1. EVALUATION
OF
NOVEL
HUMAN
RECOMBINANT
ANTI-­‐HER2/NEU
ANTIBODIES
IN
DIFFERENT
CANCER
CELL
LINES
ABSTRACT
HER2
(HER2/neu)
is
overexpressed
in
25%-­‐30%
of
patients
with
breast
cancer
and
is
associated
with
increased
metastatic
potential
and
poor
prognosis.
Blocking
the
HER2
signaling
has
been
the
focus
of
most
therapeutic
approaches
in
breast
cancer
therapy.
Although
monoclonal
antibodies
have
shown
some
success
in
the
inhibition
of
tumor
growth,
their
life-­‐threatening
side
effects
have
limited
the
use
of
these
agents.
Advances
in
recombinant
DNA
technology
facilitated
the
production
of
smaller
recombinant
antibody
fragments
such
as
single-­‐chain
Fv
(scFv)
antibodies.
Because
of
their
small
size,
these
proteins
penetrate
faster
and
deeper
into
tissues
and
clear
more
rapidly
from
the
blood
than
whole
IgG
or
Fabs.
The
scFvs
offer
potential
advantages
over
larger
antibody
molecules
for
cancer
therapy.
In
this
study
we
selected
three
specific
and
high
affinity
human
scFv
antibodies
against
three
immunodominant
epitopes
of
HER2
from
a
human
phage
display
antibody
library
and
assessed
their
effects
on
HER2
overexpressed
human
breast
cancer
cell
lines.
In
order
to
select
specific
and
high
affinity
antibodies,
four
rounds
of
panning
were
performed.
PCR
and
fingerprinting
were
done
to
check
the
existence
of
desired
insert
and
to
distinguish
the
selected
clones
with
common
patterns
respectively.
To
perform
In
vitro
assays,
three
HER2-­‐
overexpressing
cell
lines
(BT-­‐474,
SKBR-­‐3,
and
SKOV-­‐3)
along
with
a
HER2-­‐low-­‐expressing
cell
line
(MCF-­‐7)
and
a
HER2-­‐negative
cell
line
(HeLa)
were
chosen.
The
anti-­‐proliferative
effects
of
selected
scFv
antibodies
were
assessed
by
MTT
assay
on
all
the
cell
lines.
In
addition
the
effects
of
the
scFv
antibodies
on
HER2
expression
at
both
the
gene
and
protein
levels
were
investigated
in
HER2-­‐overexpressing
breast
cancer
cell
lines
(BT-­‐474
and
SKBR-­‐3)
using
real-­‐time
PCR
and
western
blotting
respectively.
The
inhibitory
effects
of
the
antibodies
on
VEGF
and
SDF-­‐1
gene
expressions
were
also
demonstrated
by
real-­‐
time
PCR
technique.

2. Twenty
clones
were
isolated
against
each
epitope.
PCR
and
fingerprinting
results
revealed
that
the
isolated
clones
against
each
epitope
have
the
insert
and
also
their
patterns
are
the
same.
One
clone
against
each
epitope
was
selected
for
further
investigations.
The
MTT
results
showed
that
the
scFvs,
separately
and
in
combination,
are
capable
of
inhibiting
the
cell
growth
of
HER2-­‐positive
cell
lines
significantly.
These
inhibitory
effects
on
BT-­‐474,
SKBR-­‐3,
and
SKOV-­‐3
cell
lines
were
80%,
75%,
and
80%
respectively
whereas
lower
inhibitory
effects
were
observed
in
MCF-­‐7
cell
line
(15%)
and
no
effect
was
observed
in
HeLa
cell
line.
Furthermore
combination
of
three
selected
scFvs
significantly
inhibited
the
cell
growth
more
efficient
than
scFvs
alone.
The
results
of
real-­‐time
PCR
and
western
blotting
demonstrated
that
our
selected
scFvs
inhibit
the
expression
of
HER2
at
both
the
gene
and
protein
levels
and
this
inhibitory
effect
was
significantly
more
effective
when
the
cells
were
treated
with
combination
of
all
three
scFv
antibodies.
Also,
the
obtained
results
from
real-­‐time
PCR
indicated
that
the
selected
scFvs
are
capable
of
down-­‐regulating
the
VEGF
gene
expression
separately
and
in
combination.
Moreover
scFv-­‐II
and
combination
of
three
scFv
antibodies
reduced
the
expression
level
of
SDF-­‐1
mRNA
in
HER2-­‐
positive
breast
cancer
cell
lines.
The
significant
inhibitory
effects
of
the
selected
scFvs
on
HER2-­‐
positive
breast
cancer
cell
lines
offer
the
potential
application
of
these
selected
libraries
in
the
treatment
of
cancers
overexpressing
HER2.
Down-­‐regulation
of
HER2
in
both
the
gene
and
protein
levels
induced
by
the
selected
scFvs
is
a
good
criterion
for
the
selection
of
these
small
antibodies
for
effective
treatment
in
cancer
immunotherapy
of
HER2-­‐positive
breast
cancer.
Moreover
the
anti-­‐angiogenic
effects
of
these
high
affinity
libraries
showed
by
reduction
in
VEGF
gene
expression
make
these
antibodies
more
attractive
for
anti-­‐cancer
therapy.
According
to
the
scFvs
combination
assays,
we
suggest
the
use
of
these
scFvs
in
combinations
form
in
order
to
down-­‐regulate
HER2,
VEGF,
and
SDF-­‐1
simultaneously
to
obtain
more
efficient
outcome
in
breast
cancer
immunotherapy.