hello I'm David
baltimore professor biology at
california institute of technology and
this is the third in a series of talks
about HIV are there ways that we can
ultimately conquer it today's talk will
be about something that we call the
grand challenge an attempt to engineer
the immune system to do things that it
ordinarily doesn't do and in particular
to see if we can adapt this form of
thinking to HIV so what do we mean by
reprogramming the immune system what we
need to do is to make vaccines or
therapies that can bring into play
antibodies or other kinds of antiviral
proteins over that amount of t-cells
that ordinarily are not there and so we
need to genetically alter cells of the
immune system by bringing in new genes
to those cells such that they will make
proteins that can can allow the immune
system to attack things it ordinarily
refuses to deal with it ordinarily is
unable to deal with an approach like
that liberates us to use modern protein
design methods to create antibody like
proteins that then we can program into
the immune system and get the immune
system to make for us the vectors that
will do use should be able to get into
the cells that make antibodies which are
called b-cells
but the root into it is probably through
the hematopoietic stem cell because that
gives rise to b-cells continually so our
overall goal for this project and it
actually is funded by the Gates
Foundation Global Health Program and is
considered by them a grand challenge the
overall goal is to direct blood stem
cells to develop into beasts
which are capable of making therapeutic
molecules against HIV and if we can do
that against other pathogens so our
immune systems are remarkable they can
make reactions against molecules that
have never been seen before
so if Union Carbide or DuPont goes out
and makes a new kind of molecular entity
never before seen on the planet you put
that into a rabbit the rabbit will make
antibodies against that molecular
determinate so it's got to be able to
learn about molecules it's never seen
before the species has never seen before
and it works it works terrifically well
the repertoire of antibodies that will
make is large but it's not infinite
there are holes in it particularly if
the antigen doesn't present itself in a
way that an antibody can see it because
the animage antigen is hidden away
inside a molecule that antibodies can't
get in there because they're just big
things and even if the immune repertoire
includes the ability to make a
particular kind of antibody we may not
be able to elicit that antibody with any
available antigen so there are lots of
situations you can imagine in which you
could do better than this remarkable
system does already so we have as a goal
in my own laboratory to engineer immune
cells to do new valuable things like
making antibodies or t-cells or using
intracellular immunogen's like RNAi I
which I talked about in the previous
talk
so this is what we call our engineering
community project and we've got three
pieces of it in fact one piece is to use
intercellular immunization with RNA I I
talked about that mainly that will be
directed against HIV and the
second is that we want to engineer
t-cells so they can react to tumor
antigens because T cells have the
ability to kill tumor cells but in fact
most tumors Altimas that grow up in
humans have eluded immune attack in one
way or another and we believe that there
are ways of programing the immune system
so that it can do things that it doesn't
do ordinarily and we are very close to
having a phase one trial of this idea
and finally and it turns out to be much
more difficult we want to engineer B
cells to make pre-specified into
monoclonal antibodies or antibody like
proteins then we'll target HIV so all of
these have a common methodology we're
making retro virus vectors generally
lentivirus vectors of the kind I
described previously to program
particular abilities into a metabolic
stem cells and then as those temps ELLs
give rise to B cells or T cells or
actually other kinds of cells they can
then carry out the specific functions
that we're interested in so it's a gene
therapy approach to stem cell
modification with the idea of generating
immunotherapy utak molecules it's this
kind of triumvirate as shown here gene
therapy leading to stem cell therapy
leading to immuno therapy and it's and
we can call it an instructive
immunotherapy it's extremely difficult
it's extremely difficult because nobody
really has gene therapy working in a
reproducible manner yet we don't have
stem cell therapies working in a
reducible reproducible manner and we
don't have immunotherapy working all
right
one of the details of this well the
details of it are that let's take the
case of attacking cancer through t-cells
we've got to make a vector which has
none of the viral genes in it but has
the G
of interest in it and in this case the
gene of interest is a gene that will
direct the specificity of t-cells way
down here to make a particular response
to a tumor we've got to put that vector
into a virus we've got to infect that
virus into a cell and there it has to
make proteins that will have this
therapeutic activity one of the cells
well we want him at a poetic stem cells
experimentally we find those in the bone
marrow of mice so we take mouse bone
marrow we expose it to viruses like this
and some fraction of the cells become
infected the red ones shown through here
we then transfer this mixture of cells
into a mouse is actually a mouse that's
been irradiated so it's own blood
forming system is gone the new cells now
replace the old cells in the in the
blood in the bones of these animals and
the animal now makes all the different
blood elements red blood cells white
blood cells platelets
but now makes them from this population
of partially genetically modified cells
and some fraction of those cells should
be lymphocytes and those lymphocytes
will be able to target themselves to
tumor cells so this I show you this not
because it has to do with HIV but
because this we have working in an
experimental system in mice and we have
been able to protect mice against really
quite lethal tumors using this form of
methodology so it's not totally
theoretical it has actually worked in at
least one experimental setting so for
HIV it's basically the same thing but
now we want to put in genes that will be
expressed in B lymphocytes to make
antibodies so we put those now into a
virus virus goes into the cells we now
imagine this in humans we take out bone
marrow cells we expose them to this
virus
get infected we put them back into the
person the person's immune system now
derives from these modified cells and we
get lymphocytes which we hope B
lymphocytes in this case which we hope
will target HIV what we would really
like to do and this is now carrying it
to another realm of technology is to get
rid of that whole intermediate stage
where we take out the cells put them in
cell culture infect them with a virus
and then put them back what we'd rather
do is put a virus into a person a viral
vector into a person so same thing we'll
make our vector
we'll put it into a virus but now we
want to put that virus directly into a
human being or to an animal and get it
to infect the appropriate cells in the
bone marrow those cells then will be
modified and exactly the same
consequence will come out at the other
end that's very difficult it's difficult
to get a virus that will find its way
into the bone but we're working on that
and there are ways of actually getting
these cells out of the bone into the
circulation so we might not have to get
the virus into the bone and there are
actually ways of getting the virus in
the bone that have worked for us we have
done this in animals and let me show you
what this took so here is our virus and
viruses have as I've described to you in
an earlier talk to abilities an ability
to bind to the specific surface of the
cell and an ability to penetrate that
cell but there's a whole class of
viruses that do that penetration in a
two-step mode so they bind here here it
is bound and then they cause the cell to
encircle the bound virus in what's
called an endosome to carry that inside
the cell so that makes a complete
membrane around it
the cell then acidifies that compartment
because that's what cells do with
endosomes they make them acid the virus
sees an acid environment and that
triggers the fusion of the virus with
the endosome membrane releasing the core
of the virus into the cell so what we've
done is to say let's separate that into
two parts one part is the binding to the
cell the second part is the fusion so we
give we viruses do this with one
molecule what we did was to do it with
two molecules and the we can then make
the binding molecule a very specific
molecule an antibody in fact and an
antibody to this surface receptor that
enables us to target the virus to
specific cells so for instance in a an
experimental system we targeted it to B
lymphocytes by making an antibody that
was specific for a protein only found on
B lymphocytes called cd20 so now this
interaction is very specific with only
one kind of cell and we showed that if
you inject the virus into a mouse and
the mouse carries human lymphocytes only
the B lymphocytes from humans will be
infected Mouse cells are not infected
and non B cells are not infected so you
can actually do this in a purse in an
animal can you do it in a person will it
work with bone marrow stem cells those
are the problems we're investigating now
how do you make such a virus that can
combine that way well what you have to
do is to get a virus packaging cell so
this is a cell that makes virus and
modify it so it has antibody on its
surface so it has a fusion molecule and
an acid fusion molecule on its surface
and we do that by making a lot of
plasmids that bind to that can be put
into the cell that encode all the
necessary proteins so a huge sort of
engineering process but it works works
at least in one
kind of cell line and we have made lots
of these kinds of viruses that have
specific targeting ability and the kind
of data that you get out is here this is
just looking at whether the the virus
comes out with all the relevant
components in it and you can see that
97% of the virus or ninety-nine point
seven percent of the virus comes out
with irrelevant components in it and I
won't go into the details of all of
these plots this is the experiment
actually that showed targeting of
primary human b-cells in vivo and what
you can see down here is that the human
B cells get infected that's because this
curve moves over human T cells do not
and all the other cells
mostly Mouse cells do not so this is a
project in the lab and it has in fact
five components to it
first of all the hardest component is
that we have to engineer antibody like
proteins that will work to inactivate
HIV and actually a colleague of mine at
Caltech Pamela Bjorkman and her
laboratory is working on this issue and
what you see here is a picture of a
four-headed antibody we're thinking
about all sorts of things things with
multiple ads things with various sorts
of modifications in them and then we
have to get them expressed so we have to
perfect the lentiviral expression system
so that it will make antibodies and
antibody like proteins will encode them
and that is a challenge in itself
we have to learn how to program him out
of poetic stem cells so they develop
into B cells that make the right
antibodies and we need experimental
systems in which we can do that the best
system would be to put human cells human
stem cells either in a mouse or in a
culture system and to develop all the
way to secreting antibody and we're
working on that problem we're also we
also need
to work on this targeting methodology so
that we can get targeting to specific
kinds of cells which will increase
safety and will allow perhaps use
directly in patients and finally we're
trying to develop a mouse actually adapt
the development of other people have
made of a mouse that will hold the human
immune system and allow us to manipulate
it in an experimental fashion what will
it take for this actually to go into
humans a lot first of all we have to
make this kind of transplant system
routine safe and reasonably priced and
reasonably priced is very important
because we've promised the Gates
Foundation that we will try to make this
kind of therapy available at a
reasonable price to people who couldn't
afford very high-tech medicine
ordinarily and then we have to be able
to show that this is safe because other
people doing these kinds of gene therapy
experiments have accidentally caused
cancer and we have to be careful that we
don't do that we think we know how to
avoid that problem but we're not sure
the logistic work to get antibody
secretion is ongoing but is is daunting
all of it is daunting but the potential
benefits are enormous because we can see
ways now of attacking cancer HIV and
other infectious diseases that might not
have been available if this technology
were not developed all of this actually
is about therapy to make them make it
into a vaccine is conceivable but then
we have to be extremely safe and we
certainly can't take out blood stem
cells into culture we have to be able to
target directly in a person and so if I
was to encapsulate everything I've said
to you here it is that we're taking big
risks in this work and I must say a lot
of junior people working in my
laboratory are taking those risks but I
don't think there's any harm in thinking
big when thinking small has led us to so
little advance in these very important
areas of targeting the immune system to
HIV and to cancer thank you very much
let me just acknowledge as finally the
people who have been involved in this
work
Pamela Berkman's laboratory as I said in
the protein work Lilly yang as the major
contributor in my laboratory we're
working very closely particularly on the
targeting experiments with pin Wong who
is at the University of Southern
California
we got initial funding for this whole
thing from the Skirball foundation when
it was really just a vague dream and
it's still mostly a dream and the Gates
Foundation has seen fit to give us a 14
million dollar grant over five years to
try to wreck it to realize this dream
thank you very much
you