Note: Descriptions are shown in the official language in which they were submitted.
CA 02420494 2003-02-28
1
CA 125 TUMOR ANTIGEN FUNCTION AND THERAPEUTIC USES THEREOF
FIELD OF THE INVENTION
This invention relates to CA 125 tumor antigen. More specifically, it relates
to the
biological function of CA 125 tumor antigen and to the use of CA 125 tumor
antigen
and its function as therapeutic targets in the treatment and prevention of
diseases
wherein CA 125 tumor antigen is overexpressed.
BACKGROUND OF THE INVENTION
l0
Ovarian cancer is one of the leading causes of death in women over 40.
Although
most patients respond to initial treatment, the majority relapses partially
due to the
appearance of chemo-resistant tumor cells. In order to improve therapy, it is
essential to understand the underlying mechanisms responsible for the
occurrence
of ovarian cancer.
CA125 antigen is the most important clinical marker of ovarian cancer
CA125 tumor antigen is the most important clinical marker of ovarian cancer as
it is
used to monitor response to chemotherapy. Rising or falling blood levels of
CA125
2 o correlate with progression or regression of the disease. CA125 antigen was
first
detected in the early 80's using the MAb OC125 which was raised against the
human
ovarian carcinoma cell line OV433 isolated from a patient with serous
papillary
cystadenocarcinoma (1). The specific reactivity of the OC125 Mab to a variety
of
human ovarian carcinoma cell lines and paraffin-embedded ovarian carcinoma
tissues has led to the development of a radioimmunoassay to detect the CA125
antigen in serum from ovarian cancer patients (2). Using this assay, rising or
falling
levels of CA125 were shown to correlate with progression or regression of
disease
demonstrating that CA 125 levels correlate with clinical course of the disease
(2-4).
It is currently employed as a predictor of clinical recurrence in ovarian
cancer and to
3 0 monitor response to chemotherapy treatment (5-8).
CA 02420494 2003-02-28
CA125 biochemical studies
Despite the widespread use of CA125 as a clinical marker of ovarian cancer,
the
biochemical and molecular nature as well as the function of this antigen are
poorly
understood. Previous biochemical studies demonstrated that the CA125 epitope
is
carried on a large glycoprotein with a M.W. in the range of 2x105-106 Da,
while others
reported that CA125 consists of many subunits of 50-200 kDa (9-14). The study
of
Lloyd et al. showed that CA125 is a high molecular weight glycoprotein having
properties of a mucin-type molecule (15). In these studies however, a definite
consensus regarding the molecular nature of CA 125 could not be elaborated and
no information about its function was provided. A partial cDNA encoding CA125
was
recently identified as MUC16. The deduced amino acid sequence proposed an
extracellular domain composed of 9 tandem repeats rich in serine, threonine
and
proline followed by a unique region, a potential transmembrane domain and a
short
cytoplasmic tail. CA125 is expressed in more than 80% of epithelial ovarian
cancer
but is not detectable in normal ovary tissues. However its role in the disease
is
unknown.
There is therefore a crucial need to identify therapeutic targets in order to
treat the
disease.
?o
SUMMARY OF THE INVENTION
An object of the present invention is to provide a therapeutic target that
satisfies the
above mentioned need.
2 .~
We developed a novel strategy to study the role of proteins that could not be
previously studied because the gene was not known or not available. Using this
strategy, we derived unique inhibitors of CA125 tumor antigen. We propose a
role
3 o for CA125 tumor antigen in the pathogenesis of ovarian cancer as well as
other
diseases where CA 125 tumor antigen is overexpressed, a non exclusive list of
which
is endometriosis, cervical cancer, fallopian tube cancer, cancer of the uterus
and
prostate cancer. Our results have lead to the identification of CA 125 tumor
antigen
CA 02420494 2003-02-28
3
and CA 125 tumor antigen function as novel therapeutic targets for the
treatment
and prevention of these diseases in mammals.
Accordingly, the present invention provides for the use of CA 125 tumor
antigen and
CA 125 tumor antigen function as therapeutic targets in the treatment and
prevention
of any disease, in mammal, wherein CA 125 is overexpressed, such as ovarian
cancer, cervical cancer, cancer of the uterus, fallopian tube cancer, prostate
cancer
and endometriosis.
1C
The present invention provides for the use of CA 125 tumor antigen as a
therapeutic
target in a method of treatment and prevention of a disease, in a mammal, in
which
CA 125 tumor antigen is overexpressed, wherein CA 125 tumor antigen is
sequestered, knocked-out, inhibited, inactivated or otherwise partially or
totally
neutralized before, during or after protein synthesis in ways that are known
to a
person skilled in the art.
The present invention also provides for the use of a CA 125 tumor antigen
function
as a therapeutic target in a method of treatment and prevention of a disease,
in a
mammal, in which CA 125 tumor antigen is overexpressed, wherein a CA 125 tumor
antigen function is sequestered, knocked-out, inhibited or otherwise partially
or totally
neutralized in ways that are known to a person skilled in the art.
The present invention also provides for the use of a CA 125 tumor antigen
function
2 ~> in a method of diagnosis of a disease, in mammal, in which CA 125 tumor
antigen
is overexpressed.
The present invention also provides for the use of a CA 125 tumor antigen
function
in a diagnostic kit for a disease, in a mammal, in which CA 125 tumor antigen
is
30 overexpressed.
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4
The present invention also provides for the use of a CA 125 tumor antigen
function
in the identification of an agent useful for the prevention and treatment of a
disease,
in a mammal, in which CA 125 tumor antigen is overexpressed.
The present invention also provides for the use of a CA 125 tumor antigen
function
in a screening method for the identification of an agent useful for the
prevention and
treatment of a disease, in a mammal, in which CA 125 tumor antigen is
overexpressed.
The present invention also provides for a pharmaceutical composition targeting
CA
125 tumor antigen or CA 125 tumor antigen function and its use in the
treatment and
prevention of a disease, in a mammal, in which CA 125 tumor antigen is
overexpressed .
The present invention also provides for a method of treatment and prevention
of a
disease, in a mammal, comprising the step of modulating cell surface
expression of
the CA 125 tumor antigen by administration of an inhibitor of CA 125 tumor
antigen
expression in mammalian cells. Preferably, the inhibitor is a single-chain
antibody
(ScFv). More preferably, the modulation consists in a downregulation of the
cell
2 o surface expression of the CA 125 tumor antigen in mammalian cells. Even
more
preferably, the ScFv sequesters CA 125 protein within organelles. Most
preferably,
organelles are the ER, the trans-golgi or cytoplasm (or any cell compartment).
In another aspect of the present invention the method of treatment and
prevention
5 comprises the use of ScFvs that are introduced to the CA 125 tumor antigen
by
subcloning them into eucaryotic expression vectors specifically targeting them
to the
ER and the trans-golgi or other specific cell compartments. Preferably, ScFvs
are
derived from the OC 125 Mab and the VK-8 Mab. More preferably, ScFvs are
OC125-3.11 and VK-8-1.9.
3G
CA 02420494 2003-02-28
r
In a further aspect of the present invention, it is provided an inhibitor of
CA 125 tumor
antigen expression which is OC125-3.11 or VK-8-1.9 for the use in the
treatment and
prevention of disease in a mammal wherein CA 125 is overexpressed.
The present invention also provides for the use of an inhibitor of CA 125
tumor
expression in the making of a medicament for the prevention or treatment of a
disease, in a mammal, wherein CA 125 is overexpressed. Preferably, the
inhibitor
is a scFv selected from the group consisting of ScFv OC125-3.11 and ScFV VK-8-
1.9.
rO
The present invention also provides for a method of treatment and prevention
of a
disease in a mammal comprising the step of identifying a modulator capable of
modulating cell surface expression of the CA 125 tumor antigen in cells of the
mammal. Preferably, the modulator is an inhibitor. More preferably, the
inhibitor is
an scFv selected from the group consisting of ScFv OC125-3.11 and ScFV VK-8-
1.9. The assay may be automated. The assay may be used for the high through-
put
screening of a number of modulators.
Other objects, advantages and features of the present invention will become
more
apparent upon reading of the following non restrictive description of
preferred
embodiments thereof, given by way of example only with reference to the
accompanying drawings.
2 5 BRIEF DESCRIPTION OF THE DRAWINGS
These and other objects and advantages of the invention will become apparent
upon
reading the description and upon referring to the drawings in which
Figure 1shows the proposed structure of the CA 125 tumor antigen.
30 Figures 2 A, B and C show the construction of an ScFv library.
CA 02420494 2003-02-28
E7
Figures 3 A and B show the selection of soluble ScFvs through a "colony lift
assay".
Figure 4 shows selection of soluble ScFvs (periplasmic extracts).
Figure 5 shows expression of ScFvs (ELISA).
Figure 6 shows expression of ScFvs in the pCantab-5E prokaryotic expression
system with and without induction.
Figure 7 shows selection of ScFvs binding to CA 125 (ELISA).
Figure 8 shows selection of ScFvs binding to CA 125 (ELISA).
Figure 9 shows cloning of ScFvs binding to CA 125 in eukaryotic expression
system.
Figures 10 A, B, C and D show Western blots showing expression of ScFvs
directed
to the golgi in OVCAR-3 and directed to the ER in PA-1.
Figure 11 shows expression of ScFv OC125 golgi 3.11 compared with expression
of CA 125.
Figure 12 show expression of ScFv VK-8 KDEL 1.9 compared with expression of CA
125.
5 Figure 13 shows expression of control linker compared with expression of CA
125.
Figure 14 shows expression of ScFv in golgi and expression of ScFv in ER
compared with expression of proteins native to golgi and ER, respectively.
3o Figure 15 illustrates construction and in vitro validation of anti-CA125
scFvs.
A) CA125 binding activity of anti-CA125 OC125, VK-8-1.9 and VK-8-4.5 scFvs
present in periplasmic extracts of bacteria as well as anti-Bcl2 4D7 scFv
CA 02420494 2003-02-28
compared to parental Mabs OC125 and VK-8 and to PBS and periplasmic extract
from bacteria, uninduced, IPTG-induced and controls.
B) Expression of scFvs from periplasmic extracts, probed with anti-Etag
antibody.
C) Immunoprecipitation and co-immunoprecipitation of Golgi- and ER-targeted
OC125-3.11 scFv from transient transfection of pSTCF.GOLGI-OC125-3.11 and
pSTCF.KDEL-OC125-3.11 in NIH:OVCAR-3 human ovarian cancer cells using
anti-c-myc, anti-CA125 Mabs OC125 and VK-8, western blot probed with anti-c-
myc 9E10 antibody.
D) Immunoprecipitation and co-immunoprecipitation of Golgi- and ER-targeted VK-
8-
1.9 scFv from transient transfection of pSTCF-GOLGI-VK8-1.9 and
pSTCF.KDEL-VK8-1.9 in NIH:OVCAR-3 human ovarian cancer cells using anti-c-
myc, anti-CA125 Mabs OC125 and VK-8, Western blot probed with anti-c-myc
9E10 antibody.
E) Immunoprecipitation and co-immunoprecipitation of ER-targeted VK-8-4.5 scFv
1 ~ from transient transfection of pSTCF.KDEL-VK8-4.5 in NIH:OVCAR-3 human
ovarian cancer cells using anti-c-myc, anti-CA125 Mabs OC125 and VK-8,
western blot prober with anti-c-myc 9E10 antibody.
Figure 16 shows localization of anti-CA125 scFvs and CA125 cell surface down
2 0 regulation.
A) NIH:OVCAR-3 cells were transiently transfected with pSTCF.Golgi-OC125-3.11
or pSTCF.KDEL-VK-8-1.9 constructs and 48hrs later the cells were fixed in ice-
cold methanol. Localization of scFvs was detected with the anti-c-myc A14
polyclonal antibody and compared with ER and Golgi residents using anti-
calreticulin PA3-900 and anti-ADP ribosylation factor MA3-060 monoclonal
antibodies, respectively. Oregon green anti-rabbit and texas red anti-Mouse
secondary antibodies were used.
B) NIH:OVCAR-3 cellswere transiently transfected with pSTCF.Golgi-OC125-3.11
j 0 or pSTCF.KDEL-VK-8-1.9 constructs and 48hrs later the cells were fixed in
ice-
cold methanol. Expression of scFvs and CA125 was detected using the anti-c-
CA 02420494 2003-02-28
myc A14 polyclonal antibody and anti-CA125 M11 monoclonal antibody. Oregon
green anti-rabbit and Texas red anti-mouse secondary antibodies were used.
Figure 17 shows Cell surface down modulation of CA125 in stable NIH:OVCAR-3
clones expressing the ER-VK-8-1.9 anti-CA125scFv and relevant control.
A) Stable transfectants expressing the ER-targeted VK-8-1.9 and VK-8-4.5 scFvs
and parental cell line NIH:OVCAR-3 were fixed in ice-cold methanol generated
and expression of CA125 at the cell surface and scFv was assesed by
immunofluorescence using anti-c-myc A14 polyclonal antibody and anti-CA125
1a M11 monoclonal antibody, respectively. Oregon green anti-rabbit and Texas
red
anti-mouse antibodies were used as secondary antibodies.
8) CA125 expression in the stable trasnfectants was analysed by FACS using
anti-
CA125 M11 monoclonal antibody and a Phyco-Erythrin-anti-mouse antibody and
compared with parental cell line NIH:OVCAR-3; Black, OVCAR-3 levels of CA125
expression at cell surface; grey, CA125 levels in stable transfectants.
Figure 18 shows decreased CA125 cell surface expression influences the
proliferation rate, cell-cell interaction and cell migration.
A) Growth curve of stable NIH:OVCAR-3 transfectants ER-VK-8-1.9#9 (positive
for
2 0 CA125 binding) and ER-VK-8-4.5#12 (negative for CA125 binding) compared to
parental cells OVCAR-3. Cells were plated in triplicate in 96-well plate and
cell
proliferation was measured every day with a XTT assay. Plot represents results
from 3 independent experiments
B) Cell aggregation assay. Cells were plated onto 0.6% agarose layer in
bacterial
25 dishes. Seventy-two hours later photomicrographs were taken (10X
magnification) to visualize the presence of cell aggregates.
C) Wound healing assay. A wound was made using a 13mm-wide razor blade in
confluent cell monolayers and 20mM hydroxy-urea was added to block cell
proliferation. Forty-eight hours later, the cells were fied in methanol and
stained
with Giemsa and microphotographs were taken (10X magnification).
D) Tumorigenic assay. Ten millions NIH:OVCAR-3 transfectants ER-VK-8-1.9#9,
CA 02420494 2003-02-28
ER-VK-8-4.5#12 and parental cells OVCAR-3 were inoculated subcutaneously in
nude mice Tumors were allowed to grow for 6 weeks after which tumorw were
excised and tumor weight was measured and plotted for each transfectant.
Figure 19
A) NEDO cDNA clone FLJ14303 encodes a part of CA125. Cos-7 cells (negative for
CA125 by Western blot and ELISA) were transfected with an expression vector
encoding the cDNA from the NEDO clone FLJ14303. Reactivity of anti-CA125
OC125 and VK-8 antibodies with the expression product of this cDNA was
1 ~'~ analysed by western blot and compared to CA125 expression in OVCAR-3
cells
as well as in mock-transfected Cos-7 cells.
B) Expression of the CA125 cytoplasmic tail fused to Gal4 DNA binding domain.
The
CA125 cytoplasmic tail was cloned in the pGBDU and pGAD for the yeast two-
hybrid system. The S.Cerevisiae strain PJ69-4a was transformed with the
pGBDU empty vector (EV) or with the vector Containing CA125 cytoplasmic tail
(Cyto). Three days after growth on appropriate media, proteins were extracted
from the 2 transfectants or the wt strain PJ69-4a, ran on 12.5% SDS-PAGE and
transfered by western blot on a PVDF membrane. The membrane was probed
with anti-Gal4 DNA binding domain antibody Gal-4-DBD RK5C1.
~~ o
Figure 20 shows cisplatin sensibility of stable NIH:OVCAR-3 clones expressing
the ER-VK-8-1.9 anti-CA125scFv and relevant controls.
Cells were plated in triplicate in 96-well plates and exposed or not to
increasing
concentrations of cisplatin. Fours days later, cell proliferation was measured
with a
2 ~ XTT assay. Percentage of survival was plotted against concentration of
cisplatin.
Curves represent results from 3 independent experiments. Red line represents
50%
survival.
3 o Figure 21 shows IC50 of cisplatin for the stable NIH:OVCAR-3 clones
expressing the ER-VK-8-1.9 anti-CA125scFv and relevant controls. Inhibitory
concentrations of cisplatin resulting in 50% survival of cells were calculated
from
CA 02420494 2003-02-28
1
curves of graph in figure 20 (red line in figure 20). .
Figure 22 shows expression of E-cadherin and av~35 integrin in NIH:OVCAR-3
cells. NIH:OVCAR-3 were fixed in ice-cold methanol generated and expression of
E-cadherin and cxv~35 integrin at the cell surface was assesed by
immunofluorescence using E-cadherin clone 36 and anti- av~5 integrin clone P1
F6
antibody and Texas red labelled secondary anti-mouse antibody.
Figure 23 shows expression of E-cadherin and scFv in NIH:OVCAR-3 stable
1 c~ transfectant expressing the ER-VK-8-1.9 anti-CA125 scFv without induction
with doxycycline. Cells were grown on glass slides for 48hrs and fixed in ice-
cold
methanol generated and expression of E-cadherin and scFv was assesed by
immunofluorescence using E-cadherin clone 36 and anti-c-myc A14 antibody and
Texas red or Oregon green-conjugated secondary anti-mouse and anti-rabbit
antibodies.
Figure 24 shows expression of E-cadherin and scFv in NIH:OVCAR-3 stable
transfectant expressing the ER-VK-8-1.9 anti-CA125 scFv when induced with
doxycycline. Cells were grown in presence of doxycycline for 48 hrs and then
fixed
in ice-cold methanol generated and expression of E-cadherin and scFv was
assessed by immunofluorescence using E-cadherin clone 36 and anti-c-myc A14
antibody and Texas red or Oregon green-conjugated secondary anti-mouse and
anti-rabbit antibodies.
Figure 25 shows expression of E-cadherin and scFv in NIH:OVCAR-3 stable
transfectant expressing the ER-VK-4.5 control scFv without induction with
doxycycline. Cells were grown on glass slides for 48hrs and fixed in ice-cold
methanol generated and expression of E-cadherin and scFv was assesed by
immunofluorescence using E-cadherin clone 36 and anti-c-myc A14 antibody and
3~~ Texas red or Oregon green-conjugated secondary anti-mouse and anti-rabbit
antibodies.
CA 02420494 2003-02-28
ll
Figure 26 shows expression of E-cadherin and scFv in NIH:OVCAR-3 stable
transfectant expressing the ER-VK-4.5 control scFv when induced with
doxycycline. Cells were grown on glass slides in the presence of doxycycline
for
48hrs and fixed in ice-cold methanol generated and expression of E-cadherin
and
scFv was assesed by immunofluorescence using E-cadherin clone 36 and anti-c-
myc
A14 antibody and Texas red or Oregon green-conjugated secondary anti-mouse and
anti-rabbit antibodies.
Figure 27 shows expression of av~5 integrin and scFv in NIH:OVCAR-3 stable
transfectant expressing the ER-VK-8-1.9 anti-CA125 scFv when induced or not
with doxycycline. Cells were grown in the absence or presence of doxycycline
for
48hrs and subsequently fixed in ice-cold methanol generated and expression of
av~35 integrin and scFv was assesed by immunofluorescence using anti- av~5
integrin clone P1 F6, anti-c-myc 9E10 antibody and Texas re or Oregron green
15 labelled secondary antibodies.
Figure 28 shows expression of av~5 integrin and scFv in NIH:OVCAR-3 stable
transfectant expressing the ER-VK-8-4.5 anti-CA125 scFv when induced or not
with doxycycline. Cells were grown in the absence or presence of doxycycline
for
48hrs and subsequently fixed in ice-cold methanol generated and expression of
av~5 integrin and scFv was assesed by immunofluorescence using anti- av(35
integrin clone P1 F6, anti-c-myc 9E10 antibody and Texas re or Oregron green
labelled secondary antibodies.
Figure 29 shows alignment of deduced amino acid sequence for anti-CA125
scFvs VK-8-1.9 and OC125-3.11 as well as control scFv VK-8-4.5 Nucleotidic
sequences encoding the anti-CA125 scFvs and their control were determined from
pCANTABSE/scFv constructs using the scFv specific primers S1 and S6 from the
pCANTAB5 sequencing primer set (Amersham Pharmacia Biotech, Piscataway, NJ).
3G Sequences were determined using the LI-COR automatic sequencing system (Bio
S&T Inc., Lachine, QUE). Amino acid sequence was deduced from the nucleotidic
CA 02420494 2003-02-28
12
sequences and aligned using the Alibee multiple alignment software available
at
www.genebee.msu.su/services/malign reduced.html. The area in boxes represent
consensus sequences of frameworks 1-4 of heavy and light chain, asterisks
correspond to differences between VK-8-1.9 and OC125-3.11 anti-CA125 scFvs
whereas arrows identify differences between VK-8-1.9 and VK-8-4.5 scFvs.
While the invention will be described in conjunction with example embodiment,
it will
be understood that it is not intended to limit the scope of the invention to
such
embodiment. On the contrary, it is intended to cover all alternatives,
modifications
and equivalents as may be included as defined by the appended claims.
DESCRIPTION
The CA125 tumor antigen is a protein associated with the majority of human
epithelial ovarian cancer, the most common form of the disease. It is also
known to
be overexpressed in other diseases such as endometriosis, cervical cancer,
cancer
of the uterus, fallopian tube cancer, prostate cancer, etc. It was recently
proposed
that CA125 is part of the mucin family of proteins. Mucins are known to play
an
important role in cell adhesion in cancer cells as well as in normal cells. In
some
cancer cells, mucins promote the metastatic process.
To elucidate the function of CA125, we developed 2 anti-CA125 single-chain
antibodies (scFvs) and show that they act as CA125 specific inhibitors. When
expressed intracellularly and retained to the ER or Golgi, the anti-CA125
scFvs
presumably entrap CA125 within the secretion pathway and therefore prevent its
proper cell surface localization in the human ovarian cancer cell line OVCAR-
3. In
addition, we have shown that stable inhibition of CA125 in OVCAR-3 cells
results in
an increased cell proliferation and reduced cell adhesion and migration and
prevent
tumor growth in nude mice. Our hypothesis is that CA125 modulates cell
proliferation, adhesion and migration through molecular mechanisms. We have
3o confirmed our observations using alternative knockout approaches such as
RNA
CA 02420494 2003-02-28
13
interference (RNAi) to ablate CA125 expression in OVCAR-3 cells and extend our
observations to a panel of human cell lines expressing various levels of CA125
such
as OV-90 and SKOV3ip1 (expressing high to low levels of CA125) and primary
ovarian cancer cells (result not shown). We also observe that loss of CA125 at
the
cell surface affects the in vivo behaviour of ovarian cancer cells in
xenograft mouse
models.
EXAMPLE I : Construction and in vitro validation of anti-CA125 scFvs
We constructed single-chain antibody libraries derived from the OC125 and VK-8
l0 hybridoma cell lines specific for CA125. The 2 scFv libraries were screened
for
CA125 binding activity by ELISA using commercially purified human CA125. ScFvs
that bound to CA125 by ELISA, OC125-3.11 and VK-8-1.9, and one that did not
bind,
VK-8-4.5 were selected (figure 15 A-B). We hypothesized that if those scFvs
(CA125
binders) once expressed intracellularly were localized to and retained within
the ER
or the Golgi then CA125 antigen would be entrapped during synthesis and thus
be
unable to localize at cell surface and interact with other intracellular
and/or
extracellular proteins to achieve its function(s). The scFvs were targeted to
the ER
or trans-median Golgi by sequence fusion with an IgK secretion leader and a
KDEL
signal or fusion with the N-terminal 81 amino acids of human beta 1,4-
2 o galactosyltransferase, a protein resident of the trans-medial Golgi (47-
49) in addition
of a c-myc tag at the C-terminus. Immunoprecipitation experiments showed that
the
anti-CA125 scFvs were immunoprecipitated with anti-c-myc antibody whereas only
OC125-3.11 and VK-8-1.9 (both positive for CA125 binding) were co-
immunoprecipitated using anti-CA125 OC125 and VK-8 MAbs (figure15 C-E). These
a 5 results demonstrate that the OC125-3.11 and VK-8-1.9 anti-CA125 scFvs bind
to
CA125 in vitro.
EXAMPLE II : Localization of anti-CA125 scFvs and cell surface down-
3 o regulation of CA125
Proper localization of our anti-CA125 scFvs in transient transfection of human
ovarian cancer cells OVCAR-3 was demonstrated by immunofluorescence. Results
CA 02420494 2003-02-28
obtained with ER-VK-8-1.9 and GOLGI-OC125-3.11 are shown in figure 16A.
Immunofluorescence studies showed that cells expressing the Golgi-targeted
OC125-3.11 or ER-targeted VK-8-1.9 lost expression of CA125 at the cell
surface.
However surrounding cells that did not express the scFvs (not transfected)
were
positive for CA125 at the cell surface. In addition, the presence of the ER-
targeted
VK-8-4.5, which did not bind CA125 by ELISA and immunoprecipitation
experiments,
did not affect expression of CA125 in the cells expressing this scFv. These
results
show that the expression and retention of ER- or GOLGI-targeted anti-CA125
scFvs
results in CA125 down-regulation at the cell surface. Anti-CA125 scFvs act
therefore
1 o act as potent inhibitors of CA125.
EXAMPLE III : Consequences of CA125 cell surface down-regulation in human
ovarian cancer cell line NIH:OVCAR-3
To determine the effects of down-modulating CA125 expression at the cell
surface,
15 we derived stable clones encoding the ER-targeted VK-8-1.9 and Golgi-OC125-
3.11
(both positive for CA125 binding) and ER-VK-8-4.5 (negative for CA125 binding)
scFvs in human ovarian cancer cell lines OVCAR-3 (high expresser of CA125), OV-
90 (moderate expresser) and SKOV3ip1 cells (low expresser). Some of the OVCAR-
3 clones have been already characterized for scFv and CA125 expression and all
of
Go the other clones (including in SKOV3ip1) have also been evaluated.
Characterization
of stable clones ER-VK-8-1.9#9 and ER-VK-8-4.5#12 is shown in figure 17. A
dramatic decrease in CA125 expression at the cell surface was observed in
stable
clone ER-VK-8-1.9#9 (positive for CA125 binding) while CA125 expression was
not
affected in the clone ER-VK-8-4.5#12 (negative for CA125 binding) although the
scFv in this clone was expressed at adequate levels (figure 17A). Similar
results
were obtained from FACS analysis (figure 17B). These results are consistent
with
results obtained previously from transient transfection experiments. Taken
together
these results demonstrate that our scFvs act as specific inhibitors of CA125
and that
our stable clones behave as unique tools to study CA125.
i
Expression of E-cadherin and av~3v integirin at the cell surface
To further characterize the stable transfectants expressing the anti-CA125
scFvs we
CA 02420494 2003-02-28
LJ
evaluated the expression of E-cadherin and ~xvw integrin at the cell surface
for each
stable transfectant. Figures 23 through 26 show that E-cadherin expression at
the
cell surface is not affected by the presence of the ER-VK-8-1.9 anti-CA125
scFv or
the control ER-VK-8-4.5 demonstrating that E-cadherin expression is not
modulated
by CA125 levels. Expression of cxv~v integrin at the cell surface of ER-VK-8-
4.5
transfectant is also not affected by the expression of the scFv (figure 28).
However,
the stable transfectant expressing the ER-VK-8-1.9 anti-CA125 scFv shows a
reduced level of av~3v integrin at the cell surface demonstrating that CA125
influences levels of cxv~3v integrin expression at the cell surface.
~~o
Cell proliferation on adhesive support
In vitro growth kinetics of ER-VK-8-1.9 and ER-VK-8-4.5 clones was evaluated
compared with that of the parental cell line using a XTT cell proliferation
assay (50).
Stable clone ER-VK-8-1.9#9 (positive for CA125 binding) grew faster than the
ER-
VK-8-4.5#12 (negative for CA125 binding) which grew at a rate similar to that
of the
parental OVCAR-3 cells (figure 18A). Stable clone ER-VK-8-1.9#9 seems to
adhere
faster to the plastic than OVCAR-3 cells or ER-VK-8-4.5#12 clone (not shown).
These results show that loss of CA125 at the cell surface affects cell
proliferation on
adhesive support.
a c;
Sensitivity to cisplatin
Consequently, sensitivity to cisplatin of the various stable transfectants was
determined. Stable clone ER-VK-8-1.9#9, the ER-VK-8-4.5#12 and the parental
cell
lines were plated in triplicate in 96-well plates and exposed or not to
increasing
concentrations of cisplatin. Fours days later, cell proliferation was measured
with a
XTT assay. Percentage of survival was plotted against concentration of
cisplatin.
Curves represent results from 3 independent experiments (figure 20). Results
showed that stable clone ER-VK-8-1.9#9 was more sensitive to cispaltin than
control
cells. IC50 were calculated and figure 21 shows that the stable clone ER-VK-8-
1.9#9
is approximately 10-fold more sensitive to cisplatin than ER-VK-8-4.5#12 and
the
parental cell lines. Similar experiments were performed using taxol and
results
showed no difference between sensitivity of stable transfectant ER-VK-8-1.9#9,
the
CA 02420494 2003-02-28
1. 6
ER-VK-8-4.5#12 and the parental cell lines confirming that the increased
sensitivity
of transfectant ER-VK-8-1.9#9 is linked to the increase in cell proliferation,
These
results demonstrate that CA125 influence cell proliferation and thereby
controls the
sensitivity to therapeutics drugs such as cisplatin.
Cell-cell interactions and anchorage independent
We also assessed the effect of CA125 cell surface down-regulation on the
ability of
the cells to mediate cell-cell interaction using a cell aggregation assay
(51). Cell-cell
interactions are measured by the ability of the cells to aggregate to each
other and
1 c grow in clumps. Transfectant ER-VK-8-4.5#12 formed small aggregates and
grew
in small clumps similarly to the parental OVCAR-3 cells (figure 18B). In
contrast,
transfectant ER-VK-8-1.9#9 (positive for CA125 binding) did not form aggregate
and
only isolated single cells were observed. In addition, the single cells
observed in this
clone did not grow and looked as if they were dead or dying. These results
show that
1 ~ loss of CA125 at the cell surface impairs the cells ability to mediate
cell-cell
interactions and to survive in anchorage-independent conditions.
Cell migration
We also determined the consequence of reducing CA125 expression levels at the
? o cell surface on cell migration. We evaluated the cell motility of ER-VK-8-
1.9#9 and
ER-VK-8-4.5#12 stable transfectants and compared to the parental cell line
using the
wound or scratch assay (52). Cells were plated in 6-well plates and when
confluent
a wound was made in the monolayer using a razor blade. To distinguish between
cell
proliferation and cell migration, cell proliferation was inhibited with 20mM
2. 5 hydroxyurea (53). Cells were incubated in the presence or the absence of
FBS. In
the absence of FBS none of the cells, neither the parental cells, were able to
migrate
and fill in the wound (not shown). This suggests that some factors present in
the
serum may be required for stimulating cell motility as showed by others in
different
tumor cell lines (54). However, in the presence of FBS, the only cells that
did not
30 migrate and fill in the wound were the cells from clone ER-VK-8-1.9#9
(positive for
CA125 binding) (figure 18C). Cells from clone ER-VK-8-4.5#12 (negative for
CA125
binding) migrated in a similar manner as the parental cells. These results
show that
CA 02420494 2003-02-28
1 ~7
CA125 affects cell migration of the OVCAR~-3 cell line.
Tumor growth
We also determined whether the loss of CA125 expression at the cell surface
affects
the in vivo behaviour of human ovarian cancer cells in tumor-bearing mice
subcutaneously or intraperitoneally. This was achieved by evaluating tumor
growth,
tumor burden, formation of ascites, presence of tumor cells in ascites,
pattern of
metastases spread and survival of mice. The tumorigenicity of each stable
transfectants was also determined in nude mice. Stable clone ER-VK-8-1.9#9,
the
1 o ER-VK-8-4.5#12 and the parental cell lines were inoculated subcutaneously
in nude
mice and tumors were allowed to grow for 6 weeks after which tumor were
excised
and tumor weight was measured. Figure 18D shows that tumor derived from stable
clone ER-VK-8-1.9#9 were significantly much smaller (if existant) than those
from the
ER-VK-8-4.5#12 and the parental cell lines demonstrating that CA125 influence
the
tumorigenic potential of ovarian cancer cells. When injected
intraperitoneally, stable
clone ER-VK-8-1.9#9 showed a significant slower growth, reduced volume of
ascites,
a decrease in the total number of viable tumor cells in suspension (in
ascites) and
therefore an overall increased in survival of mice (not shown).
These results taken together point to a role for CA125 in the pathogenesis of
ovarian
cancer by influencing tumor cell proliferation, tumor cell adhesion and
migration, and
in tumorigenesis.
EXPERIMENTAL PROCEDURES
2
Derivation of anti-CA 725 scFv constructs - The hybridoma cell line VK-8 which
express a monoclonal antibody against CA 125 tumor antigen has been described
previously and was kindly provided by K.O. L_loyd (Sloan-Kettering Memorial
Cancer
Center, New York, NY) (18). Total mRNA was extracted from VK-8 hybridoma using
3 o the PolyA-track kit from Promega (Cie, city, state). Total mRNA extracted
from
OC125 hybridoma cell line was kindly provided by R.C. Bast (MD Anderson Cancer
Center, Houston, TX). ScFvs constructs were generated using the Recombinant
CA 02420494 2003-02-28
1 ~5
Phage Antibody System (Amersham Pharmacia Biotech, Piscataway, NJ) according
to the manufacturer's instructions. Briefly, the variable heavy and light
chains (VH and
V~) were amplified from the cDNA by PCR using mouse variable region primers.
The
VH and V~ DNA fragments were linked together by overlap extension PCR using a
(GIy4Ser)3 linker to generate 750bp scFv constructs with flanking Sfil and
Notl sites.
The scFv DNA fragments were inserted into Sfil/Notl sites of the prokaryotic
expression vector pCANTABSE from the Mouse ScFv Module (Amersham
Pharmacia Biotech, Piscataway, NJ). Screening of recombinant clones expressing
a soluble scFv was accomplished by a colony lift assay as described previously
(26).
