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Module 1: Gene Delivery e Altre Varianti

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Video:

Hello everyone. Welcome to another lecture of Drug Delivery Engineering and
Principles. We are going to continue our discussion that we are having on this particular
module which is on vaccines.
(Refer Slide Time: 00:39)

So, just a quick recap of what we learnt in the last class. So, in the last class, we were
looking at gene therapy and using genes as vaccines; so, delivering the whole plasmid
containing your gene of interest into the cells itself. So, what will happen is because now
that you have delivered them, let us say, to a cell, these cells will produce the protein in
the cytoplasm and then it will signal as something that is being produced by a viral or a
cytoplasmic - more a class 1 response.
So, that was the whole idea there we learned few things in the last class in regards to
that. One was how we can use this for oral deliveries to take care of some of the food
allergies. In this case, we looked at peanut allergy, then we also talked about other
vaccines. In the other vaccines, we had several different formulations one was depot

formation. So, what we were doing is basically injecting something or implanting
something which act as a depot.
So, the antigens are here - rather than letting the antigen flow around and find the
immune cell, this particular one was asking the immune cells to come to its site only. So,
and the way this was doing it, it was releasing chemokines and because chemokines are
being released here right at this point, the highest concentration of chemokine is there.
And as it moves further away the concentration of chemokine was decreasing and this
gradient caused all these APCs to move in and survey the site. And once they come in,
there are also antigens that are loaded. This could be either in the same gel, the same as a
chemokines are loaded or they could be particles encapsulating your antigens and then
these APCs are going to come and take up your antigens which will then cause then
enhancement or signal as well as whatever response they need to activate.
So, it is acting as kind of a artificial lymph node and obviously, there are several papers
out there which actually do this lymph node mimicking and do a much much more
comprehensive sort of setup to get complete mimicking of that. But this is a preliminary
paper showing that you can get that and basically ask the APCs to come to the site, get
activated, and generate the immune response rather than actually letting the antigen find
the APCs. And then one other different type of vaccine we will talked about was using
particles and complement system activation.
So, in this particular example we saw that how the authors first of all were using size to
target lymphatics.

(Refer Slide Time: 03:46)

So, what they did, is they used 25 nanometer and 100 nanometer particles and they of
course, found the 25 nanometer is easier for diffusion in the lymphatic just because of
the size it can go into the lymphatics much easily than the 100 nanometer. And then what
they did is of this 25 nanometer, they prepared these particles from two different
conjugation chemistries; one that resulted in a hydroxyl present on the surface and one
that resulted in methoxy group present on the surface. And obviously, this is more
nucleophilic then the methoxy group which is fairly inert and what they found is this did
a much much better job in up regulating the complement system.
So, the complement system started to get activated and that act as a innate immune
response. So, the secondary response cause a lot more activation of the APCs and much
better vaccines, than the one that only use the methyl group. So, this is what we had
learnt- there are several other examples it is I mean, I can probably make a whole course
out of just these examples of various types of vaccines which are different from the
conventional vaccines as I have given you some flavor of it.

(Refer Slide Time: 05:14)

Now, we are going to talk about another type of vaccine that is particular for a disease
and these are cancer vaccines. And cancer vaccines are fairly widely used in research
phase; not so much in the clinical phase although there are some examples and then we
will talk about that. But there is a lot and lot of enthusiasm as well as hope that these
cancer vaccines in the research phase will succeed and ultimately translate to the clinics
because as we all know cancer is a big challenge that faces humanity at this point.
Pretty much all of us probably knows somebody or the other who is either suffering from
cancer or who has died because of cancer and the way our society is going the cancer
incident rate are increasing every year. And so, it is very important that we are able to
tackle this problem as quickly as possible.

(Refer Slide Time: 06:10)

So, first of all before we describe cancer vaccines, I will just give you brief overview
what cancer is I am again sure most of you know about this – it is nothing, but this
uncontrolled growth of cells which were not supposed to do that. At least not the way the
body was designed. These cancer cells actually routinely arise in our body. So, right
from your childhood all the way to the old age we have these sporadic cancer cells
coming up again and again. And the reason they are coming up because they caused by
mutations in some gene.
So, during the replication maybe one gene gets mutated. Most of the time those genes are
at some places that are benign so, they do not cause anything. Sometimes those gene lead
to a dysfunctional cell and the cell just dies. But then in some cases these mutations
either confers the ability to proliferate at a much higher pace or gives it enhance survival
capabilities. So, that can grow even in harsh environments and that these little things lead
to eventually development of cancerous cells.
Now, because there are mutations so, the proteins that are being produced are not the
sequence that they are supposed to be. So, let us say there is a mutation in some of the
gene that causes up regulation in proliferation. So, because of that mutation now your
original gene that the body knows is no longer exist there is a mutation maybe few amino
acids has changed, maybe few amino acids are replaced, the structure is different. So,
there is some antigens because now we can call them antigens because these are not

something that the body knows about. So, this is not natural; these are something that
have been mutated.
So, that is there are some antigens and these antigens are what the immune system can
detect. So, it is fairly feasible for the immune system now to be able to detect such
abbreviated or such mutated proteins. And then again this happens as I said much at a
much more rate than we know the immune system does continuously survey for these
cancerous cell that are being generated. And again almost all of the times the immune
system is very good and adept in destroying these cancer cells.
So, that is why we do not really see cancer being developed as often as it could. But then
there are few cases in which the immune system then fails to destroy them, maybe they
are not as immunogenic. These antigens may be the immune system is not able to survey
the entirety of the body and then once that happens these cancer cells can proliferate can
form tumors and that is what lead to tumor formation.
(Refer Slide Time: 09:22)

So, again and that is what we are trying to tackle here. We are saying that what if we
help the body in tackling those and again like your conventional vaccines cancer
vaccines can also be divided into two different types: One is prophylactic vaccine, which
as the name suggest and as we discussed in the past is given before the cancer has
happened. So, let us see how this how does that work. So, and then this really simple
reason is some cancers are actually caused by some oncoviruses.

So, we have previously talked about how I did know virus or retroviruses can actually go
into your genome and put in their genome and introducing very strong promoters in front
of genes. These promoters were not supposed to be there, sometimes they can go and put
these promoters in front of; let us say, a protein that helps in survival, a protein that helps
in proliferation, and that may cause the cancer to develop. So, one of the bigger example
of this is a cervical cancer. In cervical cancer, most of the time it is actually caused by a
virus and at a very high frequency actually and so, this virus goes in and because of it’s
process it develops these cervical cancer in our cells.
So, very traditionally what is being done is if you vaccinate against these viruses, these
pathogens that are leading to oncogenes being expressed, you basically get rid of cancer.
So, if these viruses do not get chance to actually go in and inject their genetic material,
then it will not be able to activate any oncogenes and our body will be protected from
such viruses.
So, again few examples here is a Hepatitis B vaccine is used to prevent liver cancer. So,
if you get a Hepatitis B infection, you have a higher tendency to get liver cancer.
However, if you get Hepatitis B vaccine, you will not get Hepatitis B disease and
because of that the liver cancer incidence will also go down. So, this is given as a
prophylactic treatment.
So, even before you are getting cancer, even before you getting Hepatitis B disease, you
have been given a vaccine that directly or indirectly leads to prevention of liver cancer.
Similarly, this cervical cancer we talked about, this is through the HPV virus which is a
Human Papilloma Virus and this virus alone is actually responsible for almost 70 percent
of cervical cancers. So, as I said the incidence of cancer infected with HPV is very very
high and almost 70 percent of the cancer of cervix is observed because of this infection.
So, if we somehow give a vaccine before this virus has chance to infect us, we not only
protect against this disease, but we will prevent the cervical cancer incidences by quite a
big margin.

(Refer Slide Time: 12:39)

So, that was on the prophylactic vaccines not much as I said this is mostly an indirect
way. At least at this point of time people have not been vaccinated directly for a cancer
cell which is not caused by any other pathogen in a prophylactic manner. So, that is
where the therapeutic vaccines then become very important because these are what will
be more clinically relevant at least in most of the cases, where a patient is coming in with
a disease already, with cancer already and then we want the body’s immune system to be
able to fight that cancer. And so, the goal overall here is to increase the immune response
against tumor antigens.
So, those tumor antigens that are caused by the mutations in your normal cell initially. If
we can get those immune responses against these antigens to go up, then the immune
system can come and kill your cancerous cell. So, at this point the major problem is that
the immune system is not detecting that these are cancer cells or not able to do much
against these because it does not think that these are cancerous or there are too many of
them or could be several reasons. So, somehow if we can strengthen the immune
response against these particular antigens, we will be able to hopefully cure cancer
through our own immune system in itself.
The problem is, it is actually very hard to generate immune response because these
antigens are very similar to our own antigens. So, now, we are talking about let us say if
this is a protein that the body already has, all that has changed is maybe one or two

amino acids here are mutated. So, the body finds it very difficult first of all to identify
that there is a change here and then secondly, there is really no other signal the body gets
to get activated for your immune response. Because when you are talking about a
bacterial cell coming in, we had talked about there is so, many different innate immune
pathways going up - the innate cells are recognizing the patterns, maybe the flagella,
maybe the LPS, maybe the DNA, the CpG islands and all.
So, all of this was a very good activator of the immune response and not only that the
proteins that these bacterial cells are secreting. Let us say if they are secreting a protein,
this protein was completely foreign. So, each and every peptide sequence that makes this
protein is new to the body. So, the body can generate immune response against all of
these regions and so, there is very high chance that you will get antibodies, you will get
cytotoxic T cells to be able to recognize, this kill whatever niche this particular bacteria
or other pathogens are surviving.
Now, but in this case first of all you do not have much much co-stimulation. Then the
antigen it itself is very very small as well this maybe only one antigen present in one
protein. So, all of those result in quite a difficult task for the immune system to be able to
mount a good response against it. And then not only that what then complicates a
problem is because these cells are now rapidly dividing sometimes the mutation could
actually be in enzymes that are supposed to take care of the fidelity of the DNA
replication. So, what I mean by that is let us say; there is an enzyme that checks whether
the DNA replication has happened correctly or not (checks correctness of DNA
replication).
So, now if this enzyme got mutated itself, then what will happen not only there is one
mutation here, but every subsequent time the cell is going to divide. There is a chance
that there is going to be more mutations or the mutations getting accumulated at a much
higher rate and so, that is what it means. You can also have a higher mutation rate that is
being developed and then; that means, that let us just say that here is one cell that was
cancerous,

(Refer Slide Time: 17:40)

So, it goes ahead and starts dividing and makes several of these. Now because this
enzyme was not working that is responsible for making sure that there is a correct DNA
replication that is happening. What will happen is as it divides further, maybe one of the
protein gets mutated and it is a being another type of cancerous cell. So, now, what will
happen is this will divide further and obviously, the red the original cancer cells will
continue to divide. And then it could again happen that another cell gets mutated through
these green line as well.
So, let us say another yellow cell gets invaded and then this is going to be surrounded by
the red original cells. So, this can actually continue to go on. And so, now, what you are
doing is not only you have a tumor because there is a mutation, but now as the time is
progressing as the tumors are becoming larger and larger, you are actually accumulating
more mutations and more different types of cells.
And so, then it becomes very hard for the body to be able to control for all this because
let us say if the immune system does come in as able to recognize one of these types. It
will still not be able to recognize all three, all ten types that might be there in that large
tumor. And so, that is why there are several variants of tumor antigens that comes up and
eventually, it becomes a harder job for the immune system to be able to handle that.

(Refer Slide Time: 19:55)

But nonetheless there are some vaccines like the other vaccines they have been classified
into different categories. So, one is the autologous tumor cell vaccine and as the name
suggest, that means, that this is from your own tumor cells. So, let us say a patient comes
in or a animal is induced a tumor, you isolate the cells from the tumor regions maybe at
the time of biopsy when it is being confirmed whether tumor is there we isolate some
part of the tumor anyways. So, we isolate some cells, we lyse them. So, when I lyse these
cells so, an initially let us say this was a cell and maybe one of the protein had this
mutation here.
So, let us say when I lyse these cell all of the proteins come out, I then mix it with some
adjuvants. So, what I can do is now I mix it with some LPS or I mix it with some
flagella; flagellin protein and then put it back in the body. So, now, what is happening is
earlier as I said that these tumor cells are not activating the immune system as much as
let us say bacterial cell would. So, now, what I have done is because I have given these
co-stimulatory signals, this is going to lead to a much enhanced response against any
mutated protein that is present in your lysate.
So, at this point you are basically not really testing as to what is the mutation, you are
just taking the whole cells, lysing them up, you hope that that cell lysate is now
containing several proteins that are mutated and you inject it back in the body along with
some adjuvants. And then let the immune system figure out what is wrong, which is the

protein that needs to target and one of the advantage here is you are actually presenting
the entire spectrum of your antigen.
So, all the antigens that may be present maybe 1, maybe 5 are being presented to the
immune system, which the immune system can then sort through and mount a response.
So, maybe let us say this one had two or three proteins that were mutated and even
though maybe only one protein is more immunogenic than the other because of the
mutations. So, then it becomes a higher chance for the immune system to at least detect
one of these different mutations and be able to kill the cell. As I said, this also causes
transduction of stimulated proteins such as IL-12 and GM-CSF. So, because of all that
you will have much better response. So, the innate immunity has kicked in a much better
way.
(Refer Slide Time: 22:41)

However, there is some risk of autoimmunity now, because as I said you are taking the
whole lysate and adding it with adjuvants and putting it back in the body. So, now, this
lysate even though it contains some of the antigens which are mutated from the normal
cell. This also contains several functioning and intact proteins that you are now
supplying with adjuvants.
So, there is a little bit of chance that if the adjuvant has worked very well or the immune
system got activated to such a high amount that some of these original proteins, some of
these original self-antigens may be recognized by the immune system and immune

system may mount the response against them. So, if that happens, then what will
happen? Then your immune system is going to go even to healthy cells, mount a
response against them because these healthy cells are producing these proteins, and by
that logic though self-antigen and that may cause even more destruction, than what you
wanted. And not to mention as I just said how do you get this tumor sample? It may
involve some surgical procedure needed to be done to get this initial isolation of the cell.
And so, it is invasive you need quite a bit of tumor sample again if you are going to go
ahead and inject it; you want to give as much antigen as possible. So, you want
substantial amount of tumor sample to be present as well.
(Refer Slide Time: 24:28)

So, here is just list of some of the tumor evasion mechanisms that have been known in
the literature. So, first of all is a physical barrier itself. So, as I said that the tumor is
growing fairly rapidly and it is filling of whatever space it can find so; obviously, this
space never existed before. This was supposed to be only a single cell, now it is
converted into many cells. So, it is just pushing around on the body, it is a very tight
network; its mechanical properties are much higher than the surrounding tissue in terms
of stiffness and all.
So, it is harder for now the immune cell. Let us say, this is an APC which is coming to
survey the area. It is harder for this cell to be able to penetrate deep inside this because it
will have to navigate through all kinds of cells and to be able to find all different antigens

that are present in this tumor microenvironment. So, there is a physical barrier just
imposed by the tumor architecture. Then you have induction of host myeloid and
lymphoid suppressor cells.
So, not only that, the tumour environment is very complex, it actually signals in such a
way that it causes suppression of your myeloid and lymphoid cells. So, they do not really
work very well. Sometimes, it causes T-regulatory cells which we discussed in the past
which are T cells that will actually regulate and tell the immune system to calm down to
be present in much higher quantity in the tumor region than the surrounding. So that
means, that even if the activated immune cell has coming, it is not going to be able to do
much because when it sees these it thinks that this is a site that it does not need to get
activated on and the tumor then can protect it itself from that.
There are direct mechanisms through which tumor binds to these T cells that are actually
immunogenic and actually blocks their function, so, they cannot really do their job.
There are expression of proteases that interfere with the cytotoxicity. So, obviously, let
us say if I try to deliver a protein that kills the cell, but there are lots of proteases in the
surrounding, then that protein that I am delivering it gets degraded in that environment.
So, it is hard for those proteins to survive.

There are certain expression of FAS ligand. So, it goes back to what I was saying earlier-
So, it expresses some of these proteins on these surfaces which causes the immune

system do not really go 100 percent against these particular tumor cells. There is a down
regulation of cytokine receptors. So, the cytokines do not really signal it very well on
these. So, one of the way the cells can dies lots of cytokines come in bind to it and the
cell functioning gets disturbed, but the tumor cells can have down regulation of such
receptors and that may not happen. I already mentioned, sometimes there are also
resistance to apoptosis.
So, maybe the mutation in such a gene that it does not die and continues to proliferate.
And so, they are much more hardy and much more adept to be able to survive in harsh
environment that immune system can present. The T cells can actually not be able to
even recognize it and that we already talked about that it is not very immunogenic as
well as the environment it itself is very tolerogenic environment, not letting the immune
system get activated in that area.

Even let us say if the body does able to mount the response; if the immune system is able
to recognize a particular antigen, as I said the mutation rate is very high. So, there will be
parts of regions of this tumor which would even mutate that and have some other
mutation that is causing now tumor. So, because of all that the body may not be able to
clear out the whole of the tumor cell even, then some of the tumor cells may survive
because they are slightly different and they then continue to proliferate and grow the
tumor.
It also down regulates some of the MHC molecules. So, it does not really process the
antigen. So, every cell has these molecules; every cell has these MHC molecules that are
presenting the antigen. But if the number of these receptors are lower so, let us say, there
is a certain density of these receptors on most healthy cells. What tumor does? It then
decreases this number. So, that not a whole lot of them are being presented. So, the
immune system can really recognize it. Then the expression of ligands for killer cells are
inhibited as well as it also shed these ligands out. So, that the immune cells are not able
to recognize this
(Refer Slide Time: 29:26)

So, here is what ideally we would like to have in cancer vaccines. So, you give these
cancer vaccines to dendritic cells or macrophages, which are antigen presenting cells,

they get activated against the antigen. So, this probably is along with some co-
stimulatory molecule and this is going to go ahead and activate the CTLs (the cytotoxic

T cells) because in this case the antibody can be useful, but it really will not do much
stuff; you want to kill the cells itself.
So, cytotoxic T cells and T helper cells are the major players and so, this is the immune
activation part of it. This is what ideally we would like and then these should go to the
tumor cells and start killing it. Using various mechanisms either by secreting granzyme
and perforin or IFN-gamma to further boost this response. However, what actually
happens is, as I said, these tumors are secreting all these several proteins that are causing
immune tolerance. So, TGF-beta, IDO, VEGF, galectin; it is causing activation of T-regs
this is also PD1, PDL1, that is also present, and is causing the tumor associated
macrophages and the different types of immune cells to not get activated. And all of this
result in modulation of this immune response and in that, suppresses the immune
response and be able to then protect this tumor cell.
Obviously, at the same time most patients also get conventional therapies such as
radiotherapy and chemotherapy, but then they suffer from various drawbacks that they
were not able to penetrate inside a dense tumor tissue. They are not able to kill all tumor
cells because of mutations. These tumor cells anyways are fairly strong in resisting cell
death and eventually, they are able to survive and obviously, there are tumor antigens in
the surrounding that you can again use for cancer vaccines. So, this is kind of how this
whole process is taking place in our body.
(Refer Slide Time: 31:56)

So, what are some of the desirable properties of cancer vaccines? So, of course, one is it
is targeting antigens preferentially that are exclusively on tumors, but not on healthy
tissue. Again if you use antigens that are present on healthy tissues, then you have
autoimmune response being generated. It should be fairly safe and non-toxic to human
cells - of course and that is true with any kind of treatment you. What about the quantity?
So, can you deliver enough to get sufficient number of your immune cells to be activated
What about the quality? the ones that are activating is being able to go and kill the cells.
You can express T cell whether they express T cell receptors with high ability to the
target antigen.
So, whether they will bind to it, give the desired function traffic to the tumor sites. So, be
able to go and find where it is and then further navigate inside the tumor as well and then
how much of the duration of response is given by these cells whether these cells are
active. So, for enough duration to kill all of the tumor cells in a large tumor. So, we will
stop here and we will continue rest in the next class.
Thank you.