Prof. Michel Revel’s 80th Birthday Symposium | 05/13 The road that led to the… – Dr Michal Izrael


The majority of the
presentations everywhere, the first slides are on behalf
of the institution like Hadassah, you put it in the end. That’s ok. Its not business wise. Now we will continue with
Dr Michal Izrael. It’s kind of continuation
to what we heard now, but this is the first presentation
actually dealing with Kadimastem, accept for mentioning
all the time Michel. This is first presentation. Michal is Vice President of
research and development for ALS and Neurodegenerative Diseases
at Kadimastem. She has been with
Kadimastem since 2009. Prior to joining Kadimastem
she performed her Doctoral work on Molecular Genetics
at Weizmann Institute of Science in the Professor Revel laboratory. Dr. Izrael was a
central part of the team that invented and developed the technology
that became the basis for Kadimastem
AstroRX Cell therapy product. Today I am going to tell you about the
road that led to the first clinical trial with astroRX. So AstroRX are
the Human embryonic stem cell derived Astrocytes that developed
at Kadimastem. The vision of Kadimastem which
is under the supervision of Prof. Revel is actually that we are
producing cells that actually are meant to replace restore or repair, and repair the functionality of
diseased and malfunctioning and organs in
various degenerative diseases. How do we do that? Our vision is actually transportation
of healthy and functional cells which can actually release patients
from the burden of degenerative diseases. We are actually producing spare parts. How do we do that? Our platform technology is by
the use of human embryonic stem cell or induced pluripotent stem cells
which are cells that can actually expand indefinitely
and they can give rise to any cell type of the human body. So we take these two features,
expand the cells, using our starting material and then
differentiate them towards two cell types. The first cell types
are human astrocytes and the second one are the
insulin producing cells which Kfir will elaborate
on that later on. This is the main idea of the company. The only time I will use the word
stem cells is only at the starting material, and we are actually producing
functioning healthy cells that can actually replace the
malfunctioning of specific diseases. So, we are focusing on ALS
and diabetes. ALS, Amyotrophic lateral sclerosis
also known as Lou Gehrig disease. Around 450,000 people
are affected worldwide. In this disease the muscle cells
that control our movement are dying due to death of motor neurons. Around 90-95% of disease, are familiar
with genetic background 5% -10%; No vice versa, 90-95% of disease, is sporadic,
with unknown genetic reason, 5%-10% of the desease is genetic with mutations such as human SOD1. We will encounter that
later on Human SOD1, there are several other mutations
such as the TDP (??) 72 etcetera. Since disease onset which usually
takes place around age 50-60, but there are cases where
the disease appears much earlier, the survival rate is
between 3-5 years from disease onset and today there is no real effective
treatment for ALS except for 2 FDA approved drugs
with very moderate effectiveness. One of them is Rilutek, and the
other one is called Radicava. The main rationale behind
using human astrocytes is that in ALS disease
the supporting cells, the astrocytes were found
to be malfunctioning and they failed to actually support
the dying motor neurons. So they failed to actually
produce neurotrophic factors that can induce the survival
,of motor neurons they failed to protect
from oxidative stress, they failed to uptake toxic factors
such as glutamate for example which is a neural transmitter
which once exists in the synaptic gap for too long time actually
can cause death in motor neurons. So the main idea is actually
to take healthy astrocytes, transplant them and replace
the malfunctioning of the endogenous human astrocytes. Ok, very simple idea.
Lets see how we do that. In Kadimastem we start with
human embryonic stem cell. We induce important stem cells
as our starting material and we mimic the embryonic
development by using all kinds of cells suspension and factors and actually
mimicking the embryonic development and we have defined a very nice protocol
where we start with these cells, we differentiate them, we obtain the astrocyte progenitor cells
that can be kept frozen as a cell bank, so we don’t need to
repeat the process all over again. So we at the
moment have clinically grade APC, astrocyte progenitor cell bank,
for our future use. These cells are kept frozen
then they are thawed, expanded a little bit, differentiated
for a very short period of time, in order to have committed astrocytes. The idea is to transplant
the cells intrathecally as Professor Tamir Ben Hur mentioned into the cerebral spinal fluid
the CSF with the idea that the cells will distribute throughout
the neural axis, they will reach the upper motor
neurons in the brain, the lower motor neurons
along the spinal cord and then they will have the
effect, the therapeutic effect. In order to do so, it’s a nice idea, we actually
need to do the process, we need to characterize the cells,
to follow all of the regulatory demands of the FDA of the Israeli
Ministry of Health, etcetera. This is how it looks like; we start
with nice xeno-free clinical graded starting material of
human brain stem cells. We did have a differentiation process
where we have the neurospheres which are attached
to very specific matrix. These cells are then harvested
and expanded as single cells and we are fully
committed to our cell product a very high purity
of cells of human astrocytes. Ok, as we said,
we need to actually follow the FDA and the Israeli ministry
of Health demands and we need to fully
characterize the cells in terms of their identity, their purity and actually have potency (??) to determine
their functionality. We differentiated the cells and we have a very nice panel
of human specific astrocytes markers, which we are obligated in terms
of acceptance criteria. So as you can see here,
we have the GLAST, the CD-44 the GFAP, the aquaporin,
as astrocytic markers so we have a very very nice purity
of these cells. In addition we need to prove
that actually the starting material the pluripotent stemcells which are
very risky they give rise to melanomas and all kinds of cell masses are not
actually present in our culture so we validated this system and we have a very nice
panel of pluripotent stem cells marker, like TRA-60, SSEA-4,
and EPCAM, etcetera. And we can actually show there are no pluripotent
stem cell markers in the culture. Next, We identified the cells
and we measured their purity and now we want to see
if they are functional in vitro. So for that aim we did several
potency isos (isolations??) One of them is the Glutomate uptake,
as we mentioned before. We put a very specific amount
of glutamate in the media We want to see if this glutamate levels
is disappearing in the media In the presence of the human astrocytes. So you can see here in the presence
of AstroRX or the human astrocytes, the glutamate actually
disappears from the media, the cells actually uptake glutamate
from the media and its very similar in comparison
to spinal cord astrocytes that we used as a control
and in terms of other cell types (??) activity
so we had it as a negative control. So next we wanted
to see whether this action actually secrete neurotrophic factors which can promote survival
of motor neurons. First we look at the classical ones
the BDNF, the GDNF, the VEGF We saw that these cells can
actually manufacture in the left bars, these factors, and these factors
are secreted to the media. Then we ask, OK,
are there additional factors which are secreted to the media, so
for that aim we did a secretome analysis, around thousands of factors
which are tested. And we can see that actually a lot
of well known neurotrophic factors are actually secreted by these cells there was a very nice correlation
with ALS for some of the factors such as TiMP1 TiMP2
OPN which are reduced in the disease and using our cells when actually
our cells secrete these factors that can actually promote
the survival of motor neurons. We wanted to see the crosstalk
between the astrocytes and the neurons. We did a co-culture
between our Human astrocytes, and the neurons
themselves and we saw that once we grew
the astrocytes with neurons that actually promoted
the survival of neurons and accelerated their neurite
outgrowth and axon outgrowth indicating their
therapeutic effect in vitro. So next we had to move to in vivo
studies to see whether these cells can actually distribute (??)
and we transplant them intrathecally. Do they survive along this time? Do they maintain their
astrocytic identity? Do they have an effect on the
progression of ALS disease? Finally, and very
importantly are these cells safe? For that aim we use the human SOD1,
the genetic type ALS model, the high copy number which
is a very severe animal model. From onset between 2-3 weeks
the animals actually die, we measured several factors
such as the survival, the grip strength, the rotarod, which are
motor performance measurements. We also measured the muscle
weight loss over the body mass once you lose your muscles your body
mass reduces and the neurological score
for the disease. The cells were
transplanted intrathecally very similar to the root
of administrations we are using for our clinical trial. These are the
results, you can see here, once the cells were transplanted there was a very significant delay
in disease onset there was a prolongation in the
survival rate of the animals and in terms of
motor performance we actually maintained motor
performance for longer time, once we measured it
with grip strength, once we measured it with the rotarod
rotation indicating their effectiveness for these kind of measurements. And it is very important to say these
studies were made in the rat model, but we also did it
with the mouse model actually transplanting the cells
intrathecally through the cisterna magna and we had very similar results. We have 2 types of models
we had effect on the cells. This is in terms of body mass,
so you can see that once the cells were
treated with AstroRX they maintained their body mass
for longer time Here is a short movie. Let’s see if it works. You can see on the left, this is the rotarod this
is the transplanted animals on the left you can
see a Sham injected animal. On the right you can see
an AstroRX injected animal and you can see the AstroRX animal maintained for longer time
on the rotarod and maintained the motor performance
for longer time. What about cell distribution? In order to actually assess
or answer this question we did intrathecal injection of
AstroRX cells into immune deficient mice. Here you can see
the brain, the spinal cord; we transplanted the cells through
the cisterna magna intrathecally and we monitored for 4, 17,
and 9 months after transplantation whether the cells survived,
do they maintain their cell mass? do they actually proliferate?
where are they located at? and as you can see here once
we transplanted the animals here, you can see different levels of the brain
and you can see along the spinal cord, the cells here are labeled
with DII, which is a fluorescent dye and you can see that the cells
actually attach to the perimeter, to the meninges and extract their
therapeutic effect to the surrounding CNS?? so we can see that actually the cells
are enveloping the CNS. In terms of cell survival we saw
that after 9 months the cells actually survived
and the cell mass, the number of cells maintained
was very similar. There was no
extraordinary proliferation; and using Ki67 which
is a proliferative marker we saw there was no proliferation
of these cells. The next question was whether
they maintained their identity, so for that aim we actually
stained with GFAP which is a human astrocyte marker. These are the images of the cells,
how they look in vitro. So this GFAP staining?? which is another astrocytes marker. You can see once the cells
are injected intrathecally they are GFAP positive,
they are astrocytes, they maintain their astrocytic identity. This is a closer look, you
can see it actually envelops the nerve bundles extracted to innervate
different muscles in their body. It was very nice to see there were
no formations of tumors or neuroteratomas in 9 months after injection
indicating the cells are safe. And this was again, under the roof,
or the umbrella of FDA guidelines. In terms of bio distribution
we wanted to know that the cells actually maintain in the CNS they
do not distribute to other cell organs or to other organs in the body. For that aim we used a PCR
a very sensitive method and we saw that they are
maintained in the CNS and they did not distribute
to other organs. After doing all this work, we manufactured the cells
we had a phase where we transitioned
from RND to GMP. We have in Kadimastem a GMP facility. We changed all of our materials
to be GMP suitable. So we have all the
chemicals manufacturing and controls we have the practical studies
indicating that the cells, that we know the identity of the cells,
we know the purity, we know how possible
mechanisms of actions and in addition we show that the cells
have an effect in the animal model and the cells are safe. Building all this regulatory package
we submit to the Israeli Ministry of Health
here in Israel, and we were approved in March 2018,
4 months ago. We were approved with phase 1 to a
clinical study at Hadassah medical center, at the Neurology department, the principle investigator
is Dr. Marc Gotkine who is an ALS expert here in Israel. There is also subinvestigator,
in the?? HCLC, the clinical trial center in Hadassah,
so everything is well supervised. We have an external CEO, which
supervises all of this clinical trial, which is KATO,
and international company. The main objective of the study,
is the phase 1, 2A, open label dose escalating clinical
study to actually evaluate the safety, this is our primary endpoint,
the tolerability and therapeutic effects of transplantation of AstroRX cells. This is the main idea.
We have 4 experimental cohorts. We start with 5 patients who are going
to be injected with 100 million cells. The next dose is 250 million cells which will be injected intrathecally
to these patients. The third cohort is two injections of
250, the mid-dose, the final cohort
is 500 million cells. As we said,
the first endpoint is safety. The second endpoint is actually
measuring some kind of efficacy, where we have 4 types of
measurement, one of them is ALS FRS, the second is a
handheld dyna-meter which can measure several sets
of muscles, the grip strength, the SVC which is
the slow vital capacity, etcetera. We hope by the end of the study
we will have results of the safety and of course some smell,
and strong efficacy data. Soo it’s very important to say, in many
neurodegenerative diseases such as Alzheimer’s, MS, etcetera, astrocytes have a pivotal role in
maintaining the wellbeing of motor neurons, or neurons in general
and also on oligodendryl cytes. We can use this technology
as a platform in order to actually inject the cells in other
neurodegenerative diseases and actually see whether we have
therapeutic effect on these diseases. This is our daily life,
how it looks in Kadimastem. We have now very strong
manufacturing activity, with some of the people
which are working in the lab. We have also R&D activity. This is actually the formulated syringe which will be used in our phase
1/2A clinical trial. At this opportunity
I would like to thank Michel, and tell you mazal tov, on behalf
of all of Kadimastem. That’s it.

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