector, "operably-
linked" is
intended to mean that the nucleotide sequence of interest is linked to the
regulatory sequences)
in a manner that allows for expression of the nucleotide sequence (e.g., in an
ih uit~o
transcription/translation system or in a host cell when the vector is
introduced into the host cell).
The term "regulatory sequence" is intended to include promoters, enhancers and
other
expression control elements (e.g., polyadenylation signals). Such regulatory
sequences are
described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN
ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences
include
those that direct constitutive expression of a nucleotide sequence in many
types of host cell and
those that direct expression of the nucleotide sequence only in certain host
cells (e.g.,
tissue-specific regulatory sequences). It will be appreciated by those skilled
in the art that the
design of the expression vector can depend on such factors as the choice of
the host cell to be
transformed, the level of expression of protein desired, etc. The expression
vectors of the
invention can be introduced into host cells to thereby produce proteins or
peptides, including
fusion proteins or peptides, encoded by nucleic acids as described herein
(e.g., gl2L proteins,
mutant forms of gl2L proteins, fusion proteins, etc.).
The recombinant expression vectors of the invention can be designed for
expression of
gl2L proteins in prolcaryotic or eukaryotic cells. For example, gl2L proteins
can be expressed
in bacterial cells such as Escherichia coli, insect cells (using baculovirus
expression vectors)
yeast cells or mammalian cells. Suitable host cells are discussed further in
Goeddel, GENE
EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego,
Calif.
49
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
(1990). Alternatively, the recombinant expression vector can be transcribed
and translated izz
vitYO, for example using T7 promoter regulatory sequences and T7 polymerase.
Expression of proteins in prokaryotes is most often carried out in Esche~ichia
coli with
vectors containing constitutive or inducible promoters directing the
expression of either fusion
or non-fusion proteins. Fusion vectors add a number of amino acids to a
protein encoded
therein, usually to the amino terminus of the recombinant protein. Such fusion
vectors typically
serve three purposes: (i) to increase expression of recombinant protein; (ii)
to increase the
solubility of the recombinant protein; and (iii) to aid in the purification of
the recombinant
protein by acting as a ligand in affinity purification. Often, in fusion
expression vectors, a
proteolytic cleavage site is introduced at the junction of the fusion moiety
and the recombinant
protein to enable separation of the recombinant protein from the fusion moiety
subsequent to
purification of the fusion protein. Such enzymes, and their cognate
recognition sequences,
include Factor Xa, thrombin and enterokinase. Typical fusion expression
vectors include pGEX
(Pharmacia Biotech Inc; Smith and Johnson, 1988. Gehe 67: 31-40), pMAL (New
England
Biolabs, Beverly, Mass.) and pRITS (Phannacia, Piscataway, N.J.) that fuse
glutathione S-
transferase (GST), maltose E binding protein, or protein A, respectively, to
the target
recombinant protein.
Examples of suitable inducible non-fusion E. coli expression vectors include
pTrc
(Amrann et al., (1988) Gene 69:301-315) and pET l 1d (Studier et al., GENE
EXPRESSION
TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif.
(1990)
60-89).
One strategy to maximize recombinant protein expression in E. coli is to
express the
protein in a host bacteria with an impaired capacity to proteolytically cleave
the recombinant
protein. See, e.g., Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN
ENZYMOLOGY
185, Academic Press, San Diego, Calif. (1990) 119-128. Another strategy is to
alter the nucleic
acid sequence of the nucleic acid to be inserted into an expression vector so
that the individual
codons for each amino acid are those preferentially utilized in E. coli (see,
e.g., Wada, et al.,
1992. Nucl. Acids Res. 20: 2111-2118). Such alteration of nucleic acid
sequences of the
invention can be carried out by standard DNA synthesis techniques.
In another embodiment, the gl2L expression vector is a yeast expression
vector.
Examples of vectors for expression in yeast Sacchaz-onzyces cerivisae include
pYepSecl
(Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz,
1982. Cell 30:
933-943), pJRY88 (Schultz et al., 1987. Gefze 54: 113-123), pYES2 (Invitrogen
Corporation,
San Diego, Calif.), and picZ (InVitrogen Corp, San Diego, Calif.).
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
Alternatively, gl2L can be expressed in insect cells using baculovirus
expression
vectors. Baculovirus vectors available for expression of proteins in cultured
insect cells (e.g.,
SF9 cells) include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3:
2156-2165) and the
pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).
In yet another embodiment, a nucleic acid of the invention is expressed in
mammalian
cells using a mammalian expression vector. Examples of mammalian expression
vectors include
pCDM8 (Seed, 1987. Natuy°e 329: 840) and pMT2PC (I~aufinan, et al.,
1987. EMBO J. 6: 187-
195). When used in mammalian cells, the expression vector's control functions
are often
provided by viral regulatory elements. For example, commonly used promoters
are derived
from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. For other
suitable
expression systems for both prokaryotic and eukaryotic cells see, e.g.,
Chapters 16 and 17 of
Sambroolc, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold
Spring
Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y., 1989.
In another embodiment, the recombinant mammalian expression vector is capable
of
directing expression of the nucleic acid preferentially in a particular cell
type (e.g., tissue-
specific regulatory elements are used to express the nucleic acid). Tissue-
specific regulatory
elements are lrnown in the art. Non-limiting examples of suitable tissue-
specific promoters
include the albumin promoter (liver-specific; Pinlcert, et al., 1987. Genes
Dev. 1: 268-277),
lymphoid-specific promoters (Calame and Eaton, 1988. Adv. Immunol. 43: 235-
275), in
particular promoters of T cell receptors (Winoto and Baltimore, 1989. EMBO J.
8: 729-733) and
immunoglobulins (Banerji, et al., 1983. Cell 33: 729-740; Queen and Baltimore,
1983. Cell 33:
741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne
and Ruddle, 1989.
P~oc. Natl. Acad. Sci. USA 86: 5473-5477), pancreas-specific promoters
(Edlund, et al., 1985.
Science 230: 912-916), and mammary gland-specific promoters (e.g., milk whey
promoter; U.S.
Pat. No. 4,873,316 and European Application Publication No. 264,166).
Developmentally-
regulated promoters are also encompassed, e.g., the marine hox promoters
(I~essel and Grass,
1990. Science 249: 374-379) and the a-fetoprotein promoter (Campes and
Tilghman, 1989.
Genes Dev. 3: 537-546).
The invention further provides a recombinant expression vector comprising a
DNA
molecule of the invention cloned into the expression vector in an antisense
orientation. That is,
the DNA molecule is operatively-liu~ed to a regulatory sequence in a manner
that allows for
expression (by transcription of the DNA molecule) of an RNA molecule that is
antisense to
gl2L mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in
the antisense
orientation can be chosen that direct the continuous expression of the
antisense RNA molecule
in a variety of cell types, for instance viral promoters and/or enhancers, or
regulatory sequences
51
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
can be chosen that direct constitutive, tissue specific or cell type specific
expression of antisense
RNA. The antisense expression vector can be in the form of a recombinant
plasmid, phagemid
or attenuated virus in which antisense nucleic acids are produced under the
control of a high
efficiency regulatory region, the activity of which can be determined by the
cell type into which
the vector is introduced. For a discussion of the regulation of gene
expression using antisense
genes see, e.g., Weintraub, et al., "Antisense RNA as a molecular tool for
genetic analysis,"
Reviews-Treyads iya Genetics, Vol. 1(1) 1986.
Another aspect of the invention pertains to host cells into which a
recombinant
expression vector of the invention has been introduced. The terms "host cell"
and "recombinant
host cell" are used interchangeably herein. It is understood that such terms
refer not only to the
particular subject cell but also to the progeny or potential progeny of such a
cell. Because
certain modifications may occur in succeeding generations due to either
mutation or
enviromnental influences, such progeny may not, in fact, be identical to the
parent cell, but are
still included within the scope of the term as used herein.
A host cell can be any prokaryotic or eukaryotic cell. For example, gl2L
protein can be
expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian
cells (such as
Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are
knov~m to those
spilled in the art.
Vector DNA can be introduced into prokaryotic or eukaryotic cells via
conventional
transformation or transfection techniques. As used herein, the terms
"transformation" and
"transfection" are intended to refer to a variety of art-recognized techniques
for introducing
foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate
or calcium
chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or
electroporation.
Suitable methods for transforming or transfecting host cells can be found in
Sambrook, et al.
(MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor
Laboratory,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and
other laboratory
manuals.
For stable transfection of mammalian cells, it is lcnown that, depending upon
the
expression vector and transfection technique used, only a small fraction of
cells may integrate
the foreign DNA into their genome. In order to identify and select these
integrants, a gene that
encodes a selectable marlcer (e.g., resistance to antibiotics) is generally
introduced into the host
cells along with the gene of interest. Various selectable markers include
those that confer
resistance to drugs, such as 6418, hygromycin and methotrexate. Nucleic acid
encoding a
selectable marker can be introduced into a host cell on the sa~.ne vector as
that encoding gl2L or
can be introduced on a separate vector. Cells stably transfected with the
introduced nucleic acid
52
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
can be identified by drug selection (e.g., cells that have incorporated the
selectable marker gene
will survive, wlule the other cells die).
A host cell of the invention; such as a prokaryotic or eukaryotic host cell in
culture, can
be used to produce (i.e., express) gl2L protein. Accordingly, the invention
further provides
methods for producing gl2L protein using the host cells of the invention. In
one embodiment,
the method comprises culturing the host cell of invention (into which a
recombinant expression
vector encoding gl2L protein has been introduced) in a suitable medium such
that gl2L protein
is produced. In another embodiment, the method further comprises isolating
gl2L protein from
the medium or the host cell.
Transgenic gl2L Animals
The host cells of the invention can also be used to produce non-human
transgenic
animals. For example, in one embodiment, a host cell of the invention is a
fertilized oocyte or
an embryonic stem cell into which gl2L protein-coding sequences have been
introduced. Such
host cells can then be used to create non-human transgenic animals in which
exogenous gl2L
sequences have been introduced into their genome or homologous recombinant
animals in which
endogenous gl2L sequences have been altered. Such aumals are useful for
studying the
function and/or activity of gl2L protein and for identifying and/or evaluating
modulators of
gl2L protein activity. As used herein, a "transgenic animal" is a non-human
animal, preferably
a mammal, more preferably a rodent such as a rat or mouse, in which one or
more of the cells of
the animal includes a transgene. Other examples of transgenic animals include
non-human
primates, sheep, dogs, cows, goats, chickens, amphibians, etc. A transgene is
exogenous DNA
that is integrated into the genome of a cell from which a transgenic animal
develops and that
remains in the genome of the mature animal, thereby directing the expression
of an encoded
gene product in one or more cell types or tissues of the transgenic animal. As
used herein, a
"homologous recombinant animal" is a non-human animal, preferably a mammal,
more
preferably a mouse, in which an endogenous gl2L gene has been altered by
homologous
recombination between the endogenous gene and an exogenous DNA molecule
introduced into a
cell of the aiumal, e.g., an embryonic cell of the animal, prior to
development of the animal.
A transgenic animal of the invention can be created by introducing gl2L-
encoding
nucleic acid into the male pronuclei of a fertilized oocyte (e.g., by
microinjection, retroviral
infection) and allowing the oocyte to develop in a pseudopregnant female
foster animal. The
human gl2L cDNA sequences of SEQ m NO:1, can be introduced as a transgene into
the
genome of a non-human animal. Alternatively, a non-human homologue of the
human gl2L
gene, such as a mouse gl2L gene (SEQ m N0:3), can be used as a transgene.
Intronic
53
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
sequences and polyadenylation signals can also be included in the transgene to
increase the
efficiency of expression of the transgene. A tissue-specific regulatory
sequences) can be
operably-linked to the gl2L transgene to direct expression of gl2L protein to
particular cells.
Methods for generating transgenic animals via embryo manipulation and microinj
ection,
particularly animals such as mice, have become conventional in the art and are
described, for
example, in U.S. Patent Nos. 4,736,866; 4,870,009; and 4,873,191; and Hogan,
1986. In:
MANIPULATING THE MousE EMBRYO, Cold Spring Harbor Laboratory Press, Cold
Spring
Harbor, N.Y. Similar methods are used for production of other transgenic
animals. A
transgenic founder animal can be identified based upon the presence of the
gl2L transgene in its
genome and/or expression of gl2L mRNA in tissues or cells of the animals. A
transgenic
founder animal can then be used to breed additional animals carrying the
transgene. Moreover,
transgenic animals carrying a transgene-encoding gl2L protein can further be
bred to other
transgenic animals carrying other transgenes.
To create a homologous recombinant animal, a vector is prepared which contains
at least
a portion of a gl2L gene into which a deletion, addition or substitution has
been introduced to
thereby alter, e.g., functionally disrupt, the gl2L gene. The gl2L gene can be
a human gene
(e.g., the cDNA of SEQ m NO:1), but more preferably, is a non-human homologue
of a human
gl2L gene. For example, a mouse homologue (SEQ m NO:3) can be used to
construct a
homologous recombination vector suitable for altering an endogenous gl2L gene
in the mouse
genome. In one embodiment, the vector is designed such that, upon homologous
recombination,
the endogenous gl2L gene is functionally disrupted (i.e., no longer encodes a
functional protein;
also referred to as a "lmoclc out" vector).
Alternatively, the vector can be designed such that, upon homologous
recombination, the
endogenous gl2L gene is mutated or otherwise altered but still encodes
functional protein (e.g.,
the upstream regulatory region can be altered to thereby alter the expression
of the endogenous
gl2L protein). In the homologous recombination vector, the altered portion of
the gl2L gene is
flanked at its 5'- and ~3'-termini by additional nucleic acid of the gl2L gene
to allow for
homologous recombination to occur between the exogenous gl2L gene carned by
the vector and
an endogenous gl2L gene in an embryonic stem cell. The additional flanlcing
gl2L nucleic acid
is of sufficient length for successful homologous recombination with the
endogenous gene.
Typically, several kilobases of flanking DNA (both at the 5'- and 3'-termini)
are included in the
vector. See, e.g., Thomas, et al., 1987. Cell 51: 503 for a description of
homologous
recombination vectors. The vector is ten introduced into an embryonic stem
cell line (e.g., by
electroporation) and cells in which the introduced gl2L gene has homologously-
recombined
with the endogenous gl2L gene are selected. See, e.g., Li, et al., 1992. Cell
69: 915.
54
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
The selected cells are then injected into a blastocyst of an animal (e.g., a
mouse) to form
aggregation chimeras. See, e.g., Bradley, 1987. I11: TERATOCARCINOMAS AND
EMBRYONIC STEM
CELLS: A PRACTICAL APPROACH, Robertson, ed. IRL, Oxford, pp. 113-152. A
chimeric embryo
can then be implanted into a suitable pseudopregnant female foster animal and
the embryo
brought to term. Progeny harboring the homologously-recombined DNA in their
germ cells can
be used to breed animals in which all cells of the animal contain the
homologously-recombined
DNA by germline transmission of the transgene. Methods for constructing
homologous
recombination vectors and homologous recombinant animals are described further
in Bradley,
1991. Curt. Opin. Biotechnol. 2: 823-829; PCT International Publication Nos.:
WO 90/11354;
WO 91/01140; WO 92/0968; and WO 93/04169.
In another embodiment, transgenic non-humans animals can be produced that
contain
selected systems that allow for regulated expression of the transgene. One
example of such a
system is the cre/loxP recombinase system of bacteriophage P1. For a
description of the
cre/loxP recombinase system, See, e.g., Lakso, et al., 1992. P~~oc. Natl.
Acad. Sci. USA 89:
6232-6236. Another example of a recombinase system is the FLP recombinase
system of
Saccha~ofnyces ceYevisiae. See, O'Gorman, et al., 1991. Scieyace 251:1351-
1355. If a cre/loxP
recombinase system is used to regulate expression of the transgene, animals
containing
transgenes encoding both the Cre recombinase and a selected protein are
required. Such animals
can be provided through the construction of "double" transgenic animals, e.g.,
by mating two
transgenic animals, one containing a transgene encoding a selected protein and
the other
containing a transgene encoding a recombinase.
Clones of the non-human transgenic animals described herein can also be
produced
according to the methods described in Wilmut, et al., 1997. Natuy-e 385: 810-
813. In brief, a cell
(e.g., a somatic cell) from the transgenic animal can be isolated and induced
to exit the growth
cycle and enter Go phase. The quiescent cell can then be fused, e.g., through
the use of electrical
pulses, to an enucleated oocyte from an animal of the same species from which
the quiescent
cell is isolated. The reconstructed oocyte is then cultured such that it
develops to morula or
blastocyte and then transferred to pseudopregnant female foster animal. The
offspring borne of
this female foster animal will be a clone of the animal from which the cell
(e.g., the somatic cell)
is isolated.
Pharmaceutical Compositions
The gl2L nucleic acid molecules, gl2L proteins, and anti-gl2L antibodies (also
referred
to herein as "active compounds") of the invention, and derivatives, fragments,
analogs and
homologs thereof, can be incorporated into pharmaceutical compositions
suitable for
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
administration. Such compositions typically comprise the nucleic acid
molecule, protein, or
antibody and a pharmaceutically acceptable carrier. As used herein,
"pharmaceutically
acceptable carrier" 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. Preferred examples of such carriers
or diluents
include, but are not limited to, water, saline, finger's solutions, dextrose
solution, and 5% human
senun albumin. 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.
A pharmaceutical composition of the invention is formulated to be compatible
with its
intended route of administration. Examples of routes of administration include
parenteral, e.g.,
intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdennal
(i.e., topical),
transmucosal, and rectal administration. Solutions or suspensions used for
parenteral,
intradermal, or subcutaneous application can 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 bisulfate; 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.
The parenteral
preparation can be enclosed in ampoules, disposable syringes or multiple dose
vials made of
glass or plastic.
Pharmaceutical compositions suitable for injectable use include sterile
aqueous solutions
(where water soluble) or dispersions and sterile powders for the
extemporaneous preparation of
sterile injectable solutions or dispersion. For intravenous administration,
suitable carriers
include physiological saline, bacteriostatic water, Cremophor ELTM (BASF,
Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must be sterile
and should be
fluid to the extent that easy syringeability exists. It must be stable under
the conditions of
manufacture and storage and must be preserved against the contaminating action
of
microorganisms such as bacteria and fungi. The carrier can be a solvent or
dispersion medium
containing, for example, water, ethanol, polyol (for example, glycerol,
propylene glycol, and
56
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
liquid polyethylene glycol, and the like), and suitable mixtures thereof. The
proper fluidity can
be maintained, for example, by the use of a coating such as lecithin, by the
maintenance of the
required particle size in the case of dispersion and by the use of
surfactants. Prevention of the
action of microorganisms can be achieved by various antibacterial and
antifungal agents, for
example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the
like. In many
cases, it will be preferable to include isotonic agents, for example, sugars,
polyalcohols such as
manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of
the injectable
compositions can be brought about by including in the composition an agent
wluch delays
absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active
compound (e.g., a
gl2L protein or anti-gl2L antibody) in the required amount in an appropriate
solvent with one
or a combination of ingredients enumerated above, as required, followed by
filtered sterilization.
Generally, dispersions are prepared by incorporating the active compound into
a sterile vehicle
that contains a basic dispersion medium and the required other ingredients
from those
enumerated above. In the case of sterile powders for the preparation of
sterile injectable
solutions, methods of preparation are vacuum drying and freeze-drying that
yields a powder of
the active ingredient plus any additional desired ingredient from a previously
sterile-filtered
solution thereof.
Oral compositions generally include an inert diluent or an edible Garner. They
can be
enclosed in gelatin capsules or compressed into tablets. For the purpose of
oral therapeutic
administration, the active compound can be incorporated with excipients and
used in the form of
tablets, troches, or capsules. Oral compositions can also be prepared using a
fluid Garner for use
as a mouthwash, wherein the compound in the fluid carrier is applied orally
and swished and
expectorated or swallowed. Pharmaceutically compatible binding agents, and/or
adjuvant
materials can be included as part of the composition. The tablets, pills,
capsules, troches and the
like can contain any of the following ingredients, or compounds of a similar
nature: a binder
such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient
such as starch or
lactose, a disintegrating agent such as alginic acid, Primogel, or corn
starch; a lubricant such as
magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a
sweetening agent
such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl
salicylate, or
orange flavoring.
For administration by inhalation, the compounds are delivered in the form of
an aerosol
spray from pressured container or dispenser that contains a suitable
propellant, e.g., a gas such
as carbon dioxide, or a nebulizer.
57
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
Systemic admiW stration can also be by transmucosal or transdermal means. For
transmucosal or transdermal administration, penetrants appropriate to the
barrier to be permeated
are used in the formulation. Such penetrants are generally known in the art,
and include, for
example, for transmucosal administration, detergents, bile salts, and fusidic
acid derivatives.
Transmucosal administration can be accomplished through the use of nasal
spxays or
suppositories. For transdermal administration, the active compounds are
formulated into
ointments, salves, gels, or creams as generally known in the art.
The compounds can also be prepared in the form of suppositories (e.g., with
conventional suppository bases such as cocoa butter and other glycerides) or
retention enemas
for rectal delivery.
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) can also be
used as pharmaceutically acceptable carriers. These can be prepared according
to methods
lcnown to those skilled in the art, for example, as described in U.S. Patent
No. 4,522,811.
It is especially advantageous to formulate oral or parenteral 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 Garner. 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 nucleic acid molecules of the invention can be inserted into vectors and
used as gene
therapy vectors. Gene therapy vectors can be delivered to a subject by, for
example, intravenous
injection, local administration (see, e.g., U.S. Patent No. 5,328,470) or by
stereotactic injection
(see, e.g., Chen, et al., 1994. P~oc. Natl. Acad. Sci. USA 91: 3054-3057). The
pharmaceutical
preparation of the gene therapy vector can include the gene therapy vector in
an acceptable
diluent, or can comprise a slow release matrix in which the gene delivery
vehicle is imbedded.
58
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
Alternatively, where the complete gene delivery vector can be produced intact
from recombinant
cells, e.g., retroviral vectors, the pharmaceutical preparation can include
one or more cells that
produce the gene delivery system.
The pharmaceutical compositions can be included in a container, pack, or
dispenser
together with instructions for administration.
Screening and Detection Methods
The isolated nucleic acid molecules of the invention can be used to express
gl2L protein
(e.g., via a recombinant expression vector in a host cell in gene therapy
applications), to detect
gl2L mRNA (e.g., in a biological sample) or a genetic lesion in a gl2L gene,
and to modulate
gl2L activity, as described further, below. In addition, the gl2L proteins can
be used to screen
drugs or compounds that modulate the gl2L protein activity or expression as
well as to treat
disorders characterized by insufficient or excessive production of gl2L
protein or production of
gl2L protein forms that have decreased or aberrant activity compared to gl2L
wild-type protein
(e.g.; metabolic disorders and diseases, such as obesity and obesity-related
disorders, diabetes,
and cachexia; cell proliferative disorders and diseases, such as hyperplasia,
cancer, and
restenosis; neurodegenerative disorders and diseases, such as Alzheimer's
Disease and
Parl~inson's Disorder; immmle disorders and diseases, such as AIDS,
inflammation, and
autoimmune diseases; and hematopoietic disorders and diseases, such as SCID,
cyclic
neutropenia, and thrombocythemia). In addition, the anti-gl2L antibodies of
the invention can
be used to detect and isolate gl2L proteins and modulate gl2L activity.
The invention further pertains to novel agents identified by the screening
assays
described herein and uses thereof for treatments as described, supra.
Screehihg Assays
The invention provides a method (also referred to herein as a "screening
assay") for
identifying modulators, i.e., candidate or test compounds or agents (e.g.,
peptides,
peptidomimetics, small molecules or other drugs) that bind to gl2L proteins or
have a
stimulatory or inhibitory effect on, e.g., gl2L protein expression or gl2L
protein activity. The
invention also includes compounds identified in the screening assays described
herein.
In one embodiment, the invention provides assays for screening candidate or
test
compounds that bind to or modulate the activity of the membrane-bound form of
a gl2L protein
or polypeptide or biologically-active portion thereof. The test compounds of
the invention can
be obtained using any of the numerous approaches in combinatorial library
methods known in
the art, including: biological libraries; spatially addressable parallel solid
phase or solution phase
59
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
libraries; synthetic library methods requiring deconvolution; the "one-bead
one-compound"
library method; and synthetic library methods using affinity chromatography
selection. The
biological library approach is limited to peptide libraries, while the other
four approaches are
applicable to peptide, non-peptide oligomer or small molecule libraries of
compounds. See, e.g.,
Lam, 1997. Asztica>zce>" Drug Design 12: 145.
A "small molecule" as used herein, is meant to refer to a composition that has
a
molecular weight of less than about 5 kD and most preferably less than about 4
kD. Small
molecules can be, e.g., nucleic acids, peptides, polypeptides,
peptidomimetics, carbohydrates,
lipids or other organic or inorganic molecules. Libraries of chemical and/or
biological mixtures,
such as fungal, bacterial, or algal extracts, are known in the art and can be
screened with any of
the assays of the invention.
Examples of methods for the synthesis of molecular libraries can be found in
the art, for
example in: DeWitt, et al., 1993. Proc. Natl. Acad. Sci. U.S.A. 90: 6909; Erb,
et al., 1994. P~oc.
Natl. Acad. Sci. U.S.A. 91: 11422; Zuckennann, et al., 1994. J. Med. Chenz.
37: 2678; Cho, et
al., 1993. Science 261: 1303; Carrell, et al., 1994. Ayzgew. Chem. Ih.t. Ed.
Engl. 33: 2059; Carell,
et al., 1994. Ahgew. Chem. Int. Ed. Eyzgl. 33: 2061; and Gallop, et al., 1994.
J. Med. Clzem. 37:
1233.
Libraries of compounds may be presented in solution (e.g., Houghten, 1992.
Biotechyaiques 13: 412-421), or on beads (Lam, 1991. NatuYe 354: 82-84), on
chips (Fodor,
1993. Natuf°e 364: 555-556), bacteria (Ladner, U.S. Patent No.
5,223,409), spores (Ladner, U.S.
Patent 5,233,409), plasmids (Cull, et al., 1992. P~oc. Natl. Acad. Sci. LISA
89: 1865-1869) or on
phage (Scott and Smith, 1990. Science 249: 386-390; Devlin, 1990. Science 249:
404-406;
Cwirla, et al., 1990. P~oc. Natl. Acad. Sci. U.S.A. 87: 6378-6382; Felici,
1991. J. Mol. Biol. 222:
301-310; Ladner, U.S. Patent No. 5,233,409.).
In one embodiment, an assay is a cell-based assay in which a cell which
expresses a
membrane-bound form of gl2L protein, or a biologically-active portion thereof,
on the cell
surface is contacted with a test compound and the ability of the test compound
to bind to an
gl2L protein determined. The cell, for example; can of mammalian origin or a
yeast cell.
Determining the ability of the test compound to bind to the gl2L protein can
be accomplished,
for example, by coupling the test compound with a radioisotope or enzymatic
label such that
binding of the test compound to the gl2L protein or biologically-active
portion thereof can be
determined by detecting the labeled compound in a complex. For example, test
compounds can
be labeled with lash 3sS, 14C, or 3H, either directly or indirectly, and the
radioisotope detected by
direct counting of radioemission or by scintillation counting. Alternatively,
test compounds can
be enzymatically-labeled with, for example, horseradish peroxidase, alkaline
phosphatase, or
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
luciferase, and the enzymatic label detected by determination of conversion of
an appropriate
substrate to product. In one embodiment, the assay comprises contacting a cell
which expresses
a membrane-bound form of gl2L protein, or a biologically-active portion
thereof, on the cell
surface with a known compound which binds gl2L to form an assay mixture,
contacting the
assay mixture with a test compound, and determining the ability of the test
compound to interact
with a gl2L protein, wherein determining the ability of the test compound to
interact with a
gl2L protein comprises determining the ability of the test compound to
preferentially bind to
gl2L protein or a biologically-active portion thereof as compared to the known
compound.
In another embodiment, an assay is a cell-based assay comprising contacting a
cell
expressing a membrane-bound form of gl2L protein, or a biologically-active
portion thereof, on
the cell surface with a test compound and determining the ability of the test
compound to
modulate (e.g., stimulate or inhibit) the activity of the gl2L protein or
biologically-active
portion thereof. Determining the ability of the test compound to modulate the
activity of gl2L
or a biologically-active portion thereof can be accomplished, for example, by
determining the
ability of the gl2L protein to bind to or interact with a gl2L target
molecule. As used herein, a
"target molecule" is a molecule with which a gl2L protein binds or interacts
in nature. A gl2L
target molecule can be a non-gl2L molecule or a gl2L protein or polypeptide of
the invention.
In one embodiment, a gl2L target molecule is a component of a signal
transduction pathway
that facilitates transduction of an extracellular signal (e.g. a signal
generated by binding of a
compound to a membrane-bound gl2L molecule) through the cell membrane and into
the cell.
The target, for example, can be a second intercellular protein that has
catalytic activity or a
protein that facilitates the association of downstream signaling molecules
with gl2L.
Determining the ability of the gl2L protein to bind to or interact with a gl2L
target
molecule can be accomplished by one of the methods described above for
determining direct
binding. In one embodiment, determining the ability of the gl2L protein to
bind to or interact
With a gl2L target molecule can be accomplished by determining the activity of
the target
molecule. For example, the activity of the target molecule can be determined
by detecting
induction of a cellular second messenger of the target (i. e. intracellular
~Ca2+, diacylglycerol, IP3,
etc.), detecting catalytic/enzymatic activity of the target an appropriate
substrate, detecting the
induction of a reporter gene (comprising a gl2L-responsive regulatory element
operatively
linked to a nucleic acid encoding a detectable marker, e.g., luciferase), or
detecting a cellular
response, for example, cell survival, cellular differentiation, or cell
proliferation.
In yet another embodiment, an assay of the invention is a cell-free assay
comprising
contacting a gl2L protein or biologically-active portion thereof with a test
compound and
determining the ability of the test compound to bind to the gl2L protein or
biologically-active
61
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
portion thereof. Binding of the test compound to the gl2L protein can be
determined either
directly or indirectly as described above. In one such embodiment, the assay
comprises
contacting the gl2L protein or biologically-active portion thereof with a
known compound
wluch binds gl2L to form an assay mixture, contacting the assay mixture with a
test compound,
and determining the ability of the test compound to interact with a gl2L
protein, wherein
determining the ability of the test compound to interact with a gl2L protein
comprises
determining the ability of the test compound to preferentially bind to gl2L or
biologically-active
portion thereof as compared to the known compound.
In still another embodiment, an assay is a cell-free assay comprising
contacting gl2L
protein or biologically-active portion thereof with a test compound and
determining the ability
of the test compound to modulate (e.g. stimulate or inhibit) the activity of
the gl2L protein or
biologically-active portion thereof. Determining the ability of the test
compound to modulate
the activity of gl2L can be accomplished, for example, by determining the
ability of the gl2L
protein to bind to a gl2L target molecule by one of the methods described
above for determining
direct binding. Tn an alternative embodiment, deternzining the ability of the
test compound to
modulate the activity of gl2L protein can be accomplished by determining the
ability of the
gl2L protein further modulate a gl2L target molecule. For example, the
catalytic/enzymatic
activity of the target molecule on an appropriate substrate can be determined
as described, supra.
In yet another embodiment, the cell-free assay comprises contacting the gl2L
protein or
biologically-active portion thereof with a known compound which binds gl2L
protein to form
an assay mixture, contacting the assay mixture with a test compound, and
determining the ability
of the test compound to interact with a gl2L protein, wherein determining the
ability of the test
compound to interact with a gl2L protein comprises determining the ability of
the gl2L protein
to preferentially bind to or modulate the activity of a gl2L target molecule.
The cell-free assays of the invention are amenable to use of both the soluble
form or a
membrane-bound form of gl2L protein. W the case of cell-free assays comprising
the
membrane-bound form of gl2L protein, it may be desirable to utilize a
solubilizing agent such
that the membrane-bound form of gl2L protein is maintained in solution.
Examples of such
solubilizing agents include non-ionic detergents such as n-octylglucoside, n-
dodecylglucoside,
n-dodecylinaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide,
Triton~ X-
100, Triton~ X-114, Thesit~, Isotridecypoly(ethylene glycol ether)n, N-dodecyl-
-N,N-dimethyl-
3-ammonio-1-propane sulfonate, 3-(3-cholamidopropyl) dimethylamminiol-1-
propane sulfonate
(CHAPS), or 3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-1-propane
sulfonate
(CHAPSO).
62
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
In more than one embodiment of the above assay methods of the invention, it
may be
desirable to immobilize either gl2L protein or its target molecule to
facilitate separation of
complexed from uncomplexed forms of one or both of the proteins, as well as to
accommodate
automation of the assay. Binding of a test compound to gl2L protein, or
interaction of gl2L
protein with a target molecule in the presence and absence of a candidate
compound, can be
accomplished in any vessel suitable for containing the reactants. Examples of
such vessels
include microtiter plates, test tubes, and micro-centrifuge tubes. In one
embodiment, a fusion
protein can be provided that adds a domain that allows one or both of the
proteins to be bound to
a matrix. For exaanple, GST-gl2L fusion proteins or GST-target fusion proteins
can be
adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or
glutatluone
derivatized microtiter plates, that are then combined with the test compound
or the test
compound and either the non-adsorbed target protein or gl2L protein, and the
mixture is
incubated under conditions conducive to complex formation (e.g., at
physiological conditions
for salt and pH). Following incubation, the beads or microtiter plate wells
are washed to remove
any unbound components, the matrix irmnobilized in the case of beads, complex
determined
either directly or indirectly, for example, as described, supra.
Alternatively, the complexes can
be dissociated from the matrix, and the level of gl2L protein binding or
activity determined
using standard techniques.
Other techniques for immobilizing proteins on matrices can also be used in the
screening
assays of the invention. For example, either the gl2L protein or its target
molecule can be
immobilized utilizing conjugation of biotin and streptavidin. Biotinylated
gl2L protein or target
molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using
techniques well-
known within the art (e.g., biotinylation kit, Pierce Chemicals, Rockford,
Ill.), and immobilized
in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
Alternatively, antibodies
reactive with gl2L protein or target molecules, but which do not interfere
with binding of the
gl2L protein to its target molecule, can be derivatized to the wells of the
plate, and unbound
target or gl2L protein trapped in the wells by antibody conjugation. Methods
for detecting such
complexes, in addition to those described above for the GST-immobilized
complexes, include
immunodetection of complexes using antibodies reactive with the gl2L protein
or target
molecule, as well as enzyme-linked assays that rely on detecting an enz~nnatic
activity
associated with the gl2L protein or target molecule.
In another embodiment, modulators of gl2L protein expression are identified in
a
method wherein a cell is contacted with a candidate compound and the
expression of gl2L
mRNA or protein in the cell is determined. The level of expression of gl2L
mRNA or protein in
the presence of the candidate compound is compared to the level of expression
of gl2L mRNA
63
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
or protein in the absence of the candidate compound. The candidate compound
can then be
identified as a modulator of gl2L mRNA or protein expression based upon this
comparison. For
example, when expression of gl2L mRNA or protein is greater (i. e.,
statistically significantly
greater) in the presence of the candidate compound than in its absence, the
candidate compound
is identified as a stimulator of gl2L mRNA~or protein expression.
Alternatively, when
expression of gl2L mRNA or protein is less (statistically significantly less)
in the presence of
the candidate compou~zd than in its absence, the candidate compound is
identified as an inhibitor
of gl2L mRNA or protein expression. The level of gl2L mRNA or protein
expression in the
cells can be determined by methods described herein for detecting gl2L mRNA or
protein.
In yet another aspect of the invention, the gl2L proteins can be used as "bait
proteins" in
a two-hybrid assay or three hybrid assay (see, e.g., U.S. Patent No.
5,283,317; Zervos, et al.,
1993. Cell 72: 223-232; Madura, et al., 1993. J. Biol. Chera2. 268: 12046-
12054; Bartel, et al.,
1993. Biotechniques 14: 920-924; Iwabuchi, et al., 1993. Ofzcogene 8: 1693-
1696; and Brent
WO 94/10300), to identify other proteins that bind to or interact with gl2L
("gl2L-binding
proteins" or "gl2L-by") and modulate gl2L activity. Such gl2L-binding proteins
are also likely
to be involved in the propagation of signals by the gl2L proteins as, for
example, upstream or
downstream elements of the gl2L pathway.
The two-hybrid system is based on the modular nature of most transcription
factors,
which consist of separable DNA-binding and activation domains. Briefly, the
assay utilizes two
different DNA constructs. In one construct, the gene that codes for gl2L is
fused to a gene
encoding the DNA binding domain of a known transcription factor (e.g., GAL-4).
In the other
construct, a DNA sequence, from a library of DNA sequences, that encodes an
unidentified
protein ("prey" or "sample") is fused to a gene that codes for the activation
domain of the known
transcription factor. If the "bait" and the "prey" proteins are able to
interact, iya vivo, forming a
gl2L-dependent complex, the DNA-binding and activation domains of the
transcription factor
are brought into close proximity. This proximity allows transcription of a
reporter gene (e.g.,
LacZ) that is operably linked to a transcriptional regulatory site responsive
to the transcription
factor. Expression of the reporter gene can be detected and cell colonies
containing the
functional transcription factor can be isolated and used to obtain the cloned
gene that encodes
the protein that interacts with gl2L.
The invention further pertains to novel agents identified by the
aforementioned screening
assays and uses thereof for treatments as described herein.
64
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
Detectio~z Assays
Portions or fragments of the cDNA sequences identified herein (and the
corresponding
complete gene sequences) can be used in numerous ways as polynucleotide
reagents. By way of
example, and not of limitation, these sequences can be used to: (i) map their
respective genes on
a chromosome; and, thus, locate gene regions associated with genetic disease;
(ii) identify an
individual from a minute biological sample (tissue typing); and (iii) aid in
forensic identification
of a biological sample. Some of these applications are described in the
subsections, below.
Chromosome Mapping
Once the sequence (or a portion of the sequence) of a gene has been isolated,
this
sequence can be used to map the location of the gene on a chromosome. This
process is called
chromosome mapping. Accordingly, portions or fragments of the gl2L sequences,
SEQ m
NOS:1 or 3, or fragments or derivatives thereof, can be used to map the
location of the gl2L
genes, respectively, on a chromosome. The mapping of the gl2L sequences to
chromosomes is
an important first step in correlating these sequences with genes associated
with disease.
Briefly, gl2L genes can be mapped to chromosomes by preparing PCR primers
(preferably 15-25 by in length) from the gl2L sequences. Computer analysis of
the gl2L,
sequences can be used to rapidly select primers that do not span more than one
exon in the
genomic DNA, thus complicating the amplification process. These primers can
then be used for
PCR screening of somatic cell hybrids containing individual human chromosomes.
Only those
hybrids containing the human gene corresponding to the gl2L sequences will
yield an amplified
fragment.
Somatic cell hybrids are prepared by fusing somatic cells from different
mammals (e.g.,
human and mouse cells). As hybrids of human and mouse cells grow and divide,
they gradually
lose human chromosomes in random order, but retain the mouse chromosomes. By
using media
in which mouse cells cannot grow, because they lack a particular enzyme, but
in which human
cells can, the one human chromosome that contains the gene encoding the needed
enzyme will
be retained. By using various media, panels of hybrid cell lines can be
established. Each cell
line in a panel contains either a single human chromosome or a small number of
human
chromosomes, and a full set of mouse chromosomes, allowing easy mapping of
individual genes
to specific human chromosomes. See, e.g., D'Eustachio, et al., 1983. Science
220: 919-924.
Somatic cell hybrids containing only fragments of human chromosomes can also
be produced by
using human chromosomes with translocations and deletions.
PCR mapping of somatic cell hybrids is a rapid procedure for assigning a
particular
sequence to a particular chromosome. Three or more sequences can be assigned
per day using a
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
single thermal cycler. Using the gl2,L sequences to design oligonucleotide
primers, sub-
localization can be achieved with panels of fragments from specific
chromosomes.
Fluorescence ih situ hybridization (FISH) of a DNA sequence to a metaphase
chromosomal spread can further be used to provide a precise chromosomal
location in one step.
Chromosome spreads can be made using cells whose division has been blocked in
metaphase by
a chemical like colcemid that disrupts the mitotic spindle. The chromosomes
can be treated
briefly with trypsin, and then stained with Giemsa. A pattern of light and
dark bands develops
on each chromosome, so that the chromosomes can be identified individually.
The FISH
technique can be used with a DNA sequence as short as 500 or 600 bases.
However, 'clones
larger than 1,000 bases have a higher lilcelihood of binding to a unique
chromosomal location
with sufficient signal intensity for simple detection. Preferably 1,000 bases,
and more
preferably 2,000 bases, will suffice to get good results at a reasonable
amount of time. For a
review of tlus technique, see, Verma, et al., HUMAN CHROMOSOMES: A MANUAL of
BASIC
TECHNIQUES (Pergamon Press, New York 1988).
Reagents for chromosome mapping can be used individually to mark a single
chromosome or a single site on that chromosome, or panels of reagents can be
used for marking
multiple sites and/or multiple chromosomes. Reagents corresponding to
noncoding regions of
the genes actually are preferred for mapping purposes. Coding sequences are
more likely to be
conserved within gene families, thus increasing the chance of cross
hybridizations during
chromosomal mapping.
Once a sequence has been mapped to a precise chromosomal location, the
physical
position of the sequence on the chromosome can be correlated with genetic map
data. Such data
are found, e.g., in McKusiclc, MENDELIAN INHERITANCE IN MAN, available on-line
through Johns
Hopkins University Welch Medical Library). The relationship between genes and
disease,
mapped to the same chromosomal region, can then be identified through linkage
analysis (co-
inheritance of physically adjacent genes), described in, e.g., Egeland, et
al., 1987. Nature, 325:
783-787.
Moreover, differences in the DNA sequences between individuals affected and
unaffected with a disease associated with the gl2L gene, can be determined. Tf
a mutation is
observed in some or all of the affected individuals but not in any unaffected
individuals, then the
mutation is likely to be the causative agent of the particular disease.
Comparison of affected and
unaffected individuals generally involves first looking for structural
alterations in the
chromosomes, such as deletions or translocations that are visible from
chromosome spreads or
detectable using PCR based on that DNA sequence. Ultimately, complete
sequencing of genes
66
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
from several individuals can be performed to confirm the presence of a
mutation and to
distinguish mutations from polymorphisms.
Tissue Typing
The gl2L sequences of the invention can also be used to identify individuals
from
minute biological samples. In this technique, an individual's genomic DNA is
digested with one
or more restriction enzymes, and probed on a Southern blot to yield unique
bands for
identification. The sequences of the invention are useful as additional DNA
markers for RFLP
("restriction fragment length polymorphisms," described in U.S. Patent No.
5,272,057).
Furthennore, the sequences of the invention can be used to provide an
alternative
technique that determines the actual base-by-base DNA sequence of selected
portions of an
individual's genome. Thus, the gl2L sequences described herein can be used to
prepare two
PCR primers from the 5'- and 3'-termini of the sequences. These primers can
then be used to
amplify an individual's DNA and subsequently sequence it.
Panels of corresponding DNA sequences from individuals, prepared in this
manner, can
provide unique individual identifications, as each individual will have a
unique set of such DNA
sequences due to allelic differences. The sequences of the invention can be
used to obtain such
identification sequences from individuals and from tissue. The gl2L sequences
of the invention
uniquely represent portions of the human genome. Allelic variation occurs to
some degree in
the coding regions of these sequences, and to a greater degree in the
noncoding regions. It is
estimated that allelic variation between individual humans occurs with a
frequency of about
once per each 500 bases. Much of the allelic variation is due to single
nucleotide
polymorphisms (SNPs), which include restriction fragment length polymorphisms
(RFLPs).
Each of the sequences described herein can, to some degree, be used as a
standard
against which DNA from an individual can be compared for identification
purposes. Because
greater numbers of polymorphisms occur in the noncoding regions, fewer
sequences are
necessary to differentiate individuals. The noncoding sequences can
comfortably provide
positive individual identification with a panel of perhaps 10 to 1,000 primers
that each yield a
noncoding amplified sequence of 100 bases. If predicted coding sequences, such
as those in
SEQ ID NOS:1 or 3, are used, a more appropriate number of primers for positive
individual
identification would be 500-2,000.
Predictive Medicine
The invention also pertains to the field of predictive medicine in which
diagnostic
assays, prognostic assays, pharmacogenomics, and monitoring clinical trials
are used for
prognostic (predictive) purposes to thereby treat an individual
prophylactically. Accordingly,
one aspect of the invention relates to diagnostic assays for determining gl2L
protein and/or
67
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
nucleic acid expression as well as gl2L activity, in the context of a
biological sample (e.g.,
blood, serum, cells, tissue) to thereby determine whether an individual is
afflicted with a disease
or disorder, or is at risk of developing a disorder, associated with aberrant
gl2L expression or
activity. The disorders include metabolic disorders and diseases, such as
obesity and obesity
related disorders, diabetes, and cachexia; cell proliferative disorders and
diseases, such as
hyperplasia, cancer, and restenosis; neurodegenerative disorders and diseases,
such as
Alzheimer's Disease and Parkinson's Disorder; immune disorders and diseases,
such as AmS,
inflarmnation, and autoimmune diseases; and hematopoietic disorders and
diseases, such as
SCm, cyclic neutropenia, and thrombocythemia. The invention also provides for
prognostic (or
predictive) assays for determining whether an individual is at risk of
developing a disorder
associated with gl2L protein, nucleic acid expression or activity. For
example, mutations in a
gl2L gene can be assayed in a biological sample. Such assays can be used for
prognostic or
predictive purpose to thereby prophylactically treat an individual prior to
the onset of a disorder
characterized by or associated with gl2L protein, nucleic acid expression, or
biological activity.
Another aspect of the invention provides methods for determining gl2L protein,
nucleic
acid expression or activity in an individual to thereby select appropriate
therapeutic or
prophylactic agents for that individual (referred to herein as
"pharmacogenomics").
Pharmacogenomics allows for the selection of agents (e.g., drugs) for
therapeutic or prophylactic
treatment of an individual based on the genotype of the individual (e.g., the
genotype of the
individual examined to determine the ability of the individual to respond to a
particular agent.)
Yet another aspect of the invention pertains to monitoring the influence of
agents (e.g.,
drugs, compounds) on the expression or activity of gl2L in clinical trials.
These and other agents are described in further detail in the following
sections.
Diagnostic Assays
An exemplary method for detecting the presence or absence of gl2L in a
biological
sample involves obtaining a biological sample from a test subject and
contacting the biological
sample with a compound or an agent capable of detecting gl2L protein or
nucleic acid (e.g.,
mR.NA, genomic DNA) that encodes gl2L protein such that the presence of gl2L
is detected in
the biological sample. An agent for detecting gl2L mRNA or genomic DNA is a
labeled
nucleic acid probe capable of hybridizing to gl2L mRNA or genomic DNA. The
nucleic acid
probe can be, for example, a full-length gl2L nucleic acid, such as the
nucleic acid of SEQ m
NOS:1 or 3, or a portion thereof, such as an oligonucleotide of at least 15,
30, 50, 100, 250 or
500 nucleotides in length and sufficient to specifically hybridize under
stringent conditions to
gl2L mRNA or genomic DNA. Other suitable probes for use in the diagnostic
assays of the
invention are described herein.
68
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
An agent for detecting gl2L protein is an antibody capable of binding to gl2L
protein,
preferably an antibody with a detectable label. Antibodies can be polyclonal,
or more
preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab
or F(ab')2) can be
used. The term "labeled", with regard to the probe or antibody, is intended to
encompass direct
labeling of the probe or antibody by coupling (i. e., physically linking) a
detectable substance to
the probe or antibody, as well as indirect labeling of the probe or antibody
by reactivity with
another reagent that is directly labeled. Examples of indirect labeling
include detection of a
primary antibody using a fluorescently-labeled secondary antibody and end-
labeling of a DNA
probe with biotin such that it can be detected with fluorescently-labeled
streptavidin. The term
"biological sample" is intended to include tissues, cells and biological
fluids isolated from a
subject, as well as tissues, cells and fluids present within a subject. That
is, the detection method
of the invention can be used to detect gl2L mRNA, protein, or genomic DNA in a
biological
sample in vitro as well as ih vivo. For example, in vitf°o techniques
for detection of gl2L mRNA
include Northern hybridizations and in situ hybridizations. In vitro
techniques for detection of
gl2L protein include enzyme linlced immunosorbent assays (ELISAs), Western
blots,
immunoprecipitations, and immunofluorescence. Ih vitro techniques for
detection of gl2L
genomic DNA include Southern hybridizations. Furthermore, in vivo techniques
for detection of
gl2L protein include introducing into a subject a labeled anti-gl2L antibody.
For example, the
antibody can be labeled with a radioactive marker whose presence and location
in a subject can
z0 be detected by standard imaging techniques.
In one embodiment, the biological sample contains protein molecules from the
test
subject. Alternatively, the biological sample can contain mRNA molecules from
the test subject
or genomic DNA molecules from the test subject. A preferred biological sample
is an adipose
tissue sample, a tumor biobsy, or a blood sample.
In another embodiment, the methods further involve obtaining a control
biological
sample from a control subj ect, contacting the control sample with a compound
or agent capable
of detecting gl2L protein, mRNA, or genomic DNA, such that the presence of
gl2L protein,
mRNA or genomic DNA is detected in the biological sample, and comparing the
presence of
gl2L protein, mRNA or genomic DNA in the control sample with the presence of
gl2L protein,
mRNA or genomic DNA in the test sample.
The invention also encompasses lcits for detecting the presence of gl2L in a
biological
sample. For example, the kit can comprise: a labeled compound or agent capable
of detecting
gl2L protein or mRNA in a biological sample; means for determining the amount
of gl2L in the
sample; and means for comparing the amount of gl2L in the sample with a
standard. The
69
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
compound or agent can be packaged in a suitable container. The kit can further
comprise
instructions for using the kit to detect gl2L protein or nucleic acid.
Prognostic Assays
The diagnostic methods described herein can furthermore be utilized to
identify subjects
having or at risk of developing a disease or disorder associated with aberrant
gl2L expression or
activity. For example, the assays described herein, such as the preceding
diagnostic assays or
the following assays, can be utilized to identify a subject having or at risk
of developing a
disorder associated with gl2L protein, nucleic acid expression or activity.
Alternatively, the
prognostic assays can be utilized to identify a subject having or at risk for
developing a disease
or disorder. Thus, the invention provides a method for identifying a disease
or disorder
associated with aberrant gl2L expression or activity in which a test sample is
obtained from a
subject and gl2L protein or nucleic acid (e.g., mRNA, genomic DNA) is
detected, wherein the
presence of gl2L protein or nucleic acid is diagnostic for a subject having or
at risk of
developing a disease or disorder associated with aberrant gl2L expression or
activity. As used
herein, a "test sample" refers to a biological sample obtained from a subject
of interest. For
example, a test sample can be a biological fluid (e.g., serum), cell sample,
or tissue.
Furthermore, the prognostic assays described herein can be used to determine
whether a
subject can be administered an agent (e.g., an agonist, antagonist,
peptidomimetic, protein,
peptide, nucleic acid, small molecule, or other drug candidate) to treat a
disease or disorder
associated with aberrant gl2L expression or activity. For example, such
methods can be used to
determine whether a subject can be effectively treated with an agent for a
disorder. Thus, the
invention provides methods for determining whether a subject can be
effectively treated with an
agent for a disorder associated with aberrant gl2L expression or activity in
which a test sample
is obtained and gl2L protein or nucleic acid is detected (e.g., wherein the
presence of gl2L
protein or nucleic acid is diagnostic for a subject that can be administered
the agent to treat a
disorder associated with aberrant gl2L expression or activity).
The methods of the invention can also be used to detect genetic lesions in a
gl2L gene,
thereby determining if a subject with the lesioned gene is at risk for a
disorder characterized by
aberrant cell proliferation and/or differentiation. In various embodiments,
the methods include
detecting, in a sample of cells from the subject, the presence or absence of a
genetic lesion
characterized by at least one of an alteration affecting the integrity of a
gene encoding a gl2L-
protein, or the misexpression of the gl2L gene. For example, such genetic
lesions can be
detected by ascertaining the existence of at least one of: (i) a deletion of
one or more nucleotides
from a gl2L gene; (ii) an addition of one or more nucleotides to a gl2L gene;
(iii) a substitution
of one or more nucleotides of a gl2L gene, (iv) a chromosomal rearrangement of
a gl2L gene;
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
(v) an alteration in the level of a messenger RNA transcript of a gl2L gene,
(vi) aberrant
modification of a gl2L gene, such as of the methylation pattern of the genomic
DNA, (vii) the
presence of a non-wild-type splicing pattern of a messenger RNA transcript of
a gl2L gene,
(viii) a non-wild-type level of a gl2L protein, (ix) allelic loss of a gl2L
gene, and (x)
inappropriate post-translational modification of a gl2L protein. As described
herein, there are a
large nmnber of assay techniques known in the art that can be used for
detecting lesions in a
gl2L gene. A preferred biological sample is an adipose tissue sample, tumor
biopsy, or a blood
sample isolated by conventional means from a subj ect. However, any biological
sample
containing nucleated cells may be used.
In certain embodiments, detection of the lesion involves the use of a
probe/primer in a
polymerase chain reaction (PCR) (see, e.g., U.S. Patent Nos. 4,683,195 and
4;683,202), such as
anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR)
(see, e.g.,
Landegran, et al., 1988. Scieyace 241: 1077-1080; and Nakazawa, et al., 1994.
P~oc. Natl. Acad.
Sci. USA 91: 360-364), the latter of which can be particularly useful for
detecting point
mutations in the gl2L-gene (see, Abravaya, et al., 1995. Nucl. Acids Res. 23:
675-682). This
method can include the steps of collecting a sample of cells from a patient,
isolating nucleic acid
(e.g., genomic, mRNA or both) from the cells of the sample, contacting the
nucleic acid sample
with one or more primers that specifically hybridize to a gl2L gene under
conditions such that
hybridization and amplification of the gl2L gene (if present) occurs, and
detecting the presence
or absence of an amplification product, or detecting the size of the
amplification product and
comparing the length to a control sample. It is anticipated that PCR and/or
LCR may be
desirable to use as a preliminary amplification step in conjunction with any
of the techniques
used fox detecting mutations described herein.
Alternative amplification methods include: self sustained sequence replication
(see,
Guatelli, et al., 1990. Pt°oc. Natl. Acad. Sci. USA 87: 1874-1878),
transcriptional amplification
system (see, Kwoh, et al., 1989. Proc. Natl. Acad. Sci. USA 86: 1173-1177);
Q[3 Replicase (see,
Lizardi, et al, 1988. BioTechuology 6: 1197), or any other nucleic acid
amplification method,
followed by the detection of the amplified molecules using techniques well
known to those of
slcill in the art. These detection schemes are especially useful for the
detection of nucleic acid
molecules if such molecules axe present in very low numbers.
In an alternative embodiment, mutations in a gl2L gene from a sample cell can
be
identified by alterations in restriction enzyme cleavage patterns. For
example, sample and
control DNA is isolated, amplified (optionally), digested with one or more
restriction
endonucleases, and fragment length sizes are determined by gel electrophoresis
and compared.
Differences in fragment length sizes between sample and control DNA indicates
mutations in
71
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
the sample DNA. Moreover, the use of sequence specific ribozymes (see, e.g.,
U.S. Patent No.
5,493,531) can be used to score for the presence of specific mutations by
development or loss of
a ribozyrne cleavage site.
In other embodiments, genetic mutations in gl2L can be identified by
hybridizing a
sample and control nucleic acids, e.g., DNA or RNA, to high-density arrays
containing hundreds
or thousands of oligonucleotides probes. See, e.g., Cronin, et al., 1996.
Hurraan Mutation 7:
244-255; I~ozal, et al., 1996. Nat. Med. 2: 753-759. For example, genetic
mutations in gl2L can
be identified in two dimensional arrays containing light-generated DNA probes
as described in
Cronin, et al., supra. Briefly, a first hybridization array of probes can be
used to scan through
long stretches of DNA in a sample and control to identify base changes between
the sequences
by malting linear arrays of sequential overlapping probes. This step allows
the identification of
point mutations. This is followed by a second hybridization array that allows
the
characterization of specific mutations by using smaller, specialized probe
arrays complementary
to all variants or mutations detected. Each mutation array is composed of
parallel probe sets,
one complementary to the wild-type gene and the other complementary to the
mutant gene.
In yet another embodiment, any of a variety of sequencing reactions known in
the art can
be used to directly sequence the gl2L gene and detect mutations by comparing
the sequence of
the sample gl2L-with the corresponding wild-type (control) sequence. Examples
of sequencing
reactions include those based on techniques developed by Maxim and Gilbert,
1977. Proc. Natl.
Acad. Sci. USA 74: 560 or Sanger, 1977. Proc. Natl. Acad. Sci. USA 74: 5463.
It is also
contemplated that any of a variety of automated sequencing procedures can be
utilized when
performing the diag~.iostic assays (see, e.g., Naeve, et al., 1995.
Biotechniques 19: 448),
including sequencing by mass spectrometry (see, e.g., PCT International
Publication No. WO
94/16101; Cohen, et al., 1996. Adv. Chromatography 36: 127-162; and Griffin,
et al., 1993.
Appl. Biochem. Biotechnol. 38: 147-159).
Other methods for detecting mutations in the gl2L gene include methods in
which
protection from cleavage agents is used to detect mismatched bases in RNA/RNA
or RNA/DNA
heteroduplexes. See, e.g., Myers, et al., 1985. Science 230: 1242. In general,
the art technique
of "mismatch cleavage" starts by providing heteroduplexes of formed by
hybridizing (labeled)
RNA or DNA containing the wild-type gl2L sequence with potentially mutant RNA
or DNA
obtained from a tissue sample. The double-stranded duplexes are treated with
an agent that
cleaves single-stranded regions of the duplex such as which will exist due to
basepair
mismatches between the control and sample strands. For instance; RNA/DNA
duplexes can be
treated with RNase and DNA/DNA hybrids treated with S1 nuclease to
enzymaticalhy digesting
the mismatched regions. In other embodiments, either DNA/DNA or RNA/DNA
duplexes can
72
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
be treated with hydroxylamine or osmium tetroxide and with piperidine in order
to digest
mismatched regions. After digestion of the mismatched regions, the resulting
material is then
separated by size on denaturing polyacrylamide gels to determine the site of
mutation. See, e.g.,
Cotton, et al., 1988. PYOG. Natl. Acad. Sci. USA 85: 4397; Saleeba, et al.,
1992. Methods
Ehzymol. 217: 286-295. In an embodiment, the control DNA or RNA can be labeled
for
detection.
In still another embodiment, the mismatch cleavage reaction employs one or
more
proteins that recognize mismatched base pairs in double-stranded DNA (so
called "DNA
mismatch repair" enzymes) in defined systems for detecting and mapping point
mutations in
gl2L cDNAs obtained from samples of cells. For example, the mutt enzyme of E.
coli cleaves
A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves
T at G/T
mismatches. See, e.g., Hsu, et al., 1994. Carcihogehesis 15: 1657-1662.
According to an
exemplary embodiment, a probe based on a gl2L sequence, e.g., a wild-type gl2L
sequence, is
hybridized to a cDNA or other DNA product from a test cell(s). The duplex is
treated with a
DNA mismatch repair enzyme, and the cleavage products, if airy, can be
detected from
electrophoresis protocols or the life. See, e.g., U.S. Patent No. 5,459,039.
In other embodiments, alterations in electrophoretic mobility will be used to
identify
mutations in gl2L genes. For example, single strand conformation polymorphism
(SSCP) may
be used to detect differences in electrophoretic mobility between mutant and
wild type nucleic
acids. See, e.g., Orita, et al., 1989. Proc. Natl. Acad. Sci. USA: 86: 2766;
Cotton, 1993. Mutat.
Res. 285: 125-144; Hayashi, 1992. GeJZ.et. Anal. Tech. Appl. 9: 73-79. Single-
stranded DNA
fragments of sample and control gl2L nucleic acids will be denatured and
allowed to renature.
The secondary structure of single-stranded nucleic acids varies according to
sequence, the
resulting alteration in electrophoretic mobility enables the detection of even
a single base
change. The DNA fragments may be labeled or detected with labeled probes. The
sensitivity of
the assay may be enhanced by using RNA (rather than DNA), in which the
secondary structure
is more sensitive to a change in sequence. In one embodiment, the subject
method utilizes
heteroduplex analysis to separate double stranded heteroduplex molecules on
the basis of
changes in electrophoretic mobility. See, e.g., Keen, et al., 1991. TYerads
Gehet. 7: 5.
In yet another embodiment, the movement of mutant or wild-type fragments in
polyacrylamide gels containing a gradient of denaturant is assayed using
denaturing gradient gel
electrophoresis (DGGE). See, e.g., Myers, et al., 1985. NatuYe 313: 495. When
DGGE is used
as the method of analysis, DNA will be modified to insure that it does not
completely denature,
for example by adding a GC clamp of approximately 40 by of high-melting GC-
rich DNA by
PCR. In a further embodiment, a temperature gradient is used in place of a
denaturing gradient
73
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
to identify differences in the mobility of control and sample DNA. See, e.g.,
Rosenbaum and
Reissner, 1987. Biophys. Chem. 265: 12753.
Examples of other techniques for detecting point mutations include, but are
not limited
to, selective oligonucleotide hybridization, selective amplification, or
selective primer extension.
For example, oligonucleotide primers may be prepared in which the known
mutation is placed
centrally and then hybridized to target DNA under conditions that permit
hybridization only if a
perfect match is found. See, e.g., Saiki, et al., 1986. Nature 324: 163;
Saiki, et al., 1989. PYOC.
Natl. Acad. Sci. USA 86: 6230. Such allele specific oligonucleotides are
hybridized to PCR
amplified target DNA or a number of different mutations when the
oligonucleotides are attached
to the hybridizing membrane and hybridized with labeled target DNA.
Alternatively, allele specific amplification technology that depends on
selective PCR
amplification may be used in conjunction with the instant invention.
Oligonucleotides used as
primers for specific amplification may carry the mutation of interest in the
center of the
molecule (so that amplification depends on differential hybridization; see,
e.g., Gibbs, et al.,
1989. Nucl. Acids Res. 17: 2437-2448) or at the extreme 3'-terminus of one
primer where, under
appropriate conditions, mismatch can prevent, or reduce polymerase extension
(see, e.g.,
Prossner, 1993. Tibteclz. 11:238). In addition it may be desirable to
introduce a novel restriction
site in the region of the mutation to create cleavage-based detection. See,
e.g., Gasparini, et al.,
1992. Mol. Cell Probes 6: 1. It is anticipated that in certain embodiments
amplification may
also be performed using Taq ligase for amplification. See, e.g., Barany, 1991.
P~oc. Natl. Acad.
Sci. USA 88: 189. In such cases, ligation will occur only if there is a
perfect match at the 3'-
terminus of the 5' sequence, making it possible to detect the presence of a
known mutation at a
specific site by looping for the presence or absence of amplification.
The methods described herein may be performed, for example, by utilizing pre-
paclcaged
diagnostic kits comprising at least one probe nucleic acid or antibody reagent
described herein,
which may be conveniently used, e.g., in clinical settings to diagnose
patients exhibiting
symptoms or family history of a disease or illness involving a gl2L gene.
Furthermore, any cell type or tissue in which gl2L is expressed may be
utilized in the
prognostic assays described herein.
Phaf°macogenomics
Agents, or modulators that have a stimulatory or inhibitory effect on gl2L
activity (e.g.,
gl2L gene expression), as identified by a screening assay described herein can
be administered
to individuals to treat (prophylactically or therapeutically) disorders (The
disorders include
metabolic disorders and diseases, such as obesity and obesity-related
disorders, diabetes, and
cachexia; cell proliferative disorders and diseases, such as hyperplasia,
cancer, and restenosis; .
74
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
neurodegenerative disorders and diseases, such as Alzheimer's Disease and
Parkinson's
Disorder; immune disorders and diseases, such as AIDS, inflammation, and
autoimmune
diseases; and hematopoietic disorders and diseases, such as SCID, cyclic
neutropenia, and
thrombocythemia.) In conjunction with such treatment, the phannacogenomics
(i.e., the study
of the relationship between an individual's genotype and that individual's
response to a foreign
compound or drug) of the individual may be considered. Differences in
metabolism of
therapeutics can lead to severe toxicity or therapeutic failure by altering
the relation between
dose and blood concentration of the pharmacologically active drug. Thus, the
pharmacogenomics of the individual permits the selection of effective agents
(e.g., drugs) for
prophylactic or therapeutic treatments based on a consideration of the
individual's genotype.
Such pharmacogenomics can further be used to determine appropriate dosages and
therapeutic
regimens. Accordingly, the activity of gl2L protein, expression of gl2L
nucleic acid, or
mutation content of gl2L genes in an individual can be determined to thereby
select appropriate
agents) for therapeutic or prophylactic treatment of the individual.
Phannacogenomics deals with clinically significant hereditary variations in
the response
to drugs due to altered drug disposition and abnormal action in affected
persons. See e.g.,
Eichelbaum, 1996. Clin. Exp. Pharmacol. Physiol., 23: 983-985; Linder, 1997.
Clisz. Chem., 43:
254-266. In general, two types of pharmacogenetic conditions can be
differentiated. Genetic
conditions transmitted as a single factor altering the way drugs act on the
body (altered drug
action) or genetic conditions transmitted as single factors altering the way
the body acts on drugs
(altered drug metabolism). These pharmacogenetic conditions can occur either
as rare defects or
as polymorphisms. For example, glucose-6-phosphate dehydrogenase (G6PD)
deficiency is a
common inherited enzymopathy in which the main clinical complication is
hemolysis after
ingestion of oxidant drugs (anti-malarials, sulfonamides, analgesics,
nitrofurans) and
consumption of fava beans.
As an illustrative embodiment, the activity of drug metabolizing enzymes is a
major
determinant of both the intensity and duration of drug action. The discovery
of genetic
polymorphisms of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT
2) and
cytochrome P450 enzymes CYP2D6 and CYP2C19) has provided an explanation as to
why
some patients do not obtain the expected drug effects or show exaggerated drug
response and
serious toxicity after taking the standard and safe dose of a drug. These
polymorphisms are
expressed in two phenotypes in the population, the extensive metabolizer (EM)
and poor
metabolizer (PM). The prevalence of PM is different among different
populations. For
example, the gene coding for CYP2D6 is highly polymorphic and several
mutations have been
identified in PM, which all lead to the absence of functional CYP2D6. Poor
metabolizers of
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
CYP2D6 and CYP2C19 quite frequently experience exaggerated drug response and
side effects
when they receive standard doses. If a metabolite is the active therapeutic
moiety, PM show no
therapeutic response, as demonstrated for the analgesic effect of codeine
mediated by its
CYP2D6-formed metabolite morphine. At the other extreme are the so called
ultra-rapid
metabolizers who do not respond to standard doses. Recently, the molecular
basis of ultra-rapid
metabolism has been identified to be due to CYP2D6 gene amplification.
Thus, the activity of gl2L protein, expression of gl2L nucleic acid, or
mutation content
of gl2L genes in an individual can be determined to thereby select appropriate
agents) for
therapeutic or prophylactic treatment of the individual. In addition,
pharmacogenetic studies can
be used to apply genotyping of polynorphic alleles encoding drug-metabolizing
enzymes to the
identification of an individual's drug responsiveness phenotype. This
knowledge, when applied
to dosing or drug selection, can avoid adverse reactions or therapeutic
failure and thus enhance
therapeutic or prophylactic efficiency when treating a subject with a gl2L
modulator, such as a
modulator identified by one of the exemplary screening assays described
herein.
Monitoring of Effects Dining Clinical Vials
Monitoring the influence of agents (e.g., drugs, compounds) on the expression
or activity
of gl2L (e.g., the ability to modulate aberrant cell proliferation and/or
differentiation) can be
applied not only in basic drug screening, but also in clinical trials. For
example, the
effectiveness of an agent determined by a screening assay as described herein
to increase gl2L
gene expression, protein levels, or upregulate gl2L activity, can be monitored
in clinical trails of
subjects exhibiting decreased gl2L gene expression, protein levels, or
downregulated gl2L
activity. Alternatively, the effectiveness of an agent determined by a
screening assay to decrease
gl2L gene expression, protein levels, or downregulate gl2L activity, can be
monitored in
clinical trails of subjects exhibiting increased gl2L gene expression, protein
levels, or
upregulated gl2L activity. hl such clinical trials, the expression or activity
of gl2L and,
preferably, other genes that have been implicated in, for example, a cellular
proliferation or
immune disorder can be used as a "read out" or markers of the immune
responsiveness of a
particular cell.
By way of example, and not of limitation, genes, including gl2L, that are
modulated in
cells by treatment with an agent (e.g., compound, drug or small molecule)
modulating gl2L
activity (e.g., identified in a screeung assay as described herein) can be
identified. Thus, to
study the effect of agents on cellular proliferation disorders, for example,
in a clinical trial, cells
can be isolated and RNA prepared and analyzed for the levels of expression of
gl2L and other
genes implicated in the disorder. The levels of gene expression (i. e., a gene
expression pattern)
can be quantified by Northern blot analysis or RT-PCR, as described herein, or
alternatively by
76
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
measuring the amount of protein produced, by one of the methods as described
herein, or by
measuring the levels of activity of gl2L or other genes. In this manner, the
gene expression
pattern can serve as a marker, indicative of the physiological response of the
cells to the agent.
Accordingly, this response state may be determined before, and at various
points during,
treatment of the individual with the agent.
In one embodiment, the invention provides a method for monitoring the
effectiveness of
treatment of a subject with an agent (e.g., an agonist, antagonist, protein,
peptide,
peptidomimetic, nucleic acid, small molecule, or other drug candidate
identified by the
screening assays described herein) comprising the steps of (i) obtaining a pre-
administration
sample from a subject prior to administration of the agent; (ii) detecting the
level of expression
of a gl2L protein, mRNA, or genomic DNA in the preadministration sample; (iii)
obtaining one
or more post-administration samples from the subject; (iv) detecting the level
of expression or
activity of the gl2L protein, mRNA, or genomic DNA in the post-administration
samples; (v)
comparing the level of expression or activity of the gl2L protein, mRNA, or
genomic DNA in
the pre-administration sample with the gl2L protein, mRNA, or genomic DNA in
the post
administration sample or samples; and (vi) altering the administration of the
agent to the subject
accordingly. For example, increased administration of the agent may be
desirable to increase the
expression or activity of gl2L to higher levels than detected, i.e., to
increase the effectiveness of
the agent. Alternatively, decreased administration of the agent may be
desirable to decrease
expression or activity of gl2L to lower levels than detected, i.e., to
decrease the effectiveness of
the agent.
Methods of Treatment
The invention provides for both prophylactic and therapeutic methods of
treating a
subject at risk of (or susceptible to) a disorder or having a disorder
associated with aberrant
gl2L expression or activity. The disorders include metabolic disorders and
diseases, such as
obesity and obesity-related disorders, diabetes, and cachexia; cell
proliferative disorders and
diseases, such as hyperplasia, cancer, and restenosis; neurodegenerative
disorders and diseases,
such as Alzheimer's Disease and Paxkinson's Disorder; immune disorders and
diseases, such as
AIDS, inflammation, and autoimmune diseases; and hematopoietic disorders and
diseases, such
as SCID, cyclic neutropenia, and thrombocythemia. These methods of treatment
will be
discussed more fully, below.
Disease ahd Disorders
Diseases and disorders that are characterized by increased (relative to a
subject not
suffering from the disease or disorder) levels or biological activity may be
treated with
77
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
Therapeutics that antagonize (i.e., reduce or inhibit) activity. Therapeutics
that antagonize
activity may be administered in a therapeutic or prophylactic manner.
Therapeutics that may be
utilized include, but are not limited to: (i) an aforementioned peptide, or
analogs, derivatives,
fragments or homologs thereof; (ii) antibodies to an aforementioned peptide;
(iii) nucleic acids
encoding an aforementioned peptide; (iv) administration of antisense.nucleic
acid and nucleic
acids that are "dysfunctional" (i.e., due to a heterologous insertion within
the coding sequences
of coding sequences to an aforementioned peptide) that are utilized to
"knockout" endogenous
function of an aforementioned peptide by homologous recombination (see, e.g.,
Capecchi, 1989.
Sciehce 244: 1288-1292); or (v) modulators ( i.e., inhibitors, agonists and
antagonists, including
additional peptide mimetic of the invention or antibodies specific to a
peptide of the invention)
that alter the interaction between an aforementioned peptide and its binding
partner.
Diseases and disorders that are characterized by decreased (relative to a
subject not
suffering from the disease or disorder) levels or biological activity may be
treated with
Therapeutics that increase (i.e., are agonists to) activity. Therapeutics that
upregulate activity
may be administered in a therapeutic or prophylactic manner. Therapeutics that
may be utilized
include, but are not limited to, an aforementioned peptide, or analogs,
derivatives, fragments or
homologs thereof; or an agonist that increases bioavailability.
Increased or decreased levels can be readily detected by quantifying peptide
and/or
RNA, by obtaining a patient tissue sample (e.g., from biopsy tissue) and
assaying it ih vitro for
RNA or peptide levels, structure and/or activity of the expressed peptides (or
mRNAs of an
aforementioned peptide). Methods that are well-known within the art include,
but are not
limited to, immunoassays (e.g., by Western blot analysis, immunoprecipitation
followed by
sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis,
immunocytochemistry, etc.)
and/or hybridization assays to detect expression of mRNAs (e.g., Northern
assays, dot blots, ifz
situ hybridization, and the like).
Prophylactic Methods
In one aspect, the invention provides a method for preventing, in a subject, a
disease or
condition associated with an aberrant gl2L expression or activity, by
administering to the
subject an agent that modulates gl2L expression or at least one gl2L activity.
Subjects at risk
for a disease that is caused or contributed to by aberrant gl2L expression or
activity can be
identified by, for example, any or a combination of diagnostic or prognostic
assays as described
herein. Administration of a prophylactic agent can occur prior to the
manifestation of symptoms
characteristic of the gl2L aberrancy, such that a disease or disorder is
prevented or,
alternatively, delayed in its progression: Depending upon the type of gl2L
aberrancy, for
example, a gl2L agonist or gl2L antagonist agent can be used for treating the
subject. The
78
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
appropriate agent can be determined based on screening assays described
herein. The
prophylactic methods of the invention are further discussed in the following
subsections.
Therapeutic Methods
Another aspect of the invention pertains to methods of modulating gl2L
expression or
activity for therapeutic purposes. The modulatory method of the invention
involves contacting a
cell with an agent that modulates one or more of the activities of gl2L
protein activity
associated with the cell. An agent that modulates gl2L protein activity can be
an agent as
described herein, such as a nucleic acid or a protein, a naturally-occurring
cognate ligand of a
gl2L protein, a peptide, a gl2L peptidomimetic, or other small molecule. In
one embodiment,
the agent stimulates one or more gl2L protein activity. Examples of such
stimulatory agents
include active gl2L protein and a nucleic acid molecule encoding gl2L that has
been introduced
into the cell. In another embodiment, the agent inhibits one or more gl2L
protein activity.
Examples of such inhibitory agents include antisense gl2L nucleic acid
molecules and anti-
gl2L antibodies. These modulatory methods can be performed in. vitro (e.g., by
culturing the
cell with the agent) or, alternatively, ifa vivo (e.g., by administering the
agent to a subject). As
such, the invention provides methods of treating an individual afflicted with
a disease or
disorder characterized by aberrant expression or activity of a gl2L protein or
nucleic acid
molecule. In one embodiment, the method involves administering an agent (e.g.,
an agent
identified by a screening assay described herein), or combination of agents
that modulates (e.g.,
up-regulates or down-regulates) gl2L expression or activity. In another
embodiment, the
method involves administering a gl2L protein or nucleic acid molecule as
therapy to
compensate for reduced or aberrant gl2L expression or activity.
Stimulation of gl2L activity is desirable in situations in which gl2L is
abnormally
downregulated and/or in which increased gl2L activity is lilcely to have a
beneficial effect. One
example of such a situation is where a subject has a disorder characterized by
aberrant cell
proliferation and/or differentiation (e.g., cancer or immune associated
disorders). Another
example of such a situation is where the subject has a gestational disease
(e.g., preclampsia).
Determination of the Biological Effect of the Therapeutic
In various embodiments of the invention, suitable in vitro or ih vivo assays
are performed
to determine the effect of a specific Therapeutic and whether its
administration is indicated for
treatment of the affected tissue.
In various specific embodiments, in vitro assays may be performed with
representative
cells of the types) involved in the patient's disorder, to determine if a
given Therapeutic exerts
the desired effect upon the cell type(s). Compounds for use in therapy may be
tested in suitable
79
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
animal model systems including, but not limited to rats, mice, chicken, cows,
monkeys, rabbits,
and the like, prior to testing in human subjects. Similarly, for ih vivo
testing, any of the animal
model system known in the art may be used prior to administration to human
subj ects.
Prophylactic and Therapeutic Uses of the Compositions of the Invention
The gl2L nucleic acids and proteins of the invention are useful in potential
prophylactic
and therapeutic applications implicated in a variety of disorders including,
but not limited to:
metabolic disorders and diseases, such as obesity and obesity-related
disorders, diabetes, and
cachexia; cell proliferative disorders and diseases, such as hyperplasia,
cancer, and restenosis;
neurodegenerative disorders and diseases, such as Alzheimer's Disease and
Parkinson's
Disorder; immune disorders and diseases, such as AmS, inflammation, and
autoimmune
diseases; and hematopoietic disorders and diseases, such as SCm, cyclic
neutropenia, and
thrombocythemia.
As an example, a cDNA encoding the gl2L protein of the invention may be useful
in
gene therapy, and the protein may be useful when administered to a subj ect in
need thereof. By
way of non-limiting example, the compositions of the invention will have
efficacy for treatment
of patients suffering from metabolic disorders and diseases, such as obesity
and obesity-related
disorders, diabetes, and cachexia; cell proliferative disorders and diseases,
such as hyperphasia,
cancer, and restenosis; neurodegenerative disorders and diseases, such as
Alzheimer's Disease
and Parkinson's Disorder; immune disorders and diseases, such as AIDS,
inflammation, aazd
autoimmune diseases; and hematopoietic disorders and diseases, such as SCID,
cyclic
neutropenia, and thrombocythemia.
Both the novel nucleic acid encoding the gl2L protein, and the gl2L protein of
the
invention, or fragments thereof, may also be useful in diagnostic
applications, wherein the
presence or amount of the nucleic acid or the protein are to be assessed. A
further use could be
as an anti-bacterial molecule (i.e., some peptides have been found to possess
anti-bacterial
properties). These materials are further useful in the generation of
antibodies that
immunospecifically-bind to the novel substances of the invention for use in
therapeutic or
diagnostic methods.
EXAMPLES
The following examples illustrate by way of non-limiting example various
aspects of the
invention.
As is the case for Spot 14, wlich is differentially regulated during BAT
differentiation,
mouse gl2L is also modulated in BAT under conditions that affect the metabolic
activity and
proliferative status of BAT. Under the experimental conditions for the
identification of
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
differentially expressed genes in BAT, a fragment of cDNA (representing an
mRNA encoding
mouse gl2L) was modulated in response to cold (4°C). The pattern of
modulation observed for
the cDNA fragment in BAT from animals raised under the experimental conditions
described
above was up-regulation in cold-acclimated vs. control, no change in warm-
acclimated vs.
control, and up-regulation in cold-acclimated vs. warm-acclimated. Over time
mouse gl2L
cDNA fragment is up-regulated, and remains up-regulated, under the condition
of cold-
acclimation. The TaqMan-derived tissue distribution of mouse gl2L shows that
the mouse
proteins expression is significantly elevated in the response to the cold in
WAT and BAT
relative to the same tissues from warm-acclimated animals.
The identification of the modulated fragment lead to the identification of a
full length
mouse cDNA (Table 2A; SEQ ID N0:3) encoding a 182 amino acid protein (Table
2B; SEQ m
N0:4) of predicted molecular weight 20355.6 D, predicted pI = 5.33, and
predicted physical
characteristics in Table 3.
A human protein homolog (Table 1B; SEQ ID N0:2) was also identified, 183 amino
acids, 20233.5 D, predicted pI=5.55, and other predicted physical
characteristics (Table 4). An
optimal cDNA was determined by assembly of 85 ESTs from public and private
databases
(Table 1A; SEQ ID NO:1). The pI's of the mouse, human homologs of the
zebrafish g12
protein, and the zebrafish g12 (predicted pI = 4.93) proteins have more
alkaline predicted pI's
than the Spot 14 family proteins (Rat Spot 14 pI = 4.61, Mouse Spot 14 pI =
4.76, Human Spot
14 pI = 4.64) due to the more extensive acid-rich domains of the Spot 14's
(Grillasca, FEBS
Lett. 1997 Jan 13;401(1):38-42) compared to the g12 family members. This and
other data
support the contention that the novel mouse and human gl2L molecules described
herein are the
true homologs of the zebrafish gastrulation-specific protein, g12.
Sequence analysis indicates that the g12 family members have a "spot box" and
an "acid
box" observed for members of the Spot 14 family members (FIG. 1). The g12
family members
have a conserved insert just after the spot box (the zebrafish insert is
significantly smaller than
the inserts for the mouse and human gl2L's) and a second highly conserved
insert just after
amino acid 54 of the mouse sequence. The g12 family members have a somewhat
less acidic
acid box (amino acids 87-114 of the mouse Spot 14 polypeptide) as compared to
the Spot 14
family members (FIG. 1). This reduced acid box likely accounts for the
relatively higher pI's of
the g12 family members compared to the Spot 14 family members.
Mouse and human gl2L are more similar to zebrafish g12 than the Spot 14's,
both at the
protein level and at the level of the nucleotide sequence.
81
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
The mouse gl2L maps to chromosome X at position DXMit89 in mouse by radiation
hybrid mapping. The human gl2L gene is found on chromosome X and is 100%
identical to
GeneBank AK001428 (Submitted (16-FEB-2000) to the DDBJ/EMBL/GenBanlc).
82
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
Table 3 Predicted physical characteristics of Mouse gl2L protein
Values assuming ALL Cys residues appear as half cystines:
~~ ~.~ 27~-nm ~. 278~nm . :279 nm.- 280 nm: ~82 nm r
~~ _ ~ _ ~ __ _ _ _ _ ~__ . ~ ._,._ ~ __ _ ~ _ ___ _.'
Extinction Coefficient !' 23595 23927 23825 23590 22900
w... ~. ~_.r.._.~. .._.~.._.....r.. .~. .... ._ _.__...._,.. .. ~._..~._~..
.~_;_ _ ._.~ ..
Optical Density 1.159 1.175 1.170 1.159 ' 1.125
.. ._~ _ _..... ... _ . ... ..._.~ _._~ _...~_.._. .. ~ _ __._. ..: _.~._._..
~ .~ ~... .~~. __._,
Values assuming NO Cys residues appear as half cystines:
83
Note: The conditions at which these equations aye valid as°e: pH 6.5,
6. 0 Mguanidium
hydrochloride, 0.02 Mplzosphate buffef°.
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
Table 4 Predicted physical characteristics of Human gl2L protein.
Values assuming NO Cys residues appear as half cystines:
276 nm ~) 278 nm fl 279 nm ~I 280 nm ~I 282 nm
Extinction Coefficient ~[ 23450 ~~ 23800 ~~ y 23705 ~ ~ 23470 ~ ' 22800
...~__ __ _ .__. 1
.. __.~._ __ ..._ ~ ~ . _ ~_ _ ~..__
Optical Density ~ 1.159 f 1.176 ~ 1.172 1.160 1.127
_. _. .__..~ _ . _ .~. . ~ .._. . ~ .____ ~ _~.
Note: The conditions at wlzich these equations are valid are: pH 6.5, 6.0
Mguanidiurn hydroclzloride, 0.02 Mplzospha
buffer.
liz surninary, the inventors have identifying novel mouse and human genes
responsive to
the thermal state of animals raised below their thermal neutral zone as a
means of identifying
such genes modulated in response to the metabolic status and responsive to
thermal conditions.
It is contemplated that such molecules relate to the endocrine nature of BAT
and its role in, and
responsiveness to, thermoregulation and metabolism.
EQUIVALENTS
Although particular embodiments have been disclosed herein in detail, this has
been
done by way of example for purposes of illustration only, and is not intended
to be limiting with
respect to the scope of the appended claims that follow. In particular, it is
contemplated by the
inventors that various substitutions, alterations, and modifications may be
made to the invention
without departing from the spirit and scope of the invention as defined by the
claims. The
choice of nucleic acid starting material, clone of interest, or library type
is believed to be a
matter of routine for a person of ordinary skill in the art with knowledge of
the embodiments
described herein. Other aspects, advantages, and modifications considered to
be within the
scope of the following claims.
84
Values assuming ALL Cys residues appear as half cystines:
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
SEQUENCE LISTING
<110> Lewin, David A.
Adams, Sean H.
<120> NOVEL GENES ASSOCIATED WITH THE THERMAL RESPONSE
<130> 10716-31
<140> Not assigned
<141> 2001-03-02
<150> 60/186,513
<151> 2000-03-02
<160> 8
<170> PatentIn Ver. 2.1
<210>1
<211>1128
<212>DNA
<213>Homo sapiens
<400> 1
gcgcgccagg ggtggccctg agcgccggcg acacctttcc tggactataa attgagcacc 60
tgggatgggt agggggccaa cgcagtcacc gccgtccgca gtcacagtcc agccactgac 120
cgcagcagcg cccttgcgta gcagccgctt gcagcgagaa cactgaattg ccaacgagca 180
ggagagtctc aaggcgcaag aggaggccag ggctcgaccc acagagcacc ctcagccatc 240
gcgagtttcc gggcgccaaa gccaggagaa gccgcccatc ccgcagggcc ggtctgccag 300
cgagacgaga gttggcgagg gcggaggagt gccgggaatc ccgccacacc ggctatagcc 360
aggcccccag cgcgggcctt ggagagcgcg tgaaggcggg catccccttg acccggccga 420
ccatccccgt gcccctgcgt ccctgcgctc caacgtccgc gcggccacca tgatgcaaat 480
ctgcgacacc tacaaccaga agcactcgct ctttaacgcc atgaatcgct tcattggcgc 540
cgtgaacaac atggaccaga cggtgatggt gcccagcttg ctgcgcgacg tgcccctggc 600
tgaccccggg ttagacaacg aggtcagcgt ggaggtaggc ggcagtggca gctgcctgga 660
ggagcgcacg accccggccc caagcccggg cagcgccaat ggaagctttt tcgcgccctc 720
ccgggacatg tacagccact acgtgctgct caagtccatc cgcaacgata ttgagtgggg 780
agtcctgcac cagccgcctc caccggctgg gagcgaggag ggcagtgcct ggaagtccaa 840
ggacatcctg gtggacctgg gccacttgga gggtgcggac gccggcgaag aagacctgga 900
acagcagttc cactaccacc tgcgcgggct gcacactgtg ctctcgaaac tcacgcgcaa 960
agccaacatc ctcactaaca gatacaagca ggagatcggc ttcggcaatt ggggccactg 1020
aggcgtggcg cccgtggctg cccagcacct tcttcgaccc atctcaccct ctctcattcc 1080
tcaaagcttt ttttttttcc ctggctgggg ggcggaaagg gcaaactg 1128
<210> 2
<211> 183
1
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
<212> PRT
<213> Homo sapiens
<400> 2
Met Met Gln Ile Cys Asp Thr Tyr Asn Gln Lys His Ser Leu Phe Asn
1 5 10 15
Ala Met Asn Arg Phe Ile Gly Ala Va1 Asn Asn Met Asp Gln Thr Val
20 25 30
Met Val Pro Ser Leu Leu Arg Asp Val Pro Leu Ala Asp Pro Gly Leu
35 40 45
Asp Asn Glu Val Ser Val Glu Val Gly Gly Ser Gly Ser Cys Leu Glu
50 55 60
Glu Arg Thr Thr Pro Ala Pro Ser Pro Gly Ser Ala Asn G1y Ser Phe
65 70 75 80
Phe Ala Pro Ser Arg Asp Met Tyr Ser His Tyr Val Leu Leu Lys Ser
85 90 95
Ile Arg Asn Asp Ile Glu Trp Gly Val Leu His Gln Pro Pro Pro Pro
100 l05 110
Ala Gly Ser Glu Glu Gly Ser Ala Trp Lys Ser Lys Asp Ile Leu Val
115 120 125
Asp Leu Gly His Leu Glu Gly Ala Asp Ala Gly Glu Glu Asp Leu Glu
130 135 140
Gln Gln Phe His Tyr His Leu Arg Gly Leu His Thr Val Leu Ser Lys
145 150 155 160
Leu Thr Arg Lys Ala Asn Ile Leu Thr Asn Arg Tyr Lys Gln Glu Ile
165 170 175
Gly Phe Gly Asn Trp Gly His
180
<210>3
<211>1927
<212>DNA
<213>Mus musculus
<400> 3
cggacgcgtg ggggaggtag gaggaggaga catcaggggt ggtcctgggc gcctgggaca 60
2
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
cctttcccgg actataaatt gagcacctgg aatgggcagg gggccggagc aaccacagtc 120
gcccttactc acagtccgat cagtgaccgc agcagcgccc ttgggcagcc accgtccgca 180
acgcaagcac tgagaaccag gggatttcgc agtgcaagag aaaaaggcta gacccagcca 240
cccaccgtca atcctgagcc aaagataaga gcagccgggc ctcacgaagg gctgagctga 300
gaaagaagca agttagagag ggcggagaag gatctgggaa tcccgtcaca ccggcttcaa 3~0
gcaggctccc ggcatcagcc tctgagagcg cttgaaggcg gcatcgccag cggtctatct 420
ccgtgtacca gcgtccctgt gtttccgcgc ccgctcggcc accatgatgc aaatctgcga 480
cacatataac cagaagcact cgctctttaa cgccatgaat cgcttcattg gcgcggtgaa 540
caacatggac cagacggtga tggtgcccag tctgctgcgc gacgtacccc tgtccgagcc 600
ggagatagac gaggtcagcg tggaggtagg cggcagtggc ggctgcctgg aggagcgcac 660
gaccccggcc ccaagcccgg gcagcgccaa tgaaagcttt ttcgcgccct cccgggacat 720
gtacagccac tacgtgctgc tcaagtccat ccgcaatgat atcgagtggg gagtcctgca 780
ccagccttcg tctccgccgg ccgggagcga ggagagcacc tggaagccca aggacatcct 840
ggtgggcctg agtcacttgg agagcgcgga tgcgggcgag gaagatctgg agcagcagtt 900
ccactaccac ctgcgcgggc tgcacaccgt gctctccaaa ctcacccgaa aagccaacat 960
cctcaccaat agatacaagc aggagatcgg cttcagtaat tggggccact gaggcggggg 1020
ctgtccccgc tgcccagcac cctctctcgg gtcggctcta ccacccctct ctttcctcca 1080
agctattttc ttcctggttg tggggcgcga agggcacact gtaaagttgg gctgtgtact 1140
tggtggggtt agtgtggaga agagggcctc atcgcgagag cagaggaaag tagtcgccag 1200
agaggggggt tcaaagaccc ccggaggggg cctactctgt gttggtggga atggaactgg 1260
gccgatgtcc ttcattcagc ctgtgccttt cttggggttt cttttctgtt tttctttccg 1320
gaagagaagg gcctgagaaa gggccatgcc agggcacagt gctgggttgc cacacatggg 1380
agggcagctt ctagccgggt gcttggggga ggcggggctc agcctcctgc tgccctgcct 1440
tgagctgcca gaggaggcct tggcgttgct aggattgcgt cagttttcct gtttgcacta 1500
tttctttttg taacagtgac cctgtcttaa gtctttcaga tctctttgct ttgaaacttc 1560
gtcgattcca ttgtgataag cgcacaaaca gcactgttgg taaccggtac tactttatta 1620
atgattttct gttacactgt acagtagtcc tgtggcaccc tatccctttc acgccacccc 1680
tcccccgccc gtgtgtgtaa actggcgatg tgccagctag gatgaagctt gccactcggc 1740
tagcgaaaat aattaacatt attatgagaa agtggattta tctaaagtgg aaccagctga 1800
cattatatct gtatcgtatg gagaatgatg aagggctcca ctgttgttat atgtcttgtt 1860
tatttaaaac tttttttaat ccagatgtag actatattct aaaaaataaa aacgcagatg 1920
tgttaac 1927
<210>4
<211>182
<212>PRT
<213>Mus musculus
<400> 4
Met Met Gln I1e Cys Asp Thr Tyr Asn Gln Lys His Ser Zeu Phe Asn
Z 5 10 15
Ala Met Asn Arg Phe I1e Gly Ala Val Asn Asn Met Asp Gln Thr Val
20 25 30
Met Val Pro Ser Leu Leu Arg Asp Val Pro Zeu Ser Glu Pro Glu Ile
35 40 45
3
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
Asp Glu Val Ser Val Glu Val Gly G1y Ser G1y Gly Cys Leu Glu Glu
50 . 55 60
Arg Thr Thr Pro Ala Pro Ser Pro Gly Ser Ala Asn Glu Ser Phe Phe
65 70 75 80
A1a Pro Ser Arg Asp Met Tyr Ser His Tyr Val Leu Leu Lys Ser Ile
85 90 95
Arg Asn Asp Ile Glu Trp Gly Val Leu His Gln Pro Ser Ser Pro Pro
100 105 110
Ala Gly Ser Glu Glu Ser Thr Trp Lys Pro Lys Asp Ile Leu Val Gly
115 120 125
Leu Ser His Leu Glu Ser Ala Asp Ala Gly Glu Glu Asp Leu Glu Gln
130 135 140
Gln Phe His Tyr His Leu Arg Gly Leu His Thr Val Leu Ser Lys Leu
145 150 155 160
Thr Arg Lys Ala Asn Ile Leu Thr Asn Arg Tyr Lys Gln Glu Ile Gly
165 170 175
Phe Ser Asn Trp Gly His
180
<210>5
<211>150
<212>PRT
<213>Mus musculus
<400> 5
Met Gln Val Leu Thr Lys Arg Tyr Pro Lys Asn Cys Leu Leu Thr Val
1 5 10 15
Met Asp Arg Tyr Ser Ala Val Val Arg Asn Met Glu Gln Val Val Met
20 25 30
Ile Pro Ser Leu Leu Arg Asp Val Gln Leu Ser Gly Pro Gly Gly Ser
35 40 45
Val Gln Asp Gly Ala Pro Asp Leu Tyr Thr Tyr Phe Thr Met Leu Lys
50 55 60
Ser Ile Cys Val Glu Val Asp His Gly Leu Leu Pro Arg Glu Glu Trp
4
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
65 70 75 80
Gln Ala Lys Val Ala Gly Asn Glu Thr Ser Glu Ala Glu Asn Asp Ala
85 90 g5 ._
Ala Glu Thr Glu Glu Ala Glu Glu Asp Arg Ile Ser Glu Glu Leu Asp
100 105 110
Leu Glu Ala Gln Phe His Leu His Phe Cys Ser Leu His His Ile Leu
115 l20 125
Thr His Leu Thr Arg Lys Ala Gln Glu Val Thr Arg Lys Tyr Gln Glu
130 135 140
Met Thr Gly Gln Val Leu
145 150
<210> 6
<211> 146
<212> PRT
<213> Rattus sp.
<400> 6
Met Gln Val Leu Thr Lys Arg Tyr Pro Lys Asn Cys Leu Leu Thr Val
1 5 10 15
Met Asp Arg Tyr Ala Ala Glu Val His Asn Met Glu Gln Val Val Met
20 25 30
Ile Pro Ser Leu Leu Arg Asp Val Gln Leu Ser Gly Pro Gly Gly Gln
35 40 45 '
A1a Gln Ala Glu Ala Pro Asp Leu Tyr Thr Tyr Phe Thr Met Leu Lys
50 55 60
Ala Ile Cys Val Asp Val Asp His Gly Leu Leu Pro Arg Glu Glu Trp
65 70 75 80
Gln Ala Lys Val Ala Gly Ser Glu Glu Asn Gly Thr Ala Glu Thr Glu
85 90 95
Glu Val Glu Asp Glu Ser Ala Ser Gly Glu Leu Asp Leu G1u Ala Gln
100 105 110
Phe His Leu His Phe Ser Ser Leu His His Ile Leu Met His Leu Thr
115 120 125
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
Glu Lys Ala Gln Glu Val Thr Arg Lys Tyr Gln Glu Met Thr Gly Gln
130 135 140
Val Trp
145
<210>7
<211>146
<212>PRT
<213>Homo sapiens
<400> 7
Met Gln Val Leu Thr Lys Arg Tyr Pro Lys Asn Cys Leu Leu Thr Val
1 5 10 15
Met Asp Arg Tyr Ala Ala Glu Val His Asn Met Glu Gln Val Va1 Met
20 25 30
Ile Pro Ser Leu Leu Arg Asp Val Gln Leu Ser Gly Pro Gly Gly Gln
35 40 45
Ala G1n Ala Glu Ala Pro Asp Leu Tyr Thr Tyr Phe Thr Met Leu Lys
50 55 60
Ala Ile Cys Val Asp Val Asp His Gly Leu Leu Pro Arg Glu Glu Trp
65 70 75 80
G1n Ala Lys Val Ala Gly Ser Glu Glu Asn Gly Thr A1a Glu Thr Glu
85 90 95
Glu Val Glu Asp Glu Ser Ala Ser Gly Glu Leu Asp Leu Glu Ala Gln
100 105 110
Phe His Leu His Phe Ser Ser Leu His His Ile Leu Met His Leu Thr
115 120 125
Glu Lys Ala Gln Glu Val Thr Arg Lys Tyr Gln Glu Met Thr Gly Gln
130 135 140
Val Trp
145
<210> 8
<211> 152
<212> PRT
<213> Danio rerio
6
CA 02401173 2002-08-22
WO 01/64884 PCT/USO1/06839
<400> 8
Met Gln Met Ser G1u Pro Leu Ser Gln Lys Asn Ala Leu Tyr Thr Ala
1 5 10 15
Met Asn Arg Phe Leu Gly Ala Val Asn Asn Met Asp Gln Thr Val Met
20 25 30
Val Pro Ser Leu Leu Arg Asp Val Pro Leu Asp Gln Glu Lys Glu Gln
35 40 45
Gln Lys Leu Thr Asn Asp Pro Gly Ser Tyr Leu Arg Glu Ala Glu Ala
50 ~ 55 60
Asp Met Tyr Ser Tyr Tyr Ser Gln Leu Lys Ser Ile Arg Asn Asn Ile
65 70 75 80
Glu Trp Gly Val Ile Arg Ser Glu Asp Gln Arg Arg Lys Lys Asp Thr
85 90 95
Ser,Ala Ser Glu Pro Val Arg Thr Glu Glu G1u Ser Asp Met Asp Leu
100 105 110
Glu Gln Leu Leu Gln Phe His Leu Lys Gly Leu His Gly Val Leu Ser
115 120 125
Gln Leu Thr Ser G1n Ala Asn Asn Leu Thr Asn Arg Tyr Lys G1n Glu
130 135 140
Ile Gly Ile Ser Gly Trp Gly Gln
145 150
7
