My name is Hidde Ploegh.
I'm a Senior Investigator at Boston Children's Hospital in the program of Cellular and Molecular Medicine.
And in this part of my presentation, I'll discuss some more recent work that describes
efforts to harness unusual features of antibodies produced by camelids.
Why would this be interesting?
These are the animals that we work with.
These are two juvenile alpacas, Bryson and Sanchez.
And the reason why these animals attracted my attention was spawned by a lecture that
I attended, in which I was misassigned to the wrong section.
And it's the first time I heard about the unusual properties of antibodies made by camelid species,
and that includes alpacas, llamas, guanacos, vicunas, camels, dromedaries --
all camelids shared this unique feature.
What I'm talking about is the fact that, unlike the typical mammalian species, which uniquely
produce antibodies composed of heavy chains and light chains, camelids also produce,
in addition to the traditional forms, heavy-chain only antibodies.
And this is of relevance because for many applications the size of a full-sized antibody
is incompatible with some of the applications we seek for them.
And it is for this reason that we try to shrink intact antibody molecules to the minimal unit
that can recognize antigen.
As I discussed in the preceding presentation, this can be done by proteolytic digestion.
Using papain, we can produce so-called Fab fragments composed of the variable regions
of the heavy chain and the light chain and one of their constant domains, respectively.
These retain antigen binding properties, but it requires considerable effort to
produce them in a proper way.
Your friend, the molecular biologist, can help you shrink these Fab fragments even further,
to create so-called single-chain Fv fragments, in which the variable regions of
the heavy chain and light chain are connected by a linker.
The problem that often arises with expression of these single-chain Fv fragments is that
they have not been through the secretory pathway of a eukaryotic cell, and consequently
they tend to be aggregation-prone and may require considerable optimization before you end up
with a stable product.
The heavy-chain only antibodies made by the camelids have no such disadvantages because
you can shrink the recognition module to just the variable region of the heavy chain.
And unlike what applies to the traditional antibodies, all features of structure required
for specific recognition of antigen are embedded within the variable regions of the heavy chain.
These fragments are called VHH fragments and are commonly referred to as nanobodies as well.
Some of the properties that make these nanobodies unusually attractive include the fact that
they can be produced in bacteria in high yield.
Many of them require neither glycosylation nor disulfide bonds for stability.
And their small size enables applications for which even single-chain Fvs would be
hard up to the task.
So, how do we make these single-chain variable region fragments?
We practice what is called multiplexed immunization, a fancy word for sticking up to two dozen
different antigens into an animal.
And after repeated boosting of the animal to achieve high affinity antibodies, we isolate
lymphocytes from peripheral blood.
And using primers designed to uniquely amplify the variable regions of
these heavy-chain only antibodies, we can create an immortalized collection of these VHH sequences.
How does one identify VHH sequences of interest?
This is done by cloning them in a phage display vector, where one can select phage that express,
as a fusion of the P3 protein, of the phage coat, a nanobody or VHH fragment.
And by performing what is called a panning reaction, by immobilizing antigen on a solid support,
we can retrieve those phage particles that express the nanobody of interest,
specific for the antigen with which we coated the plate.
So, even though we may start with animals immunized with two dozen different antigens,
by screening them individually on these antigen-coated plates we end up with nanobodies of
defined and unique specificity, which we may then produce in bacteria in high yield.
This is the conventional means of screening these so-called nanobodies.
But we've considered also the fact that these nanobodies require neither glycosylation
nor disulfide bonds, which enables their expression in the reducing environment of the cytosol.
And this has allowed us to perform functional screens to select for nanobodies that
perturb reactions inside the cytoplasm of, in my example, a mammalian cell.
So, let me give you an example.
Taking advantage of the fact that these nanobodies often require neither glycosylation nor disulfide bonds,
we devised a screening procedure that relies on the cytoplasmic expression of these nanobodies
to impede virus infection.
In this particular case, we took an animal and immunized it with disrupted influenza virus
and vesicular stomatitis virus, abbreviated as IAV and VSV, respectively, in the ensuing slides.
And upon immunization, and having extracted the RNA, instead of cloning the PCR products
into a phage display vector, as shown in the preceding slide, we clone these
amplified products in a lentiviral vector such that expression of the nanobody is under the control
of a doxycycline-inducible promoter.
Cells transduced with a lentiviral vector will produce the nanobody only when
properly exposed to doxycycline.
So, we transduce a collection of mammalian cells with these lentiviral vectors such that,
on average, each cell will receive a single antiviral construct.
Upon induction with doxycycline, that cell will produce the corresponding nanobody
in its cytoplasm, and we can then test this nanobody for its functional properties.
By exposing the transducants, as an entire collection, to a lethal challenge
-- in this case, a lytic dose of either influenza virus or vesicular stomatitis virus -- we can select
for survivors, the underlying logic being that any nanobody that impedes an aspect of
the virus replication cycle will confer a growth advantage to that cell.
We can then take the survivors and rescue the VHH or nanobody sequences, and then
validate them by independent means.
So, this is an example of such a screen, where we tested individual nanobody-producing clones
in the absence and presence of doxycycline induction.
You'll see two bars for each cell line analyzed.
The open bars indicate the situation in the absence of doxycycline induction, and we
monitor the infected state by independent means.
And as the open bars show, across the board in the absence of doxycycline induction,
each of these transduction... transductants is fully infectable by influenza virus.
But as the solid bars show, there are numerous clones, selected in this case as
protecting against influenza infect... infection, that drastically reduced the ability of the virus
to replicate.
When we test those very same clones in response to an infection with vesicular stomatitis virus,
clones that are fully protected against influenza virus are not responsive to infection
with VSV at all.
It doesn't matter whether we induce expression of the nanobody;
infection with the irrelevant virus proceeds normally.
We can conduct the converse experiment by selecting for clones that impede VSV replication,
and these in turn leave replication of influenza virus un... untouched.
Now, one of the appealing features of nanobodies, and this is one of the reasons why structural biologists
have warmed up to them, is the fact that they often serve as crystallization chaperones.
That means that for proteins that are often challenging to crystallize, inclusion of
a nanobody of the appropriate specificity will facilitate crystallization.
This of course has the tremendous advantage of identifying at atomic resolution
exactly the epitope to which the nanobody in question binds.
And this was done by Florian Schmidt and Leo Hanke, two students and postdocs in my lab.
They together solved the crystal structure of, in this case, the target of the anti-influenza
nanobodies which impede replication in complex with a representative nanobody.
In red and orange are indicated those sequences in the flu nucleoprotein that are responsible
for interaction with RNA -- because influenza virus is a negative-stranded RNA virus --
and in gold the nuclear localization signal.
And what this experiment reveals is that the nanobody binds to an area of the NP protein
not previously implicated as functionally important.
So in that sense, having this nanobody provides us with a novel angle of attack
on the virus life cycle.
Now, if one asks, by what molecular mechanism could this nanobody conceivably impede virus replication?,
we made use of a structure published for the viral ribonucleoprotein particle.
This is a viral RNA in complex with nucleoprotein.
The analogy might be of a histone binding to DNA.
And what we found is that the nanobody, when bound to the nucleoprotein, occludes the
nuclear localization signal on an adjacent NP subunit, and thus impedes import of the viral
ribonucleoprotein particle into the nucleus, which is a step essential for flu replication.
Similar experiments were done for the VSV-inhibitory nanobodies.
And in this particular case we again found that the nanobody binds to the N protein,
which is the conceptual equivalent of the NP protein in influenza virus.
The N protein complexes RNA, barely visible here as this red material found in between
the two lobes of the N protein.
The N protein forms a decameric ring.
And in the crystal structure solved for the complex between the N protein and the nanobody,
we find the presence of two such decameric rings, each subunit of which is crowned by
an individual nanobody as part of the crystal structure.
So, we have 40 subunits to the asymmetric unit.
And this is one of the largest protein structures to which my lab has contributed.
This was done together with Tom Schwartz at MIT.
If we now ask, would this particular structure help us understand exactly how the nanobody
impedes virus replication?, the answer is yes.
It turns out that the nanobody binds to a segment of the N protein to which also binds
the polymerase cofactor P.
In order for the VSV genome to be replicated,
the polymerase must interact with cofactor P to enable that reaction to proceed.
And as we observed, the nanobody competes for binding with this P protein, thus adequately
explaining why it inhibits virus replication.
And what is not shown in this slide is the fact that if we now grow virus in the presence
of this particular nanobody, we can readily select for escape variants that no longer
are susceptible to inhibition by this nanobody.
And as you might expect, the mutations that account for these escape variants map exactly
to the site to which the nanobody binds without affecting binding of cofactor P.
So we have here a complete cycle, in which we can immunize an animal with some antigenic preparation,
conduct a phenotypic screen, identify the epitope recognized at atomic resolution,
and then use an in vivo biological experiment to validate these conclusions by selecting
for escape variants.
A second example that exploits the ability of nanobodies to function in the cytoplasm
of a mammalian cell concerns nanobodies that we've made against the component
of a highly complex structure we refer to as the inflammasome.
And without going into unnecessary detail, inflammasomes are supramolecular assemblies
required for a particular aspect of innate immunity.
When an inflammasomes senses a microbial component, it oligomerizes, and it makes use of
a series of adapters to then recruit massive numbers of an enzyme called pro-caspase-1.
Once this inflammasome is properly assembled, it leads to activation of pro-caspase-1,
and this in turn allows conversion of a cytokine called pro-IL-1 into its active counterpart,
IL-1.
This is a key aspect of inflammation.
It turns out that inflammasomes occur in many molecular manifestations, two of which
are shown here.
On the left, the NLRP3 inflammasome, composed of leucine-rich repeats
-- those are these repetitive structures shown on top;
a pyrin domain via which it recruits the adapter ASC;
and the ASC protein itself has yet another protein-protein interaction domain called
a caspase activation and recruitment domain via which, as the name suggests,
caspase is recruited.
These inflammasomes occur in many different flavors.
A second one, not the only one that exists, is the NAI...
NAIP or NLRC4 inflammasome, where the involvement of the ASC adapter was considered under debate.
So, we raised nanobodies against the ASC protein, found that these antibodies could in fact
impede proper release of interleukin-1, suggesting that indeed we had an inhibitory antibody,
and again, by exploiting the fact that nanobodies serve as crystallization chaperones,
we were lucky enough to be able to determine exactly how this particularly nanobody binds to
its counterstructure, the CARD domain of the ASC protein.
What is noteworthy in this particular case is that the nanobody binds to its target
not via the complementarity determining region 3, which most often makes the key contribution
to antigen binding, but with a very important contribution of CDR2 to mediate specific interaction
with the CARD domain.
And this was work done in collaboration with Hao Wu at Boston Children's Hospital.
Now, having a nanobody that binds to this ASC protein enables us to do a variety of
different experiments.
And one such experiment involves the construction of a sensor that might report on the assembly
of inflammasome monomers and ASC monomers into the polymeric structures that are a prerequisite
for activation.
By fusing our nanobody specific for ASC to green fluorescent protein, for the first time
we can look at the distribution of this ASC protein at natural abundance.
One of the complications of inflammasome study is that overexpression of individual components
can lead to art... artifactual and premature activation.
And what we would like to know is what the behavior of these components looks like
under conditions of natural abundance.
So in this particular case, we took a monocytic human cell line capable of sustaining NLRP3 activation,
we introduced the reporter composed of the ASC-specific nanobody fused to GFP,
and in the absence of overt stimulation we looked for the distribution of ASC protein
by virtue of the fact that it's targeted by our nanobody-GFP fusion construct.
And what you see in the absence of any overt activation is this diffuse distribution
of the ASC protein.
If we now expose those very same cells to inflammasome triggers, we can begin to see
the initiation of polymerization.
We see the formation of these filaments.
The assembly of the inflammasome does not proceed to completion, which explains
the inhibitory effect of this particular nanobody, but it gives you an example of how we can
use nanobodies to report, in live cells, on phenomena that would otherwise be very difficult
to study.
Another key application which we've been interested in for a long time is to use
single-domain antibody fragments or nanobodies for imaging purposes.
If you want to develop an antibody-type reagent for imaging, it needs to meet
a certain set of criteria.
It needs to be labeled so we can detect the labeled product.
It needs to be cleared from the circulation rapidly to achieve proper signal-to-noise ratio.
And it needs to achieve adequate tissue penetration.
And this is where its small size becomes a key distinguishing feature in comparison
with full-sized antibodies.
Full-sized antibodies are about 150 kiloDaltons in molecular mass.
The nanobody fragments with which we work are approximately one-tenth the size.
And this accounts for their superior tissue penetration, their accelerated clearance
from the circulation, and thus we thought these would be ideal agents for imaging purposes.
Now, in order to label an antibody fragment, or any protein for that matter, you want to
avoid collateral damage.
Most of the chemical labeling procedures that are commonly used entail the risk of modification
of residues important for function of that protein.
And so, in the design of our imaging agents, we wish to avoid the introduction of
any chemical damage to the protein of interest.
So, to achieve that, we make use of what's called a sortase reaction.
The sortase enzyme is a transacylase that recognizes a short peptide sequence
at or close to the C-terminus of any target protein of interest.
It's essentially a thiol protease that cleaves between the threonine and glycine,
indicated in single-letter code in this slide, to result in a covalent thioacyl enzyme intermediate,
indicated in the bottom left, here.
Now, we're providing, ex vivo, a short synthetic peptide, minimally composed of a single glycine
-- ideally, two, three or more -- to which one may affix pretty much any payload of interest,
denoted here as probe.
These synthetic peptides serve as the incoming nucleophiles that attack this thioacyl enzyme
intermediate.
And it results in the formation of a covalent bond between the target protein that you've
modified with this LPXT(G)n sequence in perfectly good peptide linkage with the probe,
attached via this oligoglycine stretch.
So, this is a method that lends itself to modification of pretty much any protein of interest,
if properly optimized.
Now, the types of payloads that one can attach to proteins are limited only by what
your friend, the chemical... the chemists can produce.
In this example, I've indicated a few of the things we've successfully been able to affix
using this sortase reaction.
It includes site-specific biotinylation, the site-specific and quantitative attachment
of fluorophores, execution of protein-protein fusions, attachment of DNA molecules, and,
as will be relevant for what I have to say in the following, the attachment of radioisotopes.
The important thing to remember here is that the reaction proceeds site-specifically and
nearly stoichiometrically, which makes it very attractive for protein engineering purposes.
So, in order for us to use nanobodies against surface proteins of interest for imaging purposes,
we need to install a label that allows us to detect them.
And without... without going into the physics, we've been interested in positron emission tomography.
This is a method that can be used in the clinic to diagnose metabolically active from inactive cells.
It's a non-invasive method.
But it... it uses radioactive isotopes that are sometimes challenging to handle.
Without going into the chemical details, we've developed procedures to affix these isotopes
suitable for PET imaging to our nanobodies using the sortase reaction.
So, on the left you see some nasty chemistry that results in the formation of an F-18 labelled compound.
Fluorine 18 is the isotope suitable for PET imaging.
On the right, you see the workstream for our sortase-catalyzed reaction to install
a chemical entity that now allows... allows us to combine the two under strictly native conditions,
without inflicting any collateral damage onto the protein of interest.
So, this is how we can make nanobodies ready for imaging by positron emission tomography.
And what is shown in this little inset, here, is a mouse that we imaged with an antibody
against class-II MHC products.
These are molecules found on antigen-presenting cells.
In this case, we deal with a tumor bearing mouse where the presence of antigen-presenting cells
in the tumor can be visualized by this non-invasive imaging method.
We've also been interested, in particular, in imaging immune responses non... non-invasively.
I pointed out the fact that T lymphocytes recognize antigen by virtue of this antigen-specific
receptor that recognizes MHC products complexed with the appropriate peptide, but the reactivity
of these T cells can be mon... monit... can be modulated by the expression of
so-called costimulatory or checkpoint molecules.
And that can be either stimulatory or inhibitory.
And in order for us to image immune responses non-invasively, to focus on
these costimulatory or checkpoint molecules became a point of interest.
This is all the more important when we now know that these checkpoint molecules are important
targets for immunotherapy of cancer.
This slide is taken from the work of Jim Allison, who showed that by blocking one of these checkpoint
molecules one could achieve a remarkable improvement in an antitumor response.
In this case, you see what happens to tumor growth, measured as tumor diameter.
In the absence of any treatment, if you give an antibody that fails to impede this regulatory interaction,
no effect on tumor growth.
But if you block a checkpoint molecule that confers inhibition on T cells of
appropriate specificity, merely blocking that inhibitory molecule gives you a significant improvement
in the antitumor response.
Now, this has been wildly successful in the clinic, cures having been achieved, but only
a fraction of patients respond to such treatment and it will be very important to know
why certain people do respond to therapy and others do not.
And we think that the ability to image immune responses non-invasively may help us
provide some answers to that question.
In addition to the CTLA-4 protein, which was the focus of attention of work in the Allison lab,
much work has since focused on other numbers of the checkpoint family,
notably the PD-L1 protein.
So, let me take you through this example.
We have here a T lymphocyte that engages an antigen-presenting cell via its T cell receptor
for an antigen.
But if this antigen-presenting cell also expresses PD-L1, it engages a counterstructure
on the T cell which now confers an inhibitory signal to that T cell rendering it incapable of
exerting its usual functions, be that cytokine production or a lytic function.
If one has antibodies that block either PD-1 or PD-L1, such inhibition may be relieved.
And just like the anti-CLTA-4 example of which I showed you the graph, blocking PD-1 or PD-L1
has been shown to have a significant antitumor effect.
So, we developed nanobodies against PD-L1.
And we're able to show that the B16 melanoma, which is a frequently used mouse model of melanoma,
expresses the PD-L1 protein.
What you see here are mice injected with a radiolabeled fragment of a nanobody that recognizes
PD-L1.
These mice on their left shoulder carry a tumor.
And the presence of the PD-L1 protein on the tumor may be clearly revealed by this non-invasive
imaging method.
We always use genetic controls to infer specificity of the labeling reactions that we see.
On the right, you see a PD-L1 knockout mouse inoculated with the same tumor.
And although somewhat more faint, we can still detect the PD-L1 signal in this setting as well.
The conclusion from this experiment is that PD-L1-positivity in the tumor microenvironment
is the net result of expression of that protein on invading cells of hematopoietic origin,
as well as by expression of PD-L1 by the tumor itself.
So, while it is possible to use a radioactive isotope of fluorine, F-18, for labeling purposes,
its short half-life, 110 minutes, poses limitations.
For that reason, fortunately we have access to other isotopes that are PET-compatible,
such as zirconium-89.
And we have the chemical means to install those site-specifically as well.
In panel A, we indicate the chemical structure of the chelator required to specifically bind
zirconium-89.
And in much the same way as we use sortase to install F-18, we have chemistry that
enables us to install these zirconium-labeled compounds.
The bottom half of this slide illustrates the basic enzymatic approach we follow to
attach this zirconium.
Again, we use a sortase reaction to install the structure indicated in panel A.
And we have the added capability of modifying these structures even further through installation
of polyethylene glycol, for example, to affect the hydrophobic-hydrophilic balance of
the final product.
So, here you see an experiment in which we took a nanobody that recognizes CD8.
This is the glycoprotein marker diagnostic of cytotoxic T lymphocytes.
And on the far left you see the initial result with a nanobody simply labeled with zirconium-89.
The two massive structures seen on the bottom are the kidneys and the bladder.
And this particular nanobody, when labeled, tends to concentrate in the organs of elimination,
with only very faint labeling visible in the thymus, the lymph nodes
-- exactly where you would expect it.
But by now adding PEG spaces of 5, 10, and 20 kiloDaltons, thus improving hydrophilicity
of the labeling agent, we see a massive reduction in this uptake in the organs of elimination.
And we begin to see, with great clarity, the lymphoid structures these nanobodies
were designed to detect in the first place.
Again, we use a genetic control.
In the far right, you see a mouse that lacks any and all lymphocytes, owing to the fact
that it's mutated in the very locus responsible for VDJ rearrangement.
And the lymphoid structures so clearly visible in this panel are altogether absent in the
RAG knockout, which lacks lymphocytes altogether.
So, with this tool in hand, we can finally begin to image cytotoxic T lymphocytes
and ask the question, can we detect the influx of killer T cells into the tumor microenvironment?
What this slide shows is a mouse that's been inoculated with a pancreatic tumor cell line.
And on its left flank, you can clearly see the tumor by virtue of the fact that CD8 lymphocytes
infiltrate it, showing that we can indeed use this kind of non-invasive imag... imaging
to track the behavior of T lymphocytes in living mice.
And what's really important about this type of experiment is the fact that we no longer
need to kill the animal to examine what goes on inside it.
Typically, immunologists, in order for them to access lymphoid organs or to access lymphocytes
that reside in the tumor, have to kill the mouse, excise the organs of interest,
and then enumerate and characterize lymphocytes that are present.
With this non-invasive imaging method, albeit at modest resolution, we can now track
an animal over time and watch the immune response unfold.
So, this is our famous spinning mouse.
And once again, you can clearly see the tumor on the right flank.
In fact, you will also see that these dots represent the lymph nodes.
And lymph nodes that are in close proximity to the tumor are more intensely labeled
than lymph nodes... lymph nodes on the other side, consistent with trafficking of lymphocytes
between these different lymphoid organs.
So, how can we use this information to address one of the questions I posed earlier?
Why is it that certain tumors respond to checkpoint-blocking antibodies,
such as anti-CTLA-4 or anti-PD-1/PL-L1, and others do not?
Can we use this particular imaging method to shed some light on what happens in
that particular setting?
So, this is a complicated slide, but the concept is very simple.
We use our B16 melanoma model in a setting where some of the animals will proceed
to full-blown tumor growth and others will respond to control of the tumor in response to
checkpoint-blocking antibodies.
So in this case, we use anti- CTLA-4 antibodies, full-sized antibodies, as a therapeutic agent.
And we simply image in the tumor microenvironment what happens to the CD8 T lymphocytes
that one finds on location.
And what we find is a very strong correlation between the distribution of these CD8 T cells
and the ability of an animal to control the tumor.
If we see monofocal distribution of T lymphocytes, this is paralleled by a reduction in tumor size
and improved survival of the animals.
In animals that fail to respond to checkpoint blockade, and where the tumors grow,
we see this more heterodisperse distribution of CD8 T cells, and this becomes a predictor,
if you will, of the outcome of the final experiment.
This was validated, in collaboration with the lab of Bob Weinberg, in a breast cancer model
where two variants exist.
One is an epithelial variant that is immunogenic and is readily controlled in response to checkpoint blockade,
and there's a mesenchymal variant which is less differentiated, highly aggressive,
and poorly responsive to checkpoint blockade.
If we now image animals of either type... you see an example of the mesenchymal variant,
poorly controlled by checkpoint blockade... tumors continue to grow, and the animals
do not survive.
On the bottom, you see the epithelial variant, a monofocal distribution of CD8 T cells,
excellent response to checkpoint-blocking antibodies, and thus we think that this method for the
first time begins to shed some light on what happens in the tumor microenvironment.
We have a long way to go and there's a long list of surface molecules that we'd like to image.
There's a lot that we need to know about various aspects of the immune response.
But it will be important to measure these things in the context of a living experimental animal
and ultimately, perhaps, in the clinic.
So, the preceding segment has shown our efforts in imaging a certain aspect of adaptive immunity,
notably CD8 T cells.
But as I pointed out, host defense is an integrated system that comprises not only adaptive immunity
but innate immunity as well.
And future efforts are focused on trying to image these innate immune responses non-invasively
as well.
I think inflammation has a key aspect that would benefit from this approach.
The nanobodies that we've used have a few signature advantages:
their small size makes imaging possible;
ease of manufacture in E. coli is a plus;
the fact that they require neither glycosylation nor disulfide bonds allows us to express them
in the cytoplasm of mammalian cells and use them to conduct phenotypic screens, as I've demonstrated;
finally, their small size makes it possible to exploit the sortase reaction to
install pretty much any... any entity of choice without inflicting damage on the nanobody itself,
and that is the method we have applied to render nanobodies suitable for imaging by
positron emission tomography.
Let me finish by acknowledging the individuals who have done this work.
The virus work that I summarized was done by Florian Schmidt, a postdoc,
and Leo Hanke, a graduate student.
The nanobody platform was established by Jessica Ingram, currently on the faculty of Dana-Farber.
The radiochemistry was done by Mohammed Rashidian.
And the tumor immunology experiments were conducted with very the significant input
and participation of Mike Dougan.
I've mentioned the fact that we collaborate with Bob Weinberg on our breast cancer models,
Hao Wu on the inflammasome,
Steve Almo as a source of proteins with we... with which
we can immunize alpacas and obtain the antibodies with which we work.
Thank you very much.
