Note: Descriptions are shown in the official language in which they were submitted.
CA 02604143 2007-10-26
1
METHODS FOR DETECTING THYROID CARCINOMA CELLS
c
This application has been divided out of Canadian Application 2,329,776 filed
internationally as International Patent Application PCT/JP99/03054 filed June
7, 1999
and published as WO 99/64591 on December 16, 1999.
FIELD OF THE INVENTION
The present invention relates to the field of methods for detecting thyroid
carcinoma from thyroid cells in a clinical specimen.
BACK GROUND OF THE INVENTION
Mechanisms involving in the onset of arteriosclerosis have been gradually
elucidated in these days, and risk factors thereof have been identified.
Especially,
hypercholesterolemia, hypertension, diabetes, and smoking are recognized to be
manifest
four risk factors, thus the therapeutic treatments have been extensively
carried out.
Clinically common pathologies of these disease states are insulin resistance.
The meaning
of insulin resistance is nearly equivalent to the reduction of sensitivity to
insulin in cells,
thereby the actions of insulin upon the uptake of sugar into the cells are
deteriorated. The
insulin resistance may be caused due to the abnormalities in secretion of
insulin itself,
abnormalities of insulin receptors on target cells, abnormalities of an
intracellular signaling
system, and reduced supply of sugar to the tissue based on peripheral
circulatory disorder
that is caused hemodynamically. Reaven, 1988, reports that many pathological
states are
developed due to the insulin resistance, and designates a pathological state
as "syndrome
X" that may concurrently represent insulin resistance, glucose tolerance
abnormalities,
CA 02604143 2007-10-26
2
hyperinsulinemia, hypertriglyceridemia, hypo-HDL cholesterolemia and
hypertension, and
further suggests that the pathological state syndrome X closely participates
in the onset of
arteriosclerosis (Reaven, G. M. et al., Diabetes, 37, 1595-1607, 1988).
In addition, sugar supply to the cells is known to be generally decreased
through
insulin resistance, accompanied by enhancement of insulin secretion from
pancreas, thus
leading to hyperinsulinemia. Therefore, several problems in connection with
insulin
resistance have been raised in clinical fields. For example, insulin
resistance and
hyperinsulinemia are reported to promote- diabetic nephritis (Niskanen, L. et
al., Diabetes,
41, 736-741, 1993), and to elevate frequency of diabetic retinopathy (Yip, J.
et al., Lancet,
341, 369-370, 1993). Moreover, insulin resistance has been reported to enhance
plasminogen activator inhibitor 1 (PAI-1), to deteriorate the functions of a
blood
fibrinolytic system (Potter van Loon BJ et at, Metab. elfin. Exp., 42, 945-
954, 1993), and
to trigger arterial atherosclerosis (Sato, Y. et al., Diabetes, 38, 91-96,
1989).
Prevalence rate of diabetes accounts for 5% of the total population, and
approximately six millions of Japanese citizens are suffering from diabetes.
Diabetes
comprises insulin dependent diabetic mellitus (IDDM) and insulin independent
diabetic mellitus (NIDDM). Reportedly, IDDM accounts for about 7% of the total
diabetes cases, whilst NIDDM does about 90%. In particular, a significant
causative
factor of the onset of NIDDM that corresponds to a majority of diabetes has
been
conceived as the insulin resistance.
To date, tyrosine phosphorylation has been elucidated to play important roles
in signal transduction of insulin. Insulin receptor is a hetero-tetramer of
two
glycoprotein subunits, namely an a -subunit having a molecular weight of 135
kDa and
a 0 -subunit having a molecular weight of 95 kDa, which are bound through
disulfide
bonds resulting in a 20 2 structure. The a -subunit has an insulin binding
activity,
while the (3 -subunit has a protein tyrosine kinase (PTK) domain that is
activated by
CA 02604143 2007-10-26
3
autophosphorylation. Accordingly, when insulin is bound to thea-chain of an
insulin
receptor, certain tyrosine residues existing in the 0 -chain of the insulin
receptor are
autophosphorylated. The activity of insulin receptor tyrosine kinase is
further
promoted through the tyrosine autophosphorylation. It is reported that thus
activated
insulin receptor tyrosine kinase phosphorylates tyrosine residues of IRS
(insulin
receptor substrate), the intracellular substrates thereof, and signal
transduction is
proceeded through recognition and binding of the tyrosine-phosphorylated
insulin
receptors by AshJGrb2 or PI-3 kinase, - finally resulting in manifestation of
biological
activities of insulin, such as glucose uptake, sugar metabolism and cell
proliferation (see,
Fig. 9, Goldstein, B.J. et al., Receptor, 3, 1-15, 1993; and Kanai, F. et al.,
Biochemical and
Biophysical Research Communications, 195(2), 762-768, 1993). In this signal
transduction pathway, however, an enzyme tyrosine phosphatase, which
inactivates the
activated insulin receptors, i.e., protein tyrosine phosphatase (hereinafter
referred to as PTP),
has not been progressively studied.
The serious studies of PTPs were initiated after completion of cloning of
PTP1B
gene and elucidation of the nucleotide sequence thereof by Fischer et al in
1988, which is
cytoplasmic PTP derived from human placenta (Tonks, N. K. et al., J. Biol.
Chem., 263,
6722-6730, 1988; Charbonneau, H. et al., Proc. Natl. Acad Sci. USA, 85, 7182-
7186,
1988). Consequently, homology to PTP1B could be observed not with the known
serinelthreonine phosphatases but with two cytoplasmic regions of CD45, a
transmembranous molecule involved in a hemopoietic system. Moreover, CD45 was
also
revealed to have PTP activity (Tonks, N. K. et al., Biochemistry, 27, 8695-
8701, 1988; and
Charbonneau, H. et al., Proc. Natl. Acac Sci. USA, 85, 7182-7186, 1988).
To date, many PTPs have been cloned based on their homologies of cDNA
sequences, and new PTPs have been reported subsequently (Streuli, M. et al., I
&p. Med,
168, 1523-1530, 1988; Krueger, N. X. et al., EMBO J., 9, 3241-3252, 1990; and
CA 02604143 2007-10-26
4
Trowbridge, I. S. et al., Biochim. Biophys. Acta, 1095, 46-56, 1991). PTPs can
be
classified generally to: (1) membrane type PTPs having transmembrane region
(LCA,
leukocyte common antigen, namely CD45, as well as LAR and PTP a, f3, 'y, S, E
and Q, and
cytoplasm type PTPs without transmembrane region (PTPIB, TC-PTP, PTP-MEG,
PTPHI,
STEP and PTP 1 Q.
Many of membrane type PTPs have two PTP homologous domains inside the cell
(domain 1 and domain 2, see, Fig. 1(a) and (b)). A sequence comprising
cysteine
(signature motif), IleNal-His-Cys-Xaa-Ala-Gly-Xaa-Xaa-Arg-Ser/Thr-Gly (SEQ ID
NO:
2), has been conserved in the phosphatase domains between the PTPs reported
heretofore.
The research on crystallography of PTP1B revealed that the region forms small
pockets on
the surface of a PTP molecule, and that the cysteine residue is located to the
bottom of the
pocket, participating directly in binding of the molecule to phosphate
(Barford, D. et al.,
Science, 263, 1397-1404, 1994). In addition, it was also revealed that the
depth of the
pocket of PTP may determine the specificity of serine/threonine phosphatase
because
phosphate that is binding to serine or threonine cannot reach to the pocket of
the enzymatic
active center of PTP1B. Moreover, the importance of the above-mentioned
signature
motif in exhibiting the enzymatic activity has been elucidated (Streuli, M. et
al., EMBO 1,
9, 2399-2407, 1990). Taking into account of these observations, it has been
conceived
that the conserved cysteine in domain 1 may play an important role in
exhibiting the
enzymatic activity, and domain 2 may determine the substrate specificity of
the enzyme.
Among a group of PTPs, LAR derived from human is a prototype of receptor type
protein tyrosine phosphatases, which was cloned from human placental genome
library
using a phosphatase domain of CD45, a receptor type protein tyrosine
phosphatase, as a
probe (Streuli M. et al., J. Exp. Med., 168, 1553-1562, 1988). CD45 is
specifically
expressed on hemocytic cells, whilst LAR is expressed on the cells other than
hemocytic
cells, particularly in insulin sensitive organs such as liver and skeletal
muscle (Goldstein B.
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J., Receptor, 3, 1-15, 1993). LAR is especially interesting among many types
of receptor
type PTPs due to its similarity of the extracellular domain with cell adhesion
molecules.
The entire structure of LAR is elucidated as having 150 kDa of extracellular E-
subunit that
consists of Ig-like domains and fibronectin type III domains, and 85 kDa of P-
subunit
comprising a transmembrane region and an intracellular domain having two
phosphatase
domains, which are covalently bound immediately outside of the cell membrane
(see, Fig. 1,
Streuli M. et al., EMBO J., 11, 897-907, 1992).
A large number of functional roles of LAR have been reported to date. For
example, it was reported that: responses to neurotrophin are decreased in LAR
deficient
nerves (Yang, T. et al., 27th Annual Meeting of the Society for Neuroscience,
New
Orleans, Louisiana, USA, October 25-30, 1997, Society for Neuroscience
Abstracts, 23,
1-2, 1997); secretion of apolipoprotein B is decreased by suppression of LAR
activity
(Phung, T. L. et al., Biochemical and Biophysical Research Communications,
237(2),
367-371, 1997); and loss of expression of LAR diminishes the size of
cholinergic nerve
cells of prosencephalon basement, thus control by the cholinergic nerve cells
at
hippocampal dentate gyros is deteriorated (Yeo, T. T. et al., J. Neurosci.
Res., 47(3),
348-360, 1997). In such a manner, it has been gradually revealed that LAR is
bearing
several important roles in a living body. Furthermore at present, the most
remarkable
researches are directed to the relationships between LAR and insulin receptors
(Hashimoto,
N. et al., J. Biol. Chem., 267(20), 13811-13814, 1992).
In 1995, a literature was presented which should be noted, reporting that LAR
tyrosine phosphatase activity is abnormally increased in adipose tissue of an
obese person,
with such an increase being suggested as a cause of onset of insulin
resistance and a risk
factor of cardiovascular diseases (Ahmad, F. et al., J. Clin. Invest., 95(6)
2806-2812, 1995).
Several reports followed thereafter illustrating that LAR is closely concerned
with insulin
receptors (Mooney, R. A. et al., Biochemical and Biophysical Research
Communications,
CA 02604143 2007-10-26
6
235(3), 709-712, 1997; Orr, S. R et at., Biochemical Society Transaction,
25(3), 452S,
1997; Ahmad, F. et al., J Clin. Investigation, 100(2), 449-458, 1997; Ahmad,
F. et al., J
Biol. Chem., 272(1), 448-457, 1997; Norris, K. et al, Febs Letters, 415(3),
243-248, 1997;
and Li, P. M. et al., Cellular Signaling, 8(7), 467-473, 1996). Further, on
the basis of
such information, Ahmad, F. et al recently reported that PTPIB may be a
therapeutic
target of disease states involving in insulin resistance (Ahmad, F. et al.,
Metabolism,
Clinical and Experimental, 46(10), 1140-1145, 1997). From the researches to
date on
PTPs such as LAR, CD45 and the like, it has been elucidated that PTPs bear
extremely
important roles in an intracellular signaling system.
In 1992, Streuli el al. reported that binding between LAR E-subunit and P-
subunit
may be dissociated due to the noncovalency of their binding, and thus E-
subunit is
specifically shed from the cell membrane surface (Streuli, M. et al., E 4BO
J., 11(3),
897-907, 1992). However, because many researchers have focused the studies
using
polyclonal or monoclonal antibodies elicited against a LAR E-subunit that is
an
extracellular domain thereof, a P-subunit even solely having phosphatase
activities has been
ignored. For example, when an anti-LAR antibody is used intending measurement
of
LAR phosphatase assay, total phosphatase activity could not be measured unless
an
antibody to P-subunit is employed. In view of such circumstances, the present
inventors
started to produce antibodies that specifically bind to a LAR P-subunit,
particularly to an
intracellular domain thereof, without any specificity to CD45.
Known antibodies to protein tyrosine phosphatase include an antibody
generated using 196 amino acid residues as an antigen spanning from the
transmembrane region of CD45 to a part of phosphatase domain I (Transduction
Laboratories). However, it is unclear how these antibodies are immunospecific
to
phosphatase domains of LAR and the other protein tyrosine phosphatases.
Therefore,
it was also necessary to produce antibodies which are specific to a LAR
intracellular
CA 02604143 2007-10-26
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domain but not to CD45.
Thyroid tumors include benign adenoma and malignant carcinoma. At present,
palpation, ultrasonic diagnosis, fine needle aspiration cytology, and
diagnosis on tissue
sections are clinically carried out in order to diagnose thyroid tumors.
Thyroid
tumors can be classified into adenoma, papillary carcinoma, follicular
carcinoma,
undifferentiated (anaplastic) carcinoma, medullary carcinoma and malignant
lymphoma, whilst thyroid carcinoma (papillary and follicular cancers) can be
generally
classified into differentiated and poorly differentiated types.
On diagnosis of thyroid carcinoma, if abnormalities were found on palpation
and ultrasonic diagnosis, cytological examination with fine needle aspiration
has been
predominantly carried out because of fewer burdens to the patient, and in
difficult
cases where definite diagnosis is impossible, additional histological
diagnosis is
carried out in which thyroid tissue is excised. However, such histological
diagnosis
imposes more burdens to the patient, and there exist possibilities to excise
together
with normal tissue. In fact, discrimination by cytological examination is
often
difficult to draw exact diagnosis, thus many cases have been nevertheless
entrusted to
histological examination. Additionally, fine needle aspiration cytology does
not
result in definite diagnosis because cell-cell bindings may be destroyed in
those
specimens compared to the morphologic observation on tissue sections.
Furthermore,
in almost cases of follicular carcinoma, discrimination between benign and
malignant
tumors can be difficult even though histological diagnisis is performed as
well as
cytological examination. Accordingly, it has been strongly desired by
clinicians or
pathologists to develop new tools that can discriminate benign/malignant
tumors in
fine needle aspiration cytology even in such difficult cases for diagnosis as
in follicular
carcinoma.
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SUMMARY OF THE INVENTION
Certain exemplary embodiments provide a method for detecting thyroid
carcinoma from thyroid cells in a clinical specimen comprising the steps of:
(a) determining expression amounts of leukocyte antigen related (LAR) gene in
the
specimen as an expressed protein with anti-LAR antibody which is produced by a
hybridoma cell line with Accession No. FERM BP-6343 and has specificity to the
LAR
protein; (b) comparing the expression amount determined in step (a) with those
determined on the normal specimens; and (c) verifying thyroid carcinoma in the
clinical
specimen when the expression amount is larger than those on the normal
specimens in
step (b).
One aspect of the present invention is to provide an antibody that
specifically
binds to a phosphatase subunit of LAR, particularly an antibody that
specifically binds
to an intracellular domain of LAR. Further, in accordance with the present
invention, an
antibody is provided that specifically binds to an intracellular domain of a
LAR
phosphatase subunit, without specificity to other protein phosphatases.
The antibody may be preferably generated using a polypeptide corresponding to
an intracellular domain of LAR, encoded by a nucleotide sequence set forth in
SEQ ID
NO: 1 or any of fragments thereof as an antigen. Further, preferred antibody
may be a
monoclonal antibody because of its immunospecific property.
In accordance with another aspect of the present invention there is provided a
monoclonal antibody that specifically immunoreacts with an intracellular
domain of a
LAR phosphatase subunit, wherein the antibody does not cross-react with CD45.
In accordance with yet another aspect of the present invention there is
provided a
monoclonal antibody produced by a hybridoma cell line with Accession No. FERM
BP-6343.
In accordance with still yet another aspect of the present invention there is
provided a hybridoma cell line with Accession No. FERM BP-6343.
In accordance with still yet another aspect of the present invention there is
provided a method for generating a monoclonal antibody that specifically
immunoreacts
with an intracellular domain of a LAR phosphatase subunit and does not cross-
react
with CD45, the method comprising the steps of: immunizing an animal with a
fusion
protein comprising a LAR phosphatase domain and another protein or polypeptide
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8a
fragment; preparing a hybridoma cell line from an antibody-producing cell
obtained
from the immunized animal; and producing a monoclonal antibody from the
hybridoma
cell line, wherein the antibody specifically immunoreacts with an
intracellular domain
of a LAR phosphatase subunit, and wherein the antibody does not cross-react
with
CD45.
In accordance with still yet another aspect of the present invention there is
provided a method for generating a monoclonal antibody that specifically
immunoreacts
with an intracellular domain of a LAR phosphatase subunit and does not cross-
react
with CD45, the method comprising the steps of. immunizing an animal with a GST-
LAR phosphatase domain fusion protein; preparing a hybridoma cell line from an
antibody-producing cell obtained from the immunized animal; and producing a
monoclonal antibody from the hybridoma cell line, wherein the antibody
specifically
immunoreacts with an intracellular domain of a LAR phosphatase subunit, and
wherein
the antibody does not cross-react with CD45.
In accordance with still yet another aspect of the present invention there is
provided a method for quantitatively determining LAR and/or LAR derived
molecules
comprising the step of. determining an amount of LAR protein and/or a fragment
or a
polypeptide that comprises a LAR intracellular domain, which is contained in a
test
sample with a monoclonal antibody that specifically immunoreacts with an
intracellular
domain of a LAR phosphatase subunit and does not cross-react with CD45.
In accordance with still yet another aspect of the present invention there is
provided a method for quantitatively determining LAR and/or LAR derived
molecules
comprising the steps of: isolating LAR protein and/or a fragment or a
polypeptide that
comprises a LAR intracellular domain from a test sample with a monoclonal
antibody
that specifically immunoreacts with an intracellular domain of a LAR
phosphatase
subunit and does not cross-react with CD45; and measuring an activity of the
isolated
LAR and/or LAR derived molecules.
In accordance with still yet another aspect of the present invention there is
provided a method for producing LAR and/or LAR derived molecules comprising
the
step of: isolating LAR protein and/or a fragment or a polypeptide that
comprises a LAR
intracellular domain with a monoclonal antibody that specifically immunoreacts
with an
intracellular domain of a LAR phosphatase subunit and does not cross-react
with CD45.
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8b
In accordance with still yet another aspect of the present invention there is
provided a method for identifying LAR and/or LAR derived molecules within
tissues
comprising the step of. immunohistologically examining the tissue specimen
with a
monoclonal antibody that specifically immunoreacts with an intracellular
domain of a
LAR phosphatase subunit and does not cross-react with CD45; and detecting,
within the
tissue specimen, LAR protein and/or a fragment or a polypeptide that comprises
a LAR
intracellular domain.
In accordance with still yet another aspect of the present invention there is
provided an anti-LAR monoclonal antibody that specifically immunoreacts with
thyroid
carcinoma cells.
In accordance with still yet another aspect of the present invention there is
provided a method for detecting thyroid carcinoma from thyroid cells in a
clinical
specimen comprising the steps of. (a) determining expression amount of LAR
gene in
the specimen as an expressed LAR protein with anti-LAR antibody which has
specificity to the LAR protein; (b) comparing the expression amount determined
in
step (a) with those determined on normal specimens; and (c) verifying thyroid
carcinoma in the clinical specimen when the expression amount is larger than
those on
the normal specimens in the step (b).
In accordance with still yet another aspect of the present invention there is
provided a method for detecting thyroid carcinoma from thyroid cells in a
clinical
specimen comprising the steps of. (i) determining expression amount of LAR
gene in
the specimen as mRNA with polynucleotide which has reactivity to the mRNA
produced through transcription of the LAR gene; (ii) comparing the expression
amount
determined in step (i) with those determined on normal specimens; and (iii)
verifying
thyroid carcinoma in the clinical specimen when the expression amount is
larger than
those on the normal specimens in the step (ii).
In accordance with still yet another aspect of the present invention there is
provided a method for detecting thyroid carcinoma from thyroid cells in a
clinical
specimen through RT-PCR methodology comprising the steps of. (1) preparing a
complementary DNA from mRNA in the specimen according to reverse transcription
thereof; (2) annealing, to the complementary DNA, a polynucleotide having
reactivity to
LAR gene; (3) synthesizing double strands consisting of the LAR gene or a
fragment
CA 02604143 2011-04-28
Sc
thereof from two or more of the annealed polynucleotides under the presence of
DNA
polymerase; (4) amplifying the double strands by repeating the steps (2)-(3)
above;
(5) comparing amount of the double strands amplified at the step (4) with
those from the
normal specimens to be made according to the foregoing steps (1)-(4); and (6)
verifying
thyroid carcinoma in the clinical specimen when the expression amount is
larger than
those on the normal specimens in the step (5).
In accordance with still yet another aspect of the present invention there is
provided a method for detecting thyroid carcinoma from thyroid cells in a
clinical
specimen comprising the steps of. (a) determining expression amount of
leukocyte
antigen related (LAR) gene in the specimen as an expressed protein with anti-
LAR
antibody which has specificity to the LAR protein; (b) comparing the
expression
amount determined in step (a) with those determined on at least one normal
specimen;
and (c) verifying thyroid carcinoma in the clinical specimen when the
expression
amount is larger than those on the at least one normal specimen in step (b).
In accordance with still yet another aspect of the present invention there is
provided a method for detecting thyroid carcinoma in thyroid cells in a
clinical
specimen comprising the steps of. (a) determining an expression amount of
leukocyte
antigen related (LAR) gene in the specimen by measuring a level of mRNA with a
polynucleotide which has reactivity to the mRNA produced through transcription
of the
LAR gene; (b) comparing the expression amount determined in step (a) with
those
determined on at least one normal specimen; and (c) verifying thyroid
carcinoma in the
clinical specimen when the expression amount is larger than those in the at
least one
normal specimen in step (b).
In accordance with still yet another aspect of the present invention there is
provided a method for detecting thyroid carcinoma in thyroid cells in a
clinical
specimen comprising the steps of. (a) preparing complementary DNA from mRNA in
the specimen according to reverse transcription thereof; (b) annealing, to at
least some
of the complementary DNA, a polynucleotide primer having a sequence
corresponding
to at least a portion of leukocyte antigen related (LAR) gene; (c)
synthesizing double
stranded DNA from the primer in the presence of DNA polymerase; (d) amplifying
the
double stranded DNA by repeating steps (b)-(c) above; (e) comparing an amount
of the
double stranded DNA amplified in step (d) with those from at least one normal
CA 02604143 2011-04-28
8d
specimen obtained according to steps (a)-(d); and (f) verifying thyroid
carcinoma in the
clinical specimen when the expression amount is larger than those on the at
least one
normal specimen in step (e).
In accordance with still yet another aspect of the present invention there is
provided a method for detecting thyroid carcinoma in thyroid cells in a
clinical
specimen comprising the steps of. (a) determining expression amount of
leukocyte
antigen related (LAR) gene in the specimen as an expressed protein with an
anti-LAR
antibody which has specificity to the LAR protein; (b) comparing the
expression
amount determined in step (a) with those determined on at least one normal
specimen;
and (c) verifying thyroid carcinoma in the clinical specimen when the
expression
amount is larger than those on the at least one normal specimen in step (b).
Such an antibody may be generated using a fusion protein comprising a LAR
phosphatase domain and another protein or a polypeptide fragment, as an
antigen. As
the another protein or a polypeptide fragment to be a member of the fusion
protein, GST
(glutathione-S-transferase) may be particularly suitable, besides,
polyhistidine
(preferably 6 histidine residues), calmodulin binding peptide (CBP), protein A
may be
employed.
CA 02604143 2007-10-26
9
When polyhistidine is employed, absorption to nickel-chelating resin can be
utilized for isolation and purification of the fusion protein expressed by a
gene
recombinant process, wherein addition of EDTA or imidazole substance as well
as pH
change may be adopted for dissociating the protein from the resin. When CBP is
employed, the expressed fusion protein may be subjected to an affinity
chromatography
using calmodulin affinity resin, and then may be dissociated from the resin by
adding
EGTA. In addition, when protein A is employed, the expressed fusion protein
may be
subjected to an affinity chromatography using IgGSepharose'(e.g., IgG
Sepharose 6FF),
and then may be dissociated from the resin by changing pH.
Moreover, another candidate for a protein or a polypeptide fragment to be
TM
employed in the fusion protein may include for example, Xpress, Thioredoxin, c-
myc, V5,
HA/c-myc and the like. For isolation and purification of the intended fusion
protein with a
LAR phosphatase domain, expression of the protein may be followed by
subjecting to an
antigen-antibody affinity column.
The aforementioned preferable immunogen of the present invention, namely a
fusion protein of GST and a LAR phosphatase domain, may be suitably produced
by:
culturing Escherichia coli transformed or transfected with an expression
vector
comprising a coding region of GST gene and a coding region of a phosphatase
domain
of LAR gene at 20-30 C for 16-24 hours, preferably at 23-25 C for 18 hours;
and then
isolating the fusion protein from the culture fluid and/or bacterial cells.
Thus
obtained fusion protein may be further purified based on an affinity to a
support
carrying glutathione, e.g., glutathione sepharose beads, wherein the elution
of the
fusion protein from the glutathione sepharose beads may be performed by
boiling in
the presence of a detergent. The detergent may include sodium dodecyl sulfate,
CHAPS (3-[(3-cholamide propyl) dimethylammonio]-1-puropane sulfonate),
deoxycholic
acid, digitonin, n-dodecylmaltoside (1-O-n-dodecyl-fi -D-glucopyranosyl (1-4)
CA 02604143 2007-10-26
a-D-glucopyranoside), NonidetTM P40 (ethylphenolpoly (ethylene glycol
ether)n),
n-octylglucoside (1-O-n-octyl-(3 -D-glucopyranoside), sucrose monolaurate,
TesitTM
(dodecylpoly (ethylene glycol ether)n), Triton"' X-100 (octylphenolpoly
(ethylene glycol
ether)n), TweenTM 20 (poly (oxyethylene) n-sorbitan-monolaurate),
N-dodecyl-N,N-dimethyl-3-ammonio-l-propanesulfonate, and the like [any of `n'
represents an integer number which is more than or equal to 1]. When elution
of the
fusion protein is carried out, the resin may be boiled at 100 C for 5-10
minutes in the
presence of such detergents at a concentration that does not lead any problems
to an animal
to be administered, preferably 0.1% of sodium dodecylsulfate. Accordingly, a
purified
fusion protein, which is preferable as a contemplated immunogen, can be
obtained.
When a monoclonal antibody is generated using such a fusion protein as an
immunogen, a LAR phosphatase subunit may be employed for screening the
antibody,
however, it is more preferable to perform the screening using the fusion
protein as an
immunogen in terms of the specificity.
The exemplary monoclonal antibody of the present invention may include a
monoclonal antibody having a molecular weight of about 150 kDa that is
produced from
mouse/mouse hybridoma cells. The antibody can be applied as a tool for further
elucidation of the mechanisms of an insulin signaling system, for developing
useful
diagnostic methods of insulin resistance and NIDDM, and for prophylaxis,
therapeutics and
diagnosis of several kinds of pathological states relating to syndrome X based
on insulin
resistance. Moreover, the antibody of the present invention may be useful for
identification and acquisition of LAR related molecules, for example,
modulator, binding
protein and the like.
Further aspect of the present invention is to provide a hybridoma cell line
producing the above-mentioned monoclonal antibody. Such a hybridoma cell line
may
include mouse/mouse hybridoma cell line YU1, which was deposited on May 7,
1998, with
CA 02604143 2007-10-26
11
National Institute of Bioscience and Human-Technology, Agency of Industrial
Science and
Technology, 1-1-320, Higashi, Tsukuba, Ibaraki, JAPAN, and assigned Accession
No.
FERM BP-6343.
The antibody of the present invention has specific immunoreactivity with LAR
protein, and fragments and polypeptides that comprise at least a LAR
intracellular domain
(the fragment and polypeptide are hereinafter collectively referred to as `LAR
derived
molecules'), which was derived from natural sources, or wholly or partially
synthesized
(such as those chemically synthesized, or recombinantly synthesized).
Another aspect of the present invention is to provide a method for generating
an
antibody having specificity to a LAR phosphatase subunit, wherein the
aforementioned
fusion protein comprising a LAR phosphatase domain and another protein or a
polypeptide
fragment, preferably a GST-LAR phosphatase domain fusion protein, is employed
as an
immunogen. In this aspect of the present invention, the available another
protein or a
polypeptide fragment except GST to be a member of the fusion protein, and
purification
process of the fusion protein are as set forth above.
Further, a fusion protein comprising GST and a LAR phosphatase domain
which is a preferable immunogen may be suitably produced by: culturing
Escherichia
coli transformed or transfected with an expression vector comprising a coding
region
of GST gene and a coding region of a phosphatase domain of LAR gene at 20-30 C
for
16-24 hours, preferably at 23-25 C for 18 hours; and then isolating the fusion
protein
from the culture fluid and/or bacterial cells. Thus obtained fusion protein
may also be
further purified based on an affinity to a support carrying glutathione, e.g.,
glutathione
sepharose beads wherein the elution of the fusion protein from the glutathione
sepharose beads may be performed by boiling in the presence of a detergent, as
set
forth above, and then, for eluting the fusion protein, the resin may be boiled
at 100 C for
5-10 minutes in the presence of the detergent at a concentration which does
not lead any
CA 02604143 2007-10-26
12
problems to an animal to be administered, preferably 0.1% of sodium
dodecylsulfate.
Accordingly, the purified fusion protein, which is preferable as a
contemplated immunogen,
can be obtained.
In a method of generating a monoclonal antibody by using such a fusion protein
as
an immunogen, a LAR phosphatase subunit may be employed for screening the
antibody,
however, it is more preferable to perform the screening using the fusion
protein as an
immunogen in terms of the specificity.
The present invention further provides a method of quantitative determination
of
LAR and/or LAR derived molecules. The method is characterized by comprising
the
steps of determining an amount of LAR protein and/or a fragment or a
polypeptide that
comprises at least a LAR intracellular domain, which is contained in a test
sample using an
antibody set forth above. In this method, the antibody set forth above is used
preferably in
any of immunoblotting, immunoprecipitation and ELISA, for determining the
amount of
LAR or LAR derived molecules.
Still another aspect of the present invention is to provide a method for
quantitative determination of LAR and/or LAR derived molecules comprising the
step of
isolating LAR and/or LAR derived molecules from a test sample using the
antibody set
forth above; and measuring an activity of the isolated LAR and/or LAR derived
molecules.
In this method, in order to isolate the LAR and/or LAR derived molecules,
affinity
chromatography and/or immunoprecipitation by using a support that was bound
with the
aforementioned antibody are suitably utilized. Namely, affinity chromatography
using a
column or batch wise method, and/or immunoprecipitation may be performed
wherein the
support which was previously bound with the antibody is contacted with a test
sample to
allow specific interaction between antigen (LAR/LAR derived molecules) and
antibody,
then after washing the unbound antibody, the bound LAR/LAR derived molecules
may be
eluted.
CA 02604143 2007-10-26
13
In yet another aspect of the present invention; a method for producing LAR
and/or LAR derived molecules is provided, comprising the step of. isolating
LAR and/or
LAR derived molecules using the antibody set forth above. Isolation of the
targeted
molecules in the method for production may be suitably carried out by affinity
and/or
immunoprecipitation by using a support that was previously bound with the
antibody, as in
the aforementioned method of quantitative determination of LAR and/or LAR
derived
molecules.
Further aspect contemplated by the present invention is to provide a method
for
identifying the presence of LAR and/or LAR derived molecules within tissue
comprising
the step of. performing immunohistological examination using the
aforementioned
antibody. As the immunohistological examination, for example, in situ
immunohistological staining with a labelled antibody may be adopted, thus LAR
protein
and/or a fragment or a polypeptide that comprises at least a LAR intracellular
domain, can
be detected.
The present invention is further directed to a specific anti-LAR antibody to
thyroid
carcinoma cells. The antibodies may be those elicited using a LAR molecule as
well as
the fragment thereof, e.g., a phosphatase domain, an extracellular domain or
the like as an
antigen, and may include monoclonal and polyclonal antibodies, peptide
antibodies, single
chain antibodies, chimeric antibodies, humanized antibodies, CDR-grafted
antibodies and
the like. Particularly, the aforementioned antibodies to a LAR phosphatase
subunit, those
having immunoreactivity with thyroid carcinoma cells are provided by the
present
invention. Herein, "having immunoreactivity with thyroid carcinoma cells"
means that
almost no immunoreactivity with normal thyroid cells or benign tumor thyroid
cells (less
than or equal to 10% of the normal cells) is exhibited, whilst higher
immunoreactivity to the
thyroid carcinoma (more than or equal to 20% of the carcinoma cells) is
exhibited.
CA 02604143 2007-10-26
14
Accordingly, it makes possible to diagnose thyroid cancer through utilizing
such
an antibody, thus a method for histological diagnosis of thyroid carcinoma is
also
contemplated by the present invention. The diagnostic method is characterized
by
comprising the steps of. taking a thyroid tissue sample (specimen) from a
subject suspected
as suffering from thyroid cancer, and conducting diagnosis of thyroid cancer
through
evaluating immunoreactivity between the antibody set forth above and the
tissue specimen.
In this instance, the thyroid tissue specimens may be any of the specimens
such as those
taken by fine needle aspiration from a subject, or those prepared by excision
and extirpation
of a part of the thyroid. The diagnostic method where the specimens taken by
fine needle
aspiration are employed is more preferable in respect of lower invasiveness to
the
subject. This is an important advantage provided by the present invention
taking into
account of the nature of the diagnostic method of thyroid cancer based on the
histological observation of the tissue, where highly invasive incision
procedure has
been obliged to practice. Additionally, also in the immunohistochemical
diagnostic
method utilizing the tissue section, the present invention is more useful
because more
prominent reliability can be achieved than in the conventional method.
In the above-described diagnostic method, the specimens taken by fine needle
aspiration may be evaluated for their immunoreactivity by common in vitro
immunoassays e.g., immunoblotting, immunoprecipitation, ELISA or the like,
using
the antibody of the present invention. In contrast, when tissue sections are
used as
specimens, conventional immunohistochemical staining techniques can be
utilized to
determine the immunoreactivity based on immune responses.
Moreover, the present invention provides a composition for histological
diagnosis of thyroid carcinoma comprising the aforementioned antibody.
Markedly
reliable diagnostic method of thyroid cancer as set forth above can be
performed using
CA 02604143 2007-10-26
this composition. The composition may include excipient, carrier, buffer,
agent for
stabilizing the antibody and the like ad libitum, in addition to the antibody.
Consequently, in accordance with the present invention, specific and high
expression of LAR in thyroid carcinoma cell was revealed. Furthermore, it was
also
verified that monoclonal antibody of the present invention is useful for
diagnosis of
thyroid-cancer as illustrated in Examples. Additionally, the monoclonal
antibody of
the present invention was proven to be useful for the diagnosis of thyroid
carcinoma
where tissue sections are employed (see, Example 5, 6), and for the diagnosis
where
homogenized tissue is employed (see, Example 7). From these results, the
person
skilled in this art will comprehend that the monoclonal antibodies of the
present
invention are useful for several kinds of cytological or histological
diagnoses or biopsy.
Moreover, besides the present monoclonal antibodies, monoclonal antibodies,
polyclonal antibodies, and/or peptide antibodies that can recognize a LAR
extracellular
domain may also be utilized in such processes. Again in such cases, the
processes
may be nevertheless practiced similarly to those where the present monoclonal
antibodies are employed, however, effects resulting from release of the
extracellular
domain would be preferably considered.
It was revealed by the present invention that the antibodies to LAR can be
utilized
for diagnosis and therapy of diseases related to thyroid carcinoma. An amount
of LAR or
a fragment thereof may be determined using such an antibody on the basis of
immunological binding between them. Specifically, the method of determining an
amount of LAR or a fragment thereof may include for example, a sandwich method
wherein sandwich complex is detected that was produced by an immunoreaction of
LAR or
a fragment thereof with an antibody bound to an insoluble support and labelled
antibody;
and a method wherein an amount of LAR or a fragment thereof in a sample is
determined
utilizing a competition method by competitively immunoreacting LAR or a
fragment
CA 02604143 2007-10-26
16
thereof and labelled LAR with the antibody, and then determining an amount of
LAR or a
fragment thereof from the amount of the labelled antigen that bound to the
antibody.
When an amount of LAR or a fragment thereof is determined by the sandwich
method, a two steps method wherein an immunoreaction of an immobilized
antibody with
LAR or a fragment thereof is allowed first, then unreacted substances are
completely
removed by washing, a labelled antibody is added thereto thus a labelled
antibody-LAR or
a fragment thereof complex is formed; or a one step method wherein an
immobilized
antibody, a labelled antibody, and LAR or a fragment thereof are
simultaneously mixed.
Insoluble support used for such determination may include for example,
synthetic
resin such as polystyrene, polyethylene, polypropylene, polychlorinated vinyl,
polyester,
polyacrylate ester, nylon, polyacetal, fluorine contained resin and the like;
polysaccharides
such as cellulose, agarose and the like; glass; and metal etc. The insoluble
support may be
in several forms for example, tray-like, spherical, fibrous, cylindrical,
discal, vessel-like,
cell-like, tubular and so on. The support with the absorbed antibody is stored
in a cold
place, in the presence of an antiseptic agent such as sodium azide.
For immobilization of an antibody, a known chemical binding method or a
physical absorption method may be adopted. The chemical binding method may
include
for example, glutaraldehyde-utilizing method, maleimide method wherein N-
succinimidyl-4-(N-maleimidemethyl) cyclohexane-l-carboxylate, N-
succinimidyl-2-maleimideacetate or the like may be used, carbodiimide method
wherein
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride may be used. In
addition,
maleimidebenzoyl-N-hydroxysuccinimide ester method, N-succinimidyl-3-(2-
pyridylthio)
propionic acid method, bisdiazotized benzidine method, dipalmityllysine method
may be
included. Alternatively, a complex which was previously formed by a reaction
of a
detection test material with an antibody of which epitope is in a different
kind, may also be
captured after the third antibody to said antibody is immobilized according to
the method as
CA 02604143 2007-10-26
17
mentioned above.
The material to be used for labelling the antibody may be enzyme, fluorescent
material, luminescence material, radioactive material, metal chelate or the
like. An
enzyme may include for example, peroxidase, alkaline phosphatase, (3 -D-
galactosidase,
malate dehydrogenase, staphylococcus nuclease, delta-5-steroid isomerase,
a-glycerolphosphate dehydrogenase, triose phosphate isomerase, horseradish
peroxidase,
asparaginase, glucose oxidase, ribonuclease, urease, catalase, glucose-6-
phosphate
dehydrogenase, glucoamylase, acetylcholine esterase, and the like; and
fluorescent material
may include for example, fluorescein isothiocyanate, phycobilin protein,
rhodamine,
phycoerythrin, phycocyanin, orthophthalic aldehyde and the like; luminescence
material
may include isoluminol, lucigenin, luminol, aromatic acridiniumester,
imidazole,
acridinium salt and modified ester thereof, luciferin, luciferase, aequorin
and the like; and
radioactive material may include 1251, 1271, 131L '4C 3H, 32P 35S and the
like, but not limited
thereto as long as the material can be used in immunological determination
methods.
Further, low molecular weight hapten such as biotin, dinitrophenyl, pyridoxal
or
fluorescamine may be conjugated to the antibody. Preferably horseradish
peroxidase may
be used as a labelling enzyme. This enzyme can react with a lot of substrates,
while being
readily conjugated to the antibody by a periodic acid method.
When an enzyme is used as a labelling material, a substrate for measuring its
activity, and a color-developing agent are employed. When peroxidase is used
as an
enzyme, H202 may be used as a substrate solution, and 2,2'-azino-di-[3-
ethylbenzthiazolin
sulfonate] ammonium (ABTS), 5-aminosalicylic acid, orthophenylenediamine,
4-aminoantipyrine, 3,3',5,5'-tetramethylbenzidine or the like may be used as a
color-developing agent; when alkaline phosphatase is employed as an enzyme,
the substrate
may be orthophenylphosphate, paranitrophenylphosphate or the like;
alternatively, when
0 -D-galactosidase is used as an enzyme, the substrate may be
CA 02604143 2007-10-26
18
fluorescein-di-((3-D-galactopyranoside), 4-methyl-umbelliferyl-D-
galactopyranoside, or the
like.
The present invention further contemplates a kit, which comprises the
above-described monoclonal antibody or polyclonal antibody, and reagents.
As a crosslinking agent, N,N'-orthophenylenedimaleimide,
4-(N-maleimidemethyl) cyclohexanoyl N-succinimide ester, 6-maleimidehexanoyl
N-succinimide ester, 4,4'-dithiopyridine, orthophenylenedimaleimide,
4-(N-maleimidemethyl) cyclohexanoyl N-succinimide ester, 6-maleimidehexanoyl
N-succinimide ester, 4,4'-dithiopyridine, or other known crosslinking agents
can be utilized.
The reaction of such a crosslinking agent with the enzyme and the antibody may
be
proceeded in accordance with known methods depending upon the properties of
the
crosslinking agent that was employed.
Additionally, the antibodies to be used may be any fragments of these
antibodies
for example, Fab', Fab, F(ab')2 depending on conditions. Furthermore, an
enzymatically
labelled antibody can be obtained by a similar process in either case of
polyclonal antibody
or monoclonal antibody, whichever. When the enzymatically labelled antibody
that was
obtained by using the aforementioned crosslinking agent is purified by any
known methods,
more sensitive immunological determination system can be achieved. The
enzymatically
labelled antibody that was purified in such a manner may be mixed with a
stabilizer such as
thimerosal, glycerol or the like, alternatively, may be lyophilized, and then
stored in a cold
and dark place.
The present invention further provides a DDS (Drug Delivery System)
formulation that was targeted to thyroid carcinoma cells using the above-
described antibody
having specific immunoreactivity to thyroid carcinoma cells.
It have been elucidated that several genes are involved in thyroid carcinoma.
Mutations in tyrosine kinase domain of Ret or TRK gene were found in some of
the
CA 02604143 2007-10-26
19
patients suffering from papillary carcinoma (Fusco, A. et al., Nature, 328,
170-2, 1987).
Moreover, mutation in Ret gene was observed in 3-30% of the papillary
carcinoma patients
without any history of a radiation exposure in the past (Santoro, M. et al.,
J. Clin Invest.,
89, 1517-22, 1992; Bongdrzone, I. et al., I Clin. Endocrinol. Metab., 81, 2006-
9, 1996;
Zou, M. et al., Cancer, 73, 176-80, 1994), whilst higher incidence of 60-80%
of the Ret
mutation was observed in papillary carcinoma diagnosed, for children with
experiences of
radiation exposure on the disaster at Chernobyl nuclear power plant, or the
patients who
have case histories of radiation exposure in their childhood (Fugazzola, L. et
al., Cancer
Res., 55, 5617-20, 1995; Klugbauer, S. et al, Oncogene, 11, 2459-67, 1995;
Nikiforov, Y.
E. et al., Cancer Res., 57, 1690-4, 1997; Bounacer, A. et al., Oncogene, 15,
1263-73, 1997),
and frequency of TRK gene mutation is significantly low (Bongdrzone, I. et
al., J. Clin.
Endocrinol. Metab., 81, 2006-9, 1996). Point mutation of Ras gene is
frequently observed
in goiter and thyroid follicular carcinoma. The basis of this fact is
conceived as Ras gene
point mutation in an early stage of tumor development (Fagin, J. A., Molecular
pathogenesis. In: Braverman LE, Utiger RD, eds. Werner and Ingbar's, the
thyroid: a
fundamental and clinical text. 7th ed. Philadelphia: Lippincott-Raven, 909-16,
1996;
Challeton, C. et al., Oncogene, 11, 601-3, 1995). The mutation of genes
encoding TSH
and stimulatory G protein is reported in some cases of thyroid follicular
carcinoma
(Challeton, C. et at., Oncogene, 11, 601-3, 1995; Russo, D. et al., Oncogene,
11, 1907- 11,
1995). Further, it was also reported that mutation of tumor suppressor gene
p53 is rare in
differentiated thyroid carcinoma, however, it is frequently found in
undifferentiated
carcinoma (Fagin, J. A. et al., I Clin. Invest., 91, 179-84, 1993; Ito, T. et
al., Cancer Res.,
52, 1369-71, 1992).
According to such known information, a nucleic with the object of therapy or
diagnosis of thyroid cancer can be included in a DDS formulation targeted by
an antibody
to LAR.
CA 02604143 2007-10-26
Furthermore, it is also known that proliferation of thyroid carcinoma is
regulated by thyroid stimulating hormone (TSH), and that suppression of TSH
secretion
by administrating a thyroid hormone drug may improve recurrence, survival rate
or the
like of thyroid tumor. Accordingly, a protein, a nucleic acid or a compound
that may
inhibit TSH stimulation can be also included in the DDS formulation.
On the other hand, the present DDS formulation, which is characterized by
targeting to thyroid carcinoma cells using the aforementioned antibody having
specific
immunoreactivity with thyroid carcinoma cells, may comprise one or more
materials
which are selected from a group consisting of a nucleic acid, iodine,
radioactive iodine,
technetium and a protein, accordingly, through including such materials to the
formulation,
higher targeting ability to thyroid carcinoma is allowed, which can be
utilized for therapy or
diagnosis of thyroid cancer.
"Nucleic acid" herein refers to for example, a nucleic acid encoding a protein
that
can be expressed in a host cell, an antisense nucleic acid derived from cells,
a nucleic acid
of a decoy having a sequence of a gene encoding a binding protein of a cell-
derived
transcription factor or a sequence of a binding site of a transcription
factor, or a similar
sequence thereto.
"Antisense nucleic acid" represents a nucleic acid or a nucleic acid sequence
that
binds specifically to a nucleic acid being able to be expressed in future, at
any stage of the
gene expression, i.e., replication, transcription, translation or the like,
thus inhibits
expression of the nucleic acid which can be otherwise expressed in future.
Antisense
nucleic acid also includes an anti-gene nucleic acid resulting from a triple
strand. A
nucleic acid encoding a decoy represents a nucleic acid having a sequence of a
nucleic acid
encoding a binding protein of a cell-derived transcription factor or a
sequence of a binding
site of a transcription factor, or a similar sequence thereto, thus through
introducing the
nucleic acid into a cell as a decoy, binding of a transcription factor to its
binding site can be
CA 02604143 2007-10-26
21
inhibited, which may lead to suppression of an action of the transcription
factor, finally
suppression of a group of genes which was going to be activated may be
possibly resulted.
"Ribozyme" herein means a nucleic acid that can cut mRNA of a specified
protein, then
inhibits translation of the specified protein. A ribozyme can be designed from
a gene
sequence encoding the specified protein, which may include herein irrespective
of type of
the ribozyme, for example, a hammer head type ribozyme, a hairpin ribozyme, a
delta type
ribozyme and the like, as long as it can cut mRNA of a specified protein, thus
leading to
inhibition of translation of the specified protein. Suicide gene herein refers
to a gene that
leads a cell to death consequently, which may include programmed cell death
inducing
gene, apoptosis inducing gene, necrosis inducing gene and the like.
These nucleic acids can be selected by a person skilled in this art, and
through
including these nucleic acids into the DDS formulation, specific death of
thyroid carcinoma
cells can be achieved.
By adding radioactive iodine (1311) to the formulation, normal thyroid cells
are
destroyed, thus metastasis of carcinoma can be readily detected in systemic
radioactive
iodine scintillation test. Further, by measuring blood thyroglobulin value,
remained
carcinoma after operation, or recurrence can be identified. In addition,
radioactive iodine
therapy makes it possible to prevent from recurrence by destructing latent
carcinoma, and
systemic radioactive iodine scintillation test can be realized through using
of a lot of
radioactive iodine for therapy. This test is highly sensitive for finding
carcinoma remained.
Therefore, by using the antibody of the present invention, or the iodine
labelled or
radiolabelled antibody, usefulness in diagnosis or therapy may be further
improved.
Moreover, the protein may include antibodies, TSH (Thyroid Stimulating
Hormone), thyroid hormone and the like.
The above-mentioned DDS formulation comprising the materials as set forth
above is useful as a pharmaceutical composition for diagnosis of thyroid
cancer, or for
CA 02604143 2007-10-26
22
pharmaceutical composition for therapy of thyroid cancer.
Furthermore, a pharmaceutical composition of the present invention allows for
practicing an effective chemotherapy accompanied by less side effects, through
concentrating a drug to the focal portion utilizing an ability of this
antibody to bind to
thyroid carcinoma cells based on specific immunoreactivity, when a therapeutic
treatment of thyroid carcinoma using a chemotherapeutic agent is-intended.
Effective chemotherapeutic agent of thyroid carcinoma may include anticancer
agent such as cyclophosphamide, adriamycin, streptozotocin, 5-fluorouracil,
dacarbazine,
vincnstine and the like.
An administration process of the above-described pharmaceutical composition
may be either of which by systemic administration or topical administration.
Systemic
administration may include oral administration, intravenous administration,
subcutaneous and intramuscular injection, rectal administration, and the like,
and topical
administration may be preferably performed by direct administration into
thyroid tissue,
or administration into a vein that is connecting to thyroid tissue.
Dosages of the pharmaceutical composition of the present invention may
depend upon the known effective blood concentration level of a drug, which
should be
determined ad libitum by the skilled person in this art. Additionally, in case
of a
liposome formulation is prepared, it is important to use the antibody at a
dose that does
not hamper the liposome formation.
When particularly administered to human, the antibody to be included in the
DDS formulation may be preferably the above-described humanized antibody,
chimeric
antibody or the like without any immunogenicity to human, or with less
immunogenicity
to the at most. When a mouse monoclonal antibody is administered to a human
body,
risks of occurrence of various side effects are prospected, because such an
antibody is a
heterogeneous protein to human. Accordingly, although using a human monoclonal
CA 02604143 2007-10-26
23
antibody is desirable, fusion efficiencies may be inferior, and obtaining a
hybridoma that
is stably producing an antibody could be difficult. However, the technologies
have
been progressing currently, thus generation of human monoclonal antibodies or
chimeric
antibodies have been enabled.
Chimeric antibody may be a chimeric molecule comprising a mouse antibody
and a human antibody. Producing an antibody by immunizing a human with an
arbitrary antigen is ethically impossible. Therefore, a mouse is immunized
first, then a
gene portion of an antibody valuable region (V region) that binds to the
antigen of the
resulted mouse monoclonal antibody is excised therefrom, and this gene portion
is linked
to a gene encoding an antibody constant region (C region) from human myeloma,
to
produce a chimeric gene. When thus prepared chimeric gene is expressed in a
host cell,
a human-mouse monoclonal antibody can be produced. Because chimeric antibodies
are less immunogenic to human, it can be utilized as monoclonal antibodies to
be
administered to a human body for therapy or diagnostic imaging. Known relevant
arts
of chimeric antibodies may include Japanese provisional publication No. Hei 05-
304989,
Japanese provisional publication No. Hei 04-330295, PCT publication No.
W09106649,
Japanese provisional publication No. Sho 63-036786, Japanese publication No.
Hei
06-98021 and the like.
More recently, however, humanized antibodies were discovered, which are
reported to be more useful than chimeric antibodies. Humanized antibody is an
antibody
of which entire molecule was humanized except for CDR (Complementarity
Determining
Region) of the antibody molecule, by grafting only CDR encoding gene of the
antibody
molecule to a gene encoding humanized antibody (CDR). The humanized antibodies
have
less mouse-derived antibody portion than human-mouse chimeric antibodies, thus
they are
reported to be less antigenic and safer. In Japan, clinical tests on humanized
antibodies to
adult T-cell leukemia have been presently performed. With respect to
procedures for
CA 02604143 2007-10-26
24
producing humanized antibodies and the related arts, see, for example, PCT
publication
Nos. WO9222653, WO9845332, WO9404679, WO9837200 and WO9404679 of
Genentech, USA, and PCT publication Nos. W09429451, W09429351, W09413805,
WO9306231, WO9201059, WO9116927, WO9116928, WO9109967, WO8901974,
W08901783 of Celltech, United Kingdom and the like.
Method for producing human monoclonal antibodies may include in addition
to a cell fusion method, a transformation method with Epstein-Barr virus
(EBV), and
another fusion method of thus transformed cells and parent cells, a method of
producing a
chimeric antibody or a humanized antibody utilizing genetic engineering, and
the like. A
chimeric antibody refers to an antibody prepared by linking immunoglobulin
gene
fragments of heterologous animals, and a humanized antibody refers to an
antibody
prepared by modifying a heterologous antibody to human, such as a mouse
antibody,
wherein a primary structure except for CDR (Complementarity Determining
Region) in H
chain and L chain is substituted for corresponding primary structure of a
human antibody.
As parent cells for producing human monoclonal antibody, SHM-D 33 strain (ATCC
CRL
1668) or RF-S 1 strain may be employed, which is human/mouse heteromyeloma, so
that
high fusion efficiency that is equal to mouse parent cells can be resulted.
Hybridoma
obtained using such parent cells can be cloned without feeder cells, and can
produce IgG
type antibodies in a relatively stable manner, on a large scale. For culturing
the parent
cells, ERDF medium containing 15% FCS may be employed, and another procedures
may
be similarly carried out to the case of culture of mouse parent cells. In
addition, it is
preferable to use sufficiently sensitized human lymphocytes with the antigen,
which are
colleted from peripheral blood, for producing IgG type human monoclonal
antibodies.
When obtaining such sufficiently sensitized lymphocytes with an antigen is
difficult, in
vitro sensitization with the antigen may also be performed.
Using the methods set forth above, the present antibody can be humanized, and
CA 02604143 2007-10-26
thus it is markedly useful for administration to a human body.
In addition, usefulness may be enhanced as a diagnostic/therapeutic drug,
through
radiolabelling such an antibody with iodine, or including radiolabelled iodine
into a
pharmaceutical composition that was targeted with the antibody.
When papillary carcinoma or follicular carcinoma invades into surrounding
tissue
or metastasizes to a distal portion (especially to lung and bone) or lymph
node through
blood circulation or lymphatically, a common therapeutic strategy for
destroying the
carcinoma cells has been administration of radiolabelled (131-I) iodine.
Normal thyroid
cells incorporate iodine from the blood and concentrate it. This process is
stimulated by
TSH (Thyroid Stimulating Hormone) that is secreted from pituitary gland.
Iodine is
thereafter used to produce thyroid hormone (thyroxine T4). As set forth above,
thyroid
carcinoma or metastatic area of carcinoma normally incorporates only slight
amount of
iodine (or radioactive iodine). However, when carcinoma is under influences of
abundant
TSH, a part of thyroid carcinoma or metastatic one becomes liable to
incorporate a
significant amount of iodine upon stimulation. Consequently, a large amount of
radiation
is allowed to be directly exposed to carcinoma without injuring the
surrounding tissue.
When intact thyroid is present in a body with producing a normal level of
thyroid hormone,
TSH level that is produced may remain relatively low, however, upon decrease
of thyroid
hormone level due to removal of whole thyroid or its destruction, pituitary
gland rapidly
accelerates TSH secretion. The TSH stimulates thyroid carcinoma, leading to
incorporation of radioactive iodine. When radioactive iodine therapy is
performed for
progressed thyroid carcinoma, whole thyroid must be removed almost completely
by an
operation, and the residual tissue is required to be destroyed using
radioactive iodine.
Once this procedure is carried out, patients having carcinoma cells remained
in the neck
area or those having metastatic carcinoma to a distal place are subjected to
scanning when
their TSH level is high enough, using a test amount of radioactive iodine
(normally 2-10
CA 02604143 2007-10-26
26
mCi). If substantial amount of radioactive iodine was proved to assemble to a
region of
thyroid carcinoma, yet more therapeutic amount of radioactive iodine (normally
100-200
mCi: 3700-7400 MBq) is administered in an attempt of destruction of the
carcinoma cells.
Because radioactive iodine is safe and effective also to the patients having
more invasive
thyroid carcinoma, many physicians have learned to use radioactive iodine
routinely for
less invasive papillary carcinoma or follicular carcinoma.
Therefore, by labelling the antibody that binds to LAR using iodine, or by
including radioactive iodine into a pharmaceutical composition targeted with
the antibody,
specificity to thyroid carcinoma cells can be further enhanced, thus
therapeutic or
diagnostic utilization may be enabled.
Accordingly, an anti-LAR antibody that specifically recognizes thyroid
carcinoma
cells is also useful in a Drug Delivery System (DDS). Drug Delivery System
(Mitsuru
Hashida, Drug Delivery System, New Challenges to Manufacturing Drugs and
Therapy,
Kagaku Doujin, (1995)) is a novel technique related to drug administration
aiming at:
contriving administration routes or forms of drugs; delivering the drugs
selectively to
targeted sites by controlling pharmacokinetics of the drugs in a body;
achieving the optimal
therapeutic effects as a result; and minimizing the adverse effects exerted by
the drugs.
Although various DDS formulations have been developed heretofore, liposome
formulations (Hiroshi Terada, Tetsuro Yoshimura eds., Experiment Manual of
Liposome in
Life Science, Springer-Verlag, Tokyo, (1992)) are among all highlighted in
supplementation of a deficient enzyme, administration of carcinostatic agent
and antibiotics,
as well as in gene therapy.
Liposome is a closed small vesicle composed of a lipid bilayer of which basis
is
phospholipid that constructs a biomembrane, which is known to be safe with a
superior
function as a drug carrier, because it can capsulate various drugs
irrespective of their
solubility whether the drugs may be lipid soluble or water soluble, according
to their
CA 02604143 2007-10-26
26/1
composition comprising a lipid membrane and an aqueous layered part.
CA 02604143 2007-10-26
27
In addition, it is well known that a targeting ability can be imparted to a
liposome
through binding an antibody, peptide or the like on the surface of the
liposome (Kazuo
Man yama, Tomoko Takizawa, Motoharu Iwatsuru et al., Biochimica et Biophysica
Acta
1234, 74 (1995; Jlbao Zhao, Shunsaku Kimura, Yukio Imanishi, Biochimica et
Biophysica
Acta 1283, 37 (1996)). Accordingly, anti-LAR antibodies can be used for the
purpose of
improving specificity to thyroid carcinoma cells of various liposome
formulations.
Further, characteristic features of the liposomes are their abilities to
produce a variety of
carriers (vectors) of which properties are distinct by alteration of a kind of
the lipid, or
modifying with polyethylene glycol: for example, temperature sensitive
liposomes (Sakae
Unezaki, Kazuo Maruyama, Motoharu Iwatsuru et al., Pharmaceutical Research 11,
1180
(1994)), liposomes with stability in blood (Kazuo Maruyama, Tsutomu Yuda,
Motoharu
Iwatsuru et al., Biochimica et Biophysica Acta 1128, 44 (1992)), cationic
liposomes as
plasmid introducing vector (Xiang Gao, Daniel Jaffurs, Leaf Huang et al.;
Biochemical and
Biophysical Research Communications 200, 1201 (1994)), and the like can be
prepared.
However, liposomes are usually incorporated into cells by an endocytotic
pathway
followed by incorporation into early endosomes proximal to the cell menbrane.
Then, the
liposomes are delivered to late endosomes in a deeper part of the cell, and
finally
transferred to lysosomes. The liposomes that were transferred to lysosomes are
degraded
by actions of hydrolytic enzymes, and the drugs capsulated in the liposomes
are
simultaneously metabolized, therefore, there exists a problem that the
accessible rate of the
drugs that are kept unchanged into the cytoplasm may be extremely low.
Currently, a method for introducing drugs and the like directly into the
cytoplasm
without any injury against a cell membrane that is a barrier of a cell has
been studied. For
example, when liposomes gain an ability to fuse with a membrane, drugs
introduced
thereinto would be able to be delivered directly into the cytosol without
transfer via
lysosomes. Methods for fusion of the liposome with a cell have been studied
heretofore
CA 02604143 2007-10-26
28
which may include: pH sensitive liposomes (Kenji Kono, Ken-ichi Zenitani,
'Toru
Takabishi, Biochimica et Biophysica Acta 1193, 1(1994)); and reconstituted
liposomes that
are liposomes incorporated with an envelope protein of virus thereinto
(Sangeeta Bagai,
Debi P. Sarkar, The Journal of Biological Chemistry 269, 1966(1994)).
Recently, a fusiogenic liposome (HVJ-liposome) was reported, which is a
liposome with an imparted ability of Sendai virus (Hemagglutinating Virus of
Japan) to
fuse with a membrane (Yoshio Okada, Currenitopics in Membranes and Transport
32, 297
(1988)). Sendai virus (HVJ) is a pioneering virus for genetics in which animal
cells were
employed, based on observation of an intercellular fusion event (Y. Maeda, J.
Kim, Y.
Okada et al., Experimental Cell Research 108, 108 (1977)). Furthermore, HVJ
can also
fuse with liposomes (Mahito Nakanishi, Tsuyoshi Uchida, Yoshio Okada et al.,
Experimental Cell Research 159, 399 (1985)), and the fusion body (HVJ-
liposome) can in
turn fuse with a cell membrane. Namely, HVJ-liposome that was prepared by a
direct
reaction between a liposome and HVJ is a so-called hybrid vector, carrying a
cavity inside
which is derived from the liposome, and an outside spike structure identical
to that of a viral
envelope. HVJ-liposomes can introduce any substances as long as they can be
capsulated
into liposomes, such as proteins, chemical substances, genes and the like,
into cells at high
efficiencies that are equivalent to Sendai virus (Tetsuhiko Nakagawa, Hiriyuki
Mizuguchi,
Tadanori Mayumi, Drug Delivery System 11, 411 (1996)). Additionally, an
improved
type of HVJ-liposome was proposed wherein introducing efficiencies may be
enhanced by
co-introduction with DNA and a nuclear protein HMG-1 (Non-histone chromosomal
protein, High Mobility Group-1) having a DNA binding ability (Yasufumi Kaneda
et al,
J.Molec. Medicine 73, 289 (1995)).
Another example of a menbrane-fused liposome that can be used is a liposome
formulation in which VSV (Vesicular Stomatitis Virus; Yoshiyuki Nagai, Akira
Ishihama
ed, Protocols for Experiments of Virus, Medical View (1995)) is utilized (J.
Virol., 72(7),
CA 02604143 2007-10-26
29
6159-63, 1998; Exp. Cell. Res., 200(2), 333-8, 1992; Proc. Natl. Acad Sci.
USA, 87(7),
2448-51, 1990; and Biochim. Biophys. Acta, 987(1), 15-20, (1989)). VSV is a
single
strand RNA (-) virus belonging to genus Vesiculovirus in family Rhabdovirus,
having G
protein that is an envelope protein on a membrane surface (Akihiko Kawai,
Journal of
Virology 24, 826 (1977)). Infection mechanisms of VSV to cells may proceed via
an
endocytotic pathway similarly to liposomes. However, to be distinct from
liposomes,
because VSV has a characteristic to fuse with an endosome membrane, VSV
introduces its
own gene into cytoplasm without degradation by hydrolytic enzymes contained in
lysosome. So far, it has been noted that VSV has an ability to fuse with a
menbrane, and
that any hemolytic action is not exhibited against human erythrocyte by VSV
(Carole A.
Bailey, Douglas K. Miller, John Lenard, Virology 133, 11](1984)). Further,
because
VSV utilizes ubiquitously existing phosphatidylserine in lots of tissue cells
as a receptor,
wide variety of hosts may be allowed (Michael J. Clague, Christian Schoch,
Robert
Blumenthal, Biochemistry 29, 1303 (1990)), and propagation of this virus
quickly proceeds,
thus characteristics of VSV may be that the virus can be readily collected at
a large amount.
On the other hand, it is reported that VSV and liposome may result in fusion
(Satoshi
Yamada, Shunichi Ohnishi, Biochemistry 25, 3703 (1986)).
As explained in detail, anti-LAR antibodies can be utilized for the purpose of
enhancement of targeting abilities of any kinds of liposome formulations,
including
membrane-fused liposomes, pH sensitive liposomes, reconstitution liposomes,
cationic
liposomes and the like, or modified types thereof.
Besides, substantive reports on methods for enhancing targeting abilities
using
monoclonal antibodies and their usefulness have been found (Hum. Antibodies,
9(1),
61-5, 1999; J Clin Pharm. Ther., 22(1), 7-19, 1997; J Int. Med Res., 25(1), 14-
23, 1997;
Proc. Natl. Acad Sci. USA, 93 (24), 14164-9, 1996; Hepatology, 22(5), 1482-7,
1995;
Hepatology, 22(5), 1527-37, 1995, Proc. Natl. Acad. Sci. USA, 92(15), 6986-90,
1995;
CA 02604143 2007-10-26
Immunomethods, 4(3), 259-72,1994; J. Drug Target, 2(4), 323-31, 1994; Cancer
Res ,
57(10), 1922-8, 1997; Crit. Rev. Biotechnol., 17(2), 149-69, 1997; Methods
Find Exp. Clin.
Pharmacol., 16(7), 505-12, 1994; Trends Biotechnol., 12(6), 234-9, 1994; and
Bioconjug,
Chem., 4(1), 94-102, 1993), thus monoclonal antibodies to LAR can be used in
therapeutic
treatment of thyroid carcinoma according to the above-described literatures or
known
techniques.
Further in addition, anti-LAR antibodies of the present invention can be
utilized
with contemplation in targeting to thyroid carcinoma, for gene therapy with
viral vectors, or
for DDS formulations wherein polyacid-glycolic acid microsphere, lipid
microsphere,
polyethylene glycol-modified enzyme or the like is used.
In still another embodiment of the present invention, high expression of LAR
in thyroid carcinoma cells can bring comprehension that high rate of
transcription from
a LAR molecule-encoding nucleic acid sequence to mRNA followed by translation
is
conducted in those cells. Accordingly, persons skilled in this art can readily
diagnose
carcinoma through measuring an expression level of LAR mRNA by using probes
for
the mRNA
Furthermore, the present invention can contribute substantially to molecular
biological studies on transcription factors, promoters, enhancers, or the like
that may
accelerate the transcription of LAR in thyroid carcinoma cells.
BRIEF DESCRIPTION OF THE DRAWING
Figure 1 is a schematic drawing depicting a subunit structure of LAR (a); and
a
schematic drawing illustrating the mutated LAR phosphatase domain structures
inside the
membrane (b) prepared as demonstrated in Examples.
Figure 2 represents immunoblots illustrating time dependent tyrosine
phosphorylation induced by insulin stimulation in COS cells that were
cotransfected
CA 02604143 2007-10-26
31
with LAR/CS and wild type insulin receptor.
Figure 3 represents immunoblots illustrating phosphorylation-dephosphorylation
in COS cells that were cotransfected with wild type or mutants of LAR, and
wild type
insulin receptor.
Figure 4 represents an immunoblot illustrating dephosphorylation of a n-chain
of
insulin receptor by wild type or mutants of LAR.
Figure 5 represents an immunoblot illustrating tyrosine phosphorylation of
insulin receptor and LAR in COS cells that were cotransfected with wild type
or mutant
of insulin receptor, and LAR/CS.
Figure 6 represents SDS-polyacrylamide gel, showing a molecular weight of
the antibody YU1 of the present invention.
Figure 7 represents immunoblots showing immunospecificity of the antibody
YU1 of the present invention.
Figure 8 represents immunoblots showing tyrosine phosphorylation of LAR by
tyrosine kinase of insulin receptor.
Figure 9 is a schematic drawing depicting a signal transduction cascade of
insulin
that is controlled by phosphorylation-dephosphorylation in which insulin
receptor and LAR
participate.
Figure 10 represents immunoblots of thyroid normal and carcinoma tissues with
the antibody YUI of the present invention, demonstrating the specific
immunoreactivity
with human thyroid carcinoma tissue.
Figures 11-13 represent photos showing positive immunostaining of thyroid
carcinoma cells, but not in normal follicular cells using the antibody YU1 of
the present
invention.
Figure 14 represents results of immunoblotting demonstrating tissue
distribution
of LAR in mouse using the antibody YU 1 of the present invention.
CA 02604143 2007-10-26
32
BEST EMBODIMENT FOR CARRYING OUT THE INVENTION
Experimental Example 1: Tyrosine phosphorylation of insulin receptors by LAR
mutants
and studies on association between LAR and insulin receptors
First, in order to elucidate signal transduction controlling mechanisms of
insulin
by LAR, analysis was performed with a strategy in which mutated LAR is used
that was
prepared by substitution of cysteine with serine, which exists in a catalytic
center of PTP
domain of LAR.
A) Expression vector of LAR and insulin receptors
Three kinds of LAR expression vectors were used, i.e., (a) LAR WT: human wild
type LAR (SEQ ID NO: 3); (b) LAR C/S: mutated LAR, having substitution of
cysteine in
a catalytic center of LAR-PTP domain I (amino acid residue position 1552 of
SEQ ID NO:
3) for serine by substituting nucleotide G, position 4983 of,SEQ ID NO: 3,
with C; and (c)
LAR DC/S: further mutated one in addition to LAR C/S, with substitution of
cysteine in
LAR-PTP domain 2 (amino acid residue position 1813 of SEQ ID NO: 3) for serine
by
substituting nucleotide G with C, position 5856 of SEQ ID NO: 3 (see, Fig. 1
(b)), each of
which was incorporated into pMT expression vector (see, Streuli M. et al.,
EMBO J, 11,
897-907, 1992; and Streuli M. et al., FMBO J, 9, 2399-2407, 1990).
Meanwhile, insulin receptor expression vectors used were: (a) IR WT: wild
type; and (b) IR K1018M: mutated insulin receptor having substitution of
lysine of the
position 1018 of ATP binding site of wild type insulin receptor, with
methionine resulting
in deficiency of tyrosine kinase activity, each of which cDNA was incorporated
downstream of SRa promoter (see, Kanai F. et al., Biochemical and Biophysical
Research Communications, 195, 762-768, 1993).
B) Transfection into COS-7 cells
COS-7 cells were seeded into RPMI 1640 medium (Nissui Pharmaceutical Co.,
CA 02604143 2007-10-26
33
LTD.) supplemented with 10% fetal calf serum at 1.0 x 106 cells/8 mL/90 4)
dish, then after
16 hours incubation, expression vectors of LAR C/S and IR WT were
cotransfected into
COS-7 cells using DEAE-dextran method. The LAR C/S employed was a vector that
was
revealed to include complete deficiency in tyrosine phosphatase activities in
vitro (Streuli
M. el al., FMBO J., 9, 2399-2407, 1990) according to mutation as mentioned
above in
paragraph A, (b).
Cotransfection was performed according to the following procedure. Initially,
40
g I of 10 mM chloroquine was added to 4 ml of RPMI 1640 medium (10.2 g/L of
RPMI
1640 (Nissui Pharmaceutical Co., LTD.) containing 0.3 g of glutamine and 0.1 g
of
kanamycin, pH 7.4 that was adjusted with 10% NaHCO3).
To 2 ml of this solution, 5 g of LAR expression vector and 1 g of IR
expression
vector were added, on the other hand, 16 g 1 of 100 mg/ml DEAE-dextran was
added to 2
ml of the remaining solution. Then, both solutions were mixed thoroughly with
stirring.
Thus prepared 3.75 ml of solution of expression vector was plated at 1.0 x 106
cells/8
ml/dish, and was added to COS-7 cells that had been precultured for 16 hours
at 37 C, in a
5% CO2 incubator. Following 4 hours culture under the similar conditions to
the
preculture, the cells were treated with 10% DMSO solution for 2 minutes, then
washed
with PBS (137 mM NaCl, 2.7 mM KCI, 4.3 mM Na2HPO4.12H2O, 1.4 mM KH2PO4),
thereafter, 8 ml of.RPMI1640 containing 10% FCS was added thereto, and
cultured for 48
hours at 37 C within an incubator that was adjusted to 5% CO2.
C) Insulin stimulation and preparation of cell lysate
COS-7 cells after completing transfection were incubated for 16 hours in serum
free RPMI 1640 culture medium, followed by stimulation with 10"' M insulin
(Seikagaku
Corporation) for determined periods, i.e., 0, 1, 5, 15 and 30 minutes.
Stimulation for 0
minute was conducted by standing on ice without incubating at 37 C, although
insulin was
added similarly. After each of the time elapsed from the beginning of insulin
stimulation,
CA 02604143 2007-10-26
34
culture fluid was entirely aspirated from the cells, and 5 ml of PBS w/Inh_
(PBS containing
tyrosine phosphatase inhibitors: 1 mM sodium vanadate, 5 mM sodium fluoride, 5
mM
sodium pyrophosphate, 5 mM EDTA-2Na, 137 mM NaCl, 2.7 mM KCI, 4.3 mM
Na2HPO4.12H20, 1.4 mM KH2PO4) was immediately added.
Following washes of the whole cells with PBS w/Inh., the fluid was removed by
aspiration, and 1 ml of lysis buffer (1% Nonidet P-40, 150 mM NaCl, 50 mM Tris-
HCI
(pH 7.4), 5 mM EDTA, 10 mM iodoacetamide, 10 mM sodium fluoride, 10 mM sodium
pyrophosphate, 0.4 mM sodium vanadate, 0.1 mM oxidized phenylarsine, 1 mM
benzamidine, 1 mM phenylmethylsulfonyl fluoride) was added to the cells, which
were
thereafter collected with a cell scraper. The cell suspension was transferred
to a 1.5 ml
tube, and then incubated at 4 C for 30 minutes to result in complete lysis of
the cells.
Supernatant, which was obtained by centrifuge of the fluid at 12,000 rpm, 4 C
for 10
minutes following incubation was employed as a cell lysate in the experiments
set forth
below.
D) Immunoprecipitation
Immunoprecipitation was performed for the cell lysate obtained as above
paragraph C, with an anti-LAR E-subunit antibody (a mixture of 7.5 g of 11.1
A and 7.5
g of 75.3A, see, Streuli M. et al., EMBO J., 11, 897-907, 1992). To 1 ml of
the above
cell lysis solution, 15 g of MOPC 21 (mouse IgGI x : Sigma Corporation) as a
mock was
added, then the solution was incubated at 4 C for one hour, added 20 I of -y
-bind
TM
(GammaBind Plus Sepharose: Pharmacia Biotech Inc.) thereto, and further
incubated for
one hour at 4 C to execute preabsorption_ The solution was centrifuged at 4 C,
12,000
rpm for 10 minutes, then 950 g I of the supernatant was transferred to another
tube. Next,
15 p g of anti-LAR E-subunit antibody was added to the supernatant, then the
solution was
incubated at 4 C for one hour, added 20 g I ofy -bind thereto, and further
incubated for one
hour at 4 C.
CA 02604143 2007-10-26
After centrifuge at 4 C, 12,000 rpm for 10 minutes, the precipitate was washed
with I ml of lysis buffer twice, then once with PBS w/Inh., and suspended in
20 g I of SDS
sample buffer. The suspension was heated for 5 minutes in a boiling water bath
to prepare
a sample for electrophoresis.
E) Immunoblotting
The above-mentioned sample was subjected to electrophoresis using 7.5%
SDS-polyacrylamide gel, followed by transfer to a nitrocellulose membrane
(Schleicher &
Schuell) using a transfer device at 400 mA for 4 hours. Then blocking was
conducted by
incubating the membrane in 3% bovine serum albumin solution for longer than 30
minutes at a room temperature. After washing with sufficient volume of TBS-T
(TBS
with Tween 20: 10 mM Tris-HCI (pH 7.4), 150 mM NaCl, 0.1 % Tween 20) for 10
minutes
more than twice, an anti-phosphotyrosine antibody (4G10, UBI) that was 50,000-
fold
diluted with TBS-T, the anti-LAR E-subunit antibody or an anti-insulin
receptor 0 -chain
antibody (UBI) was added thereto, then the mixture was shaken for one hour at
a room
temperature. After washing with sufficient volume of TBS-T for 5 minutes more
than
three times, 15 ml of TBS-T solution containing HRP-labelled anti-mouse IgG
antibody
(horseradish peroxidase-labelled anti-mouse IgG: Santa Cruz Biotechnology,
Inc.) 1.5 ml
was added thereto, and shaken for one hour at a room temperature. After
washing with
sufficient volume of TBS-T for 5 minutes more than three times, bands of the
protein were
detected that can bind to each of the antibodies, by means of
chemiluminescence using a kit
of luminescence reagents (Wako Pure Chemical Industries, Ltd-)-
F) Results
As results of immunoblotting with the anti-phosphotyrosine antibody following
to immunoprecipitation with the anti-LAR E-subunit antibody of cell lysate
prepared
after stimulation with insulin for determined time periods of cotransfected
COS-7 cells
with LAR C/S and IR WT in the above-described manner, tyrosine phosphorylation
of an
CA 02604143 2007-10-26
36
insulin receptor (3 -chain as well as a 85 kDa protein could be observed with
the insulin
stimulation for 1 minute. Such tyrosine phosphorylation could also be
successively
observed with the insulin stimulation for 30 minutes (see, Fig. 2A).
Furthermore, results from the immunoblotting with the anti-LAR E-subunit
antibody (Fig. 2B), the anti-insulin receptor (3 -chain antibody (Fig. 2C) and
the
anti-phosphotyrosine antibody (Fig. 2A) demonstrated that LAR and insulin
receptor may
associate depending on the presence or absence of tyrosine phosphorylation of
the insulin
receptor.
Experimental Example 2: Studies on tyrosine dephosphorylation of insulin
receptor
by various LAR (1)
Next, COS-7 cells were similarly cotransfected with LAR WT; LAR C/S or
LAR DC/S, and IR WT followed by stimulation with insulin for 5 minutes,
immunoprecipitation with the anti-LAR E-subunit antibody, and then
immunoblotting with
various types of antibodies for the. precipitates was carried out.
Consequently, tyrosine
phosphorylation of the insulin receptor (3 -chain or the 85 kDa protein could
not be detected
for the cells cotransfected with insulin receptor and LAR WT, in comparison
with the cells
cotransfected with LAR C/S or LAR DC/S (see, Fig. 3 A).
Additionally in these experiments, amounts of expression of LAR (Fig. 3C)
and the insulin receptor (Fig. 3D) were almost identical in both of the
cotransfectants,
therefore LAR WT was suggested to dephosphorylate the phosphorylated tyrosine
of the
insulin receptor 13 -chain as well as the 85 kDa protein (Fig. 3B).
Further, when the immunoprecipitates with the anti-LAR E-subunit antibody were
immunoblotted using the anti-insulin receptor 0 -chain antibody, the
cotransfectant with
LAR DC/S showed a weaker band of an insulin receptor R -chain, compared to the
cotransfectant with LAR WT or LAR C/S.
CA 02604143 2007-10-26
37
These results indicate that the association between insulin receptor and LAR
DC/S
is weaker, when compared with that of LAR WT or LAR C/S. The only one
difference
between LAR C/S and LAR DC/S is one amino acid residue position 1813 of
phosphatase
domain 2, accordingly, this domain 2, which was postulated to involve in
binding with
substrates without tyrosine phosphatase activity, was proved to be playing a
role in binding
between LAR and insulin receptor.
Experimental Example 3: Studies on tyrosine dephosphorylation of insulin
receptor
by various LAR (2)
In order to further study as to whether tyrosine dephosphorylation of insulin
receptor occurs only in cases where LAR was bound, or in every insulin
receptor, cell
lysate of the cotransfectant was subjected to electrophoresis, and then
immunoblotted with
the anti-phosphotyrosine antibody. Consequently, tyrosine dephosphorylation of
insulin
receptor was markedly found only in cells that had been cotransfected with LAR
WT.(see,
Fig. 4).
Experimental Example 4: Studies on tyrosine phosphorylation of insulin
receptor
in the presence of LAR C/S
In order to elucidate whether tyrosine phosphorylation of the 85 kDa protein
is
effected by a tyrosine kinase activity of insulin receptor, COS-7 cells were
produced that
were cotransfected with LAR C/S, and IR WT or IR K1018M(IR MT) having a
deficiency
in tyrosine kinase of insulin receptor. Following insulin stimulation of the
cells for 5
minutes, immunoprecipitation was performed with the anti-LAR E-subunit
antibody, and
immunoblotting with the anti-phosphotyrosine antibody was carried out (see,
Fig. 5).
Consequently, the cells cotransfected with IR WT showed tyrosine
phosphorylation of an
insulin receptorp -chain and the 85 kDa protein upon stimulation with insulin,
however, the
CA 02604143 2007-10-26
38
cells cotransfected with IR K1018M showed no such phosphorylation at all.
From these results, it was revealed that rapid tyrosine phosphorylation of
insulin
receptor occurs upon binding of insulin to insulin receptor; and that the
insulin receptor
tyrosine kinase leads tyrosine phosphorylation of the 85 kDa protein.
The 85 kDa protein was therefore speculated as a P-subunit of LAR of which
binding to insulin receptor was demonstrated.
Example 1: Generation of antibodies to an intracellular domain of a LAR P-
subunit
Antibodies to an intracellular domain of LAR were generated according to the
following procedures.
A) Preparation of immunogen
Glutathione-S-transferase-LAR fusion protein (GST-LAR) was employed as an
immunogen. E. coli AD202 was transformed with an expression vector, pGEX-2T
vector
(Pharmacia Biotech Inc.), which was incorporated to its BamHI/EcoRI site with
cDNA
corresponding to 607 amino acids spanning from the end of a transmembrane
region of a
LAR P-subunit to the entire cytoplasmic region (SEQ ID NO: 1, 3467 bp)
according to a
general procedure. After the E. coli was incubated overnight in LB (Amp. +)
agar medium
(LB (Amp. +) described below containing 7.5 g of agar), single colony was
inoculated to
50 ml of LB (Amp. +) medium (containing triptone 10 g/L, yeast extract 5 g/L,
NaCl 5 g/L,
N NaOH 0.2 miL, and ampicillin 50 g / ml), and further incubated overnight.
Then
the E. coli was inoculated to 500 ml of LB (Amp. +) medium, and incubated at
37 C until
absorbance at 600 nm reaches to approximately 1.0, followed by addition of 50
1 of 1 M
IPTG (isopropyl-(3 -D(-)-thiogalactopyranoside, Wako Pure Chemical Industries,
Ltd.) and
an incubation at 25 C overnight. Thus resulted culture was centrifuged at
3,000 rpm,
4 C for 15 minutes, and the precipitated bacterial bodies were suspended in 50
ml of
NETN (0.5 % Nonidet P-40, 1 mM EDTA, 20 mM Tris-HC1 pH 8.0, 100 mM NaCl).
CA 02604143 2007-10-26
39
Thereafter, the suspension was subjected to twice repeated treatments of
ultrasonication for
1 minute and standing on ice for one minute, and then centrifuged at 14,000
rpm, 4 C for
20 minutes to obtain the supernatant. To 10 ml of the lysate of the E.coh, 100
l of
suspension of glutathione sepharose beads (Glutathione Sepharose 4B (Pharmacia
Biotech
Inc.) that had been prepared by washing three times, and suspended in 50%
NETN) was
added, and then incubated for 30 minutes at a room temperature. Thus resulted
suspension was centrifuged at 3,000 rpm, 4 C for 5 minutes, and supernatant
was
removed. The precipitated glutathione sepharose beads were washed twice with
NETN,
then once with PBS, thereafter 100 1 of SDS sample buffer (125 mm Tris-HC1
pH 6.8,
0.1% sodium dodecylsulfate, 5% 2-mercaptoethanol) was added thereto, and
heated in a
boiling water bath for 10 minutes to elute the GST-LAR fusion protein. The
eluate from
TM
which the beads were eliminated was concentrated by centrifuge using Centricon-
10
(Amicon) at 3,000 rpm, 4 C for 45 minutes.
One ml of PBS was added to the concentrate in order to bufferize the solution,
and
the solution was concentrated again by centrifuge at 3,000 rpm, 4 C for 45
minutes. This
process for bufferization was repeated twice, and thus resulted solution was
employed as an
immunogen solution. Purification and concentration of the antigenic protein
were
confirmed by SDS-polyacrylamide gel electrophoresis.
Meanwhile, on a final immunization, the antigen solution was prepared in a
different process because it should be administered intravenously. The lysate
of the
above-described E. coil that is expressing GST-LAR fusion protein was
incubated with
glutathione sepharose beads, and after centrifuge, the precipitated beads were
washed twice
with NETN, and three times with PBS. Next, 100 p. 1 of GSH elution buffer (20
mM
glutathione, IM Tris-HCI, pH 9.6) was added thereto, and the mixture was
gently stirred for
minutes at a room temperature to accomplish the elution of GST-LAR. After
repeating
the steps of centrifuge at 3,000 rpm, 4 C for 5 minutes and recovering the
supernatant
CA 02604143 2007-10-26
three times, the total eluate was dialyzed in saline at 4 C for 2 days, then
thus obtained
solution was employed as an immunogen solution for intravenous administration.
B) Immunization
Eight female Balb/c mice of 6 weeks old received intraperitoneal
administration
of pristane (2,6,10,14-tetramethylpentadecane, Sigma Corporation) at 0.5
ml/animal.
After 2 weeks passed, the antigen solution for intraperitoneal immunization
that was
emulsified by blending with Freund's complete adjuvant (GIBCO) at a ratio of
1:1 was
intraperitoneally administered at about 10 p. g of GST-LAR fusion protein per
one mouse.
Thereafter, the antigen solution for intraperitoneal immunization. that was
admixed with
Freund's incomplete adjuvant (GIBCO) at a ratio of 1:1 was prepared to be
about 30-70 g
of GST-LAR per one mouse, and the mixture was intraperitoneally administered
approximately once every 2 weeks. On day 4 after the fourth immunization,
blood was
collected from ocular fundus vein, and an antibody titer in the serum was
determined by
ELISA method.
C ELISA
Protein solutions of GST-LAR and GST alone that were prepared similarly to the
procedure of preparation of the antigen for intravenous immunization were
respectively
dialyzed against purified water at 4 C overnight. These solutions were
adjusted to 0.5 g /
ml in PBS, and subjected to absorption to an ELISA plate (Falcon 3911
MicroTest Tu
Flexible Assay Plate) at 50 Dwell for one hour. After five times washes with
wash buffer
(PBS containing 0.05% Tween20), blocking with 5% skim milk (prepared by
dissolving
2.5 g of skim milk in 50 ml of PBS) was conducted. Following washes, the serum
as
obtained in the above section B was diluted to 16,000 fold with dilution
buffer (PBS
containing 0.25% BSA), and was added to the wells at 50 g Dwell, and then
incubated for
one hour in a wet box. After washing the plate, HRP-labelled anti-mouse IgG
antibody
that was diluted to 1,000 fold was added to the plate at 50 p Dwell, and
incubated for one
CA 02604143 2007-10-26
41
hour. Following washes with wash buffer four times and once with PBS, a
substrate
solution of o-phenylenediamine (Wako Pure Chemical Industries, Ltd.) that was
dissolved
in a citrate buffer (prepared by dissolving 5.6325 g of citric acid
monohydrate and 18.35 g
of Na2HPO4.12H2O in purified water to make 500 ml in total) at a concentration
of 1 mg
/ml was added at 50 Vwell, allowed for reaction for 30 minutes, and then 50
g I of 10%
H2SO4 was added to terminate the reaction. Fifty p I of the solution was
transferred to
each well of a 96-well plate (Sumitomo Bakelite Co., LTD.) for measurement,
and then
absorbance at 450 nm was measured.
D) Cell fusion
Two mice that showed elevation of the antibody titers to GST-LAR in accordance
with the results of the above ELISA were finally immunized, and spleen was
excised
therefrom on the third day to prepare splenocytes according to an ordinary.
procedure.
Parent cells employed for cell fusion were Balb/c mouse-derived myeloma cell
strain NS I that was previously selected in a medium containing 20Fi g /ml 8-
azaguanine,
and confirmed as hypoxanthine, guanine, phosphoribosyl transferase (HGPRT)
deficient
strain. Cell fusion, HAT selection and cloning were-performed with 2 x 107
ofNSI cells
and I x 10" of splenocytes, using CIonaCellTM HY Hybridoma Cloning Kit
(StemCell
Technologies Inc.).
Screening of the supernatant from the culture of the cloned hybridoma was
carried
out according to ELISA method described in section C above, with 50 I of the
supernatant
of hybridoma culture using plates bound with 0.5 .t g/ml protein solution of
GST,
GST-LAR or GST-CD45 (Furukawa, T. et al., Proc. Natl. Acad Sci. USA,
91,10928-10932,1994) prepared by the similar method for preparation of the
antigen for
intravenous immunization as described above. In this ELISA method, hybridoma
was
selected, which did not show any immune response to the wells bound with GST
or
GST-CD45, but showed an immune response only to the wells bound with GST-LAR.
CA 02604143 2007-10-26
42
Passage culture of the cloned hybridoma was conducted with RPMI 1640 medium
(Nissui
Pharmaceutical Co., LTD.) containing 10% fetal bovine serum (GIBCO).
Through screening by ELISA method of the culture supernatant in this manner
from the hybridoma that was HAT selected, a clone YUI having specificity to
LAR
intracellular domain, with stable antibody producibility and proliferation
ability could be
obtained.
This hybridoma cell line YU1 was deposited on May 7, 1998, with National
Institute of Bioscience and Human-Technology, Agency of Industrial Science and
Technology, 1-1-320, Higashi, Tsukuba, Ibaraki, JAPAN, and assigned Accession
No.
FERM BP-6343.
E) Typing of monoclonal antibody
Supernatant of 0.5 ml from culture of hybridoma YUI obtained in the above
section D was diluted with 4.5 ml of TBS-T, and isotype was determined for 3
ml of the
diluted solution using mouse monoclonal antibody isotyping kit (Amersham
International
plc.). As a result, the isotype of the antibody was proved to be IgG2bx .
F) Generation and purification of monoclonal antibody
Balb/c mice of 6 weeks old received intraperitoneal administration of pristane
at
0.5 ml/animal, and after 10 days, hybridoma cell YU 1 that was obtained by
cloning in
section D above was intraperitoneally injected at 2.5 x 106- 1.3 x 107
cells/0.5 ml/animal.
Abdominal hypertrophy was observed approximately 10 days thereafter,
accordingly,
ascites fluid was collected using a 20-gauge injection needle several times.
Thus
collected ascites fluid was centrifuged at 1,000 rpm, 4 C for 5 minutes to
separate
supernatant and precipitate. The supernatant was incubated at 37 C for 30
minutes, and
stood at 4 C overnight. Following centrifuge at 12,000 rpm, 4 C for 10
minutes, the
monoclonal antibody YU 1 was purified using an affinity column HiTrap ProteinG
(Pharmacia Biotech Inc.) from the resulted 1.5 ml of supernatant.
Concentration of the
CA 02604143 2007-10-26
43
antibody was calculated from molecular extinction coefficient of mouse IgG,
based on the
measured absorbance at 280 nm of the antibody solution thus obtained.
In addition, a molecular weight of the monoclonal antibody YUl was estimated
from mobility on SDS-polyacrylamide gel electrophoresis. The results are shown
in Fig. 6.
As is clear from the Fig. 6, monoclonal antibody YUI comprises H-chain of
about 50 kDa
and L-chain of about 25 kDa, having a total molecular weight of about 150 kDa.
Example 2: Studies on specificity of monoclonal antibody
An expression vector of LAR WT was transfected into COS-7 cells according
to the procedures described in Example 1, sections. A and B. Following
immunoprecipitation of the cell lysate with the purified monoclonal antibody
obtained
in Example 1, immunoblotting was carried out. As a control on
immunoprecipitation,
MOPC 21 for the antibodies belonging to IgGI subclass (the anti-LAR E-subunit
antibody
(supra) and an anti-CD45 antibody (Santa Cruz Biotechnology, . Inc., 35-Z6)),
or mouse
IgG2b K (MOPC 195, CAPPEL) for the monoclonal antibody YUI was employed.
From the analyses using the LAR enforced expression system in COS-7 cells, the
monoclonal antibody YU1. recognized proteins of 85 kDa that corresponds to a
LAR
P-subunit and of about 200 kDa that corresponds to a precursor, after
immunoprecipitation
with the anti-LAR E-subunit antibody (see, Fig. 7B).
Moreover, upon immunoblotting with an antibody that recognizes a LAR
E-subunit after immunoprecipitation of cell extract of COS-7 cells transfected
with LAR
using these antibodies (IgGI, IgG2b, or YU1), detection of proteins of 150 kDa
that
corresponds to a LAR E-subunit and of about 200 kDa that corresponds to a
precursor was
restricted only to that immunoprecipitated with the antibody YUI (see, Fig.
7A). From
the results above, it was revealed that the monoclonal antibody YU1 could be
utilized for
immunoprecipitation and immunoblotting of a LAR P-subunit.
CA 02604143 2007-10-26
44
Example 3: Phosphorylation of LAR by insulin receptor tyrosine kinase
Experimental Example 4 suggested a possibility that a tyrosine phosphorylated
85 kDa band that was detected with cotransfection of insulin receptor and LAR
may be a
P-subunit of LAR.
Accordingly, studies were conducted using the monoclonal antibody YU1,
which was generated in Example 1, as to whether the 85 kDa protein of which
tyrosine
was phosphorylated by insulin receptor tyrosine kinase, was a LAR P-subunit
according
to a similar procedure described in Example 1.
Cell lysate of COS-7 cells stimulated with insulin for 1 minute following
cotransfection of LAR WT or LAR C/S with IR, was immunoprecipitated with the
anti-LAR E-subunit antibody, and then immunoblotted with a mixture of the anti-
LAR
E-subunit antibody and the antibody YU I, thus a precursor of LAR and each
subunit were
detected.
Further reprobe of this blot with the anti-phosphotyrosine antibody showed
agreement of the 85 kDa tyrosine phosphorylated band with a band of a LAR P-
subunit
(see, Fig. 8). These results illustrate that LAR is one of the substrates of
insulin receptor.
In addition, because the tyrosine phosphorylation of a LAR P-subunit was not
detected for the cotransfectant with LAR WT, LAR was supposed to conduct
autodephosphorylation (see, Fig. 3).
As shown in Fig. 9, when insulin binds to an insulin receptor a -chain,
tyrosine
kinase activity is elevated through autophosphorylation of the insulin
receptor 0 -chain-
This activity of tyrosine kinase finally results in occurrence of insulin
actions such as
glucose uptake, glucose metabolism, and cell proliferation. The activated
insulin receptor
was indicated to be back to an inactive state through tyrosine
dephosphorylation by LAR
Additionally, it was proved that insulin receptor kinase phosphorylates
tyrosine of
CA 02604143 2007-10-26
a LAR intracellular domain, and the phosphorylation was speculated to
participate in
determination of substrate specificity of the LAR intracellular domain or
elevation of
phosphatase activity. Besides, LAR is conceived as controlling its enzymatic
activity
through autodephosphorylation of the phosphorylated tyrosine.
From the results set forth above, possibility could be illustrated on a
molecular
level, that stimulation of enzymatic activity of LAR may be responsible for
insulin
resistance.
Example 4: Tissue distribution of LAR in mouse.
To one gram of each of the organs that were excised from male C57BL/6
mouse of seven weeks old was added with 3 ml of cold cell lysis buffer (the
same buffer as
described in Experimental Example 1, section C) followed by homogenization on
ice, and
incubation for 30 minutes on ice. After centrifuge at -4 C, 15,000 x g for 20
minutes, the
supernatant was recovered, additionally obtained supernatant by the same
centrifuge
condition was recovered, then total supernatant was employed as a tissue
sample. Protein
determination was performed according to a manual of DC Protein Assay (Bio-
Rad).
Thus obtained each supernatant (corresponding to 0.2 mg of protein) was
electrophoresed, followed by immunoblotting with YUl according to a procedure
described in Experimental Example 1, E.
The results of the immunoblotting are shown in Fig. 14. YU1 can also
recognize mouse LAR, thereby expression in thymus and brain could be
identified.
Slight expression could be found in kidney and liver as well.
Example 5: Immunohistochemical staining of thyroid carcinoma tissue section
with YU 1
Thyroid tissue was fixed in 10% neutral phosphate buffered formaldehyde
solution, and embedded in a paraffin block to prepare a specimen on a slide.
The
CA 02604143 2007-10-26
46
detailed procedures are described below.
1) Deparaffination
The fixed paraffin block of the tissue section was immersed in 100% xylene for
5
minutes twice, and then serially immersed in 100% ethanol, 90% ethanol, and
70% ethanol
for 3 minutes respectively. Finally, the section was immersed in 10 mM citrate
buffer (pH
6.0). In order to make antigen determinants exposed in this state, the
specimen was
subjected to an autoclave treatment at 100 C, for 5 minutes.
2) Immunostaining
The section was washed with 50 mM Tris-HCI buffer (pH 7.6) containing
0.15M NaCI (Tris solution), and then immersed in this Tris solution.
Thereafter, the liquid
was wiped away from the slide glass, then the section was dropped with 3%
aqueous
hydrogen peroxide, and stood for 3 minutes in order to eliminate endogenous
peroxidase..
Following sufficient washes with water and additional sufficient washes with
Tris
solution, the excess liquid was wiped away, and. the section was incubated
with 50 mm
Tris-HC1 buffer (pH 7.6) solution containing a carrier protein (2% BSA),
0.015M sodium
azide and 0. 15M NaCI for 15 minutes to effect blocking.
Next, after the excess liquid was wiped away without washing, primary
antibody YU1 (1000 fold dilution of the stock solution) was added on the
section, and
incubated for 90 minutes in a wet box.
The tissue section was then washed sufficiently with 50 mM Tris-HCI buffer
(pH 7.6) containing 0. 15M NaCl, and secondary antibody (biotinylated anti-
mouse
immunoglobulin) was added, followed by incubation for 45 minutes.
Thereafter, the section was sufficiently washed with 50 mM Tris-HCI buffer
(pH 7.6) containing 0. 15M NaCl, dropped with streptavidin conjugated
horseradish
peroxidase, and then stood for 25 minutes.
Next, after sufficient washes of the section with 50 mM Tris-HC1 buffer (pH
CA 02604143 2007-10-26
47
7.6) containing 0.15M NaCl, then 0.05% DAB (3,3'-diaminobenzidine
tetra-hydrochloride) solution in 50 mM Tris-HCI buffer (pH 7.6) containing
0.02%
hydrogen peroxide and 0.15M NaCI was added, followed by confirmation of color
development under microscopy, and then the reaction was terminated by
immersion of
the slide glass into water.
Following termination of the reaction, the specimen was soaked in Mayer's
hematoxylin for 5-10 seconds for counter staining. Thereafter, followed by
washes of the
specimen with water, and soaking in 100% ethanol for 1 minute twice, next in
100% xylene
for 1 minute twice, inclusion with Malinol and observation were carried out.
In these experiments, blocking, secondary antibody, and streptavidin-
peroxidase
solutions were from LSAB kit available from DAKO Japan Co. Ltd., (Kyoto), and
DAB
was a commercially available reagent from Dojindo (Kumamoto), Malinol was from
Muto
Pure Chemicals Ltd., (Tokyo), and Mayer's hematoxylin employed was prepared by
the
present inventor.
Thus resulted immunohistochemical staining of thyroid papillary carcinoma
cells
are shown in Figures 11-13. These Figures demonstrate selective reactivity of
YU1
antibody to thyroid carcinoma cells (brown color stained part), without any
reactivity to
normal follicular cells and stroma of tumor tissue (blue color stained part).
Accordingly, it was proved that diagnosis of thyroid carcinoma using
immunohistochemical staining with the present antibody can be accomplished,
and that the
present antibody can also be useful in a DDS system comprising anticancer
agents
(chemotherapeutic agents).
Example 6: Immunohistochemical staining of another benign tumor cells and
carcinoma cells
CA 02604143 2007-10-26
48
According to the similar procedure in Example 5, immunostaining of various
benign tumor and carcinoma cells (that were derived from human) shown in Table
I below
was examined.
Positive results were estimated as appearances of staining based on reactivity
to
YU1 antibody, and each positivity is presented in Table I below.
Table I
Number Number of
Tumor Of Cases Positive Cases Positivity
Benign Meningioma 10 0 0
Thyroid adenoma 10 0 0
Malignant Thyroid carcinoma 21 21 100
Glioma 13 1 7.7
Gastric carcinoma 16 1 6.3
Colon carcinoma 26 13 50
Lung carcinoma 20 2 10
Breast carcinoma 20 3 15
Liver carcinoma 8 0 0
Kidney carcinoma 21 0 0
Prostate carcinoma 32 2 6.3
Consequently, it was obviously shown that the positivity in thyroid carcinoma
was 100%, to the contrary, benign tumor and carcinoma originated from another
organs showed lower positivity or completely negative. Although comparative
CA 02604143 2007-10-26
49
higher positivity was shown in colon cancer, normal grandular epithelia were
also
weakly positive, therefore, positive staining presented in colon carcinoma was
distinct
from that in thyroid carcinoma, accordingly, specific immunoreactivity of YU1
to
thyroid carcinoma cells was suggested, with which remarkable staining were
observed.
Example 7: Specific immunoreaction of thyroid carcinoma with YUI :
Studies on feasibility of utilizing immunoassays
To one gram of human thyroid carcinoma and normal tissues that were used in
Example 5, 3 ml of cold cell lysis buffer (set forth above in Example 5) was
added, and
homogenized using Polytron thereafter, the homogenate was incubated for 30
minutes
on ice. Following centrifuge at 4 C, 15,000 x g for 20 minutes, the
supernatant was
recovered, and additionally obtained supernatant by the same centrifuge
condition was
recovered, then total supernatant was employed as a tissue sample. Protein
determination
was performed according to a manual of DC Protein Assay (Bio-Rad).
Thus obtained supernatant (corresponding to I mg of protein), or
immunoprecipitates of COS-7 cells transfected with LAR using the anti-LAR
antibody
as a positive control (prepared according to the procedures in Example I A-C)
were
electrophoresed, followed by immunoblotting with YU1, according to a procedure
described in Experimental Example 1, section E. For detection, Immuno Star
Reagents (Wako Pure Chemical Industries, Ltd.) was employed.
The results obtained in these experiments are shown in Fig. 10. As is clear
from Fig. 10, it was evident that YUI specifically recognizes the thyroid
carcinoma
cells, distinct from the normal thyroid cells. Accordingly, it was proved that
tissue
samples obtained in fine needle aspiration cytology of thyroid could be
utilized for
diagnosis of thyroid cancer.
CA 02604143 2007-10-26
INDUSTRIAL APPLICABILITY
The antibodies to a LAR phosphatase subunit that is provided by the present
invention can specifically recognize an intracellular domain of LAR having
phosphatase
activity. Therefore, the antibodies can be extremely useful tools for
elucidating signal
transduction mechanisms of insulin, and for identifying, obtaining LAR
modulators,
binding proteins, and the like. Furthermore, the antibodies can be applied for
developing useful diagnostic methods of insulin resistance and NIDDM, for
prophylaxis
and diagnosis of various disease states of syndrome X that is based on insulin
resistance,
and for prophylaxis and diagnosis of onset of arteriosclerosis and cardiac
diseases.
Additionally, because the antibodies of the present invention have specific
immunoreactivity to thyroid carcinoma, they are useful in diagnosis of thyroid
carcinoma
using fine needle aspiration cytology or tissue sections, and in
pharmaceutical compositions
that utilize DDS for thyroid carcinoma therapy, while they can be helpful to
molecular
biological studies on transcription of LAR molecules in thyroid carcinoma
cells and
regulation factors of expression at a translational level.
