Notes 3: CAR-T and CAR-NK Cells
Chimeric Antigen Receptor (CAR) T cell Therapy
car means chimeric antigen receptor
so a chimeric antigen receptor is an engineered molecule that carries these minimal components.
Eventually it's a transmembrane protein where the most external, component part is the FV, the variable fragment of an antibody.
Remember the antibodies, heavy chain, light chain.
We're talking about the variable part of the antibody and we're looking at a single chain.
We're not looking at both components.
We're looking in at the single chain variable fragment.
Then there's a linker and this linker is just a sequence of peptides that are not particularly folded and they're just needed so that this thing can flip around basically.
Then there's another piece, there's a transmembrane domain and then the internal domain of this car is the intracellular domain of the CD three zeta.
So what is CD three?
This is the T cell receptor, CD three is part of the T cell receptor complex and it's this intracellular domain that triggers the signal once the T cell receptor has bound the antigen on the other side, a signal is triggered that is transduced inside the cell and produces a cascade of intracellular responses, including activation, basically of the of the T lymphocytes.
As long as all the other co-stimulatory signals are there.
As long as the inhibitory signals are not there and blah blah blah,
but as long as everything is in place, it is this part that actually transduces the signal to activate the T cell.
And by activation, we mean a colonal expansion, secretion of cytokines that promote both its own expansion and promote expansion of other T cells and other cells. A bunch of things.
So this intracellular domain is present in the car as well.
Constantly improving CAR design
Now, what is the idea here?
first, I should say there are various generations where probably even beyond the fifth generation here
But basically people have been adding a bunch of different components to potentiate the function of the car
so the function of the car is really with the with the single chain variable fragment to recognize an antigen that is expressed on the cancer cells, a new antigen.
And again, it depends, there are some antigens that we know are always expressed by a certain type of tumors.
There are antigens that are expressed by the tumors and also by normal cells.
it really depends on the kind of tumor.
But let's pretend that we're looking at a tumor where a certain antigen is only expressed in the tumor cells.
This SCFV, this single chain variable fragment will recognize that antigen.
then it's attached through this linker and hinge is attached to a transmembrane domain whose function is just to stick it through the plasma membrane and then the CD3 zeta intracellular domain.
This is the first generation we've just gone through.
In the second generation they added, a co-stimulatory molecule. They added a piece of CD 28 or other molecules that are also co-stimulatory receptors like CD 28. So that they could further potentate the one CD, this whole thing is engaged and CD three triggers the activation of the T lymphocyte where this is expressed. Then it could further do so because there is also a piece of the co-stimulatory molecule.
In 3rd gen, Then they added another one CD 27.
In 4th gen, And then they add a bit that actually interacts directly with the DNA. This is a domain from NFAT is a transcription factor that is involved in promoting transcription of certain cytokines like IL-12, IL-13, which is pro inflammatory cytokines
In 5th gen, and then they thought, we might as well also add the domain of interlukein to receptor beta that interacts with JAL/STAT again, same concept.
So basically, they've been adding bits and pieces to the intracellular domain. So that when this whole thing is introduced in a T cell, if the T cell engages a cancer antigen through this variable domain from an antibody that recognizes that antigen, then a cascade of events is triggered intracellular that leads to activation of the T cell, a massive activation of the T cell and a massive activation of the immune system against the cells that express the antigen recognized by this bit outside the cell.
Now, the question is how do we get this beautiful car, this beautiful chimeric antigen receptor to be expressed in the T cells of a patient.
This is an engineered. This is all made in the lab does not exist in nature. Now, we need to take this thing and put it in the T cells of the patient.
CAR T-Cell therapy
Here's how we do it.
We take blood from the patient.
We isolate T cells and in the lab, we transduce the T cells that we have taken from the patient and we introduce an artificial gene.
Remember transduction, we talked about transduction, right?
It could be transfection with electroporation, it could be transduction.
I believe it's usually transduction because it's higher efficiency.
We introduce somehow this artificial gene in the cells that expresses this whole thing.
This car that the cell makes the car exports it to the plasma membrane.
We amplify the cells in vitro in the lab.
So we make millions of them and then we infuse them again into the patient.
Now, these are the patients own T cells which have been potentiated by addition of a receptor that a recognizes its own tumor.
before we have taken the tumor out of the patient or a biopsy of the tumor, we know what tumor, it is, what antigens it expresseses. And so which kind of variable fragment we should use to recognize those antigens expressed by that tumor we put them back in and now this patient has in addition to its own immune system.
Also this amazing, super powerful car T cells that attack the cancer cells and kill them.
None of that is happening because this is just the artificial gene, what you introduce, usually it's not the whole gene. Like our recombination happens at the genomic level, right at the level of the whole gene.
Whereas we say we introduce an artificial gene, which is an artificial gene, but it's just the coding sequence.
So it's already just basically the cDNA of the mRNA that encodes this whole thing.
So there's no splicing, there's nothing happening.
It's ready to go, it's transcribed and then it's ready to be translated right away.
CAR T-Cell Therapy
Does anybody know why we're taking the patient's own cells and not a donor's cell cells?So we don't want reaction again.
You said against someone else's cells, but it could be even the other way around.
So what do you say that the recipient could react against somebody else's cells?
That is what we call host versus graft disease. And it's what we normally get when we receive an organ transplant.
For example, we receive an organ from another person is immediately recognized as non self unless we receive massive doses of immunosuppressants and we choose a donor that is as much compatible with us as possible.
So that is close to us in the genetic makeup of the antigens that they express like could be.
The ideal case is a identical twin, not everybody has an identical twin. In fact, it's only a very small percentage of people.
So we go for another sibling or a parent or a close relative.
That's why, you know, when we transplant organs, we go for, for close relatives and things like that because we want them to be genetically as close as possible. So the antigens that they express are as similar as possible and not attacked by the host, receiving the organ that is host versus graft disease. And that could happen if we use T cells from another donor.
But what could happen also? And it's actually more likely to happen in this case is the opposite is the graft versus host disease because these are T cells, their job is to recognize non self.
If they're coming from Nicholas here, if he gives me his T cells, his T cells, once they are in my body, they're gonna go. Whoa, what's this? I don't know any of this. I'm gonna attack everything and remember they're powerful. These T cells are crazy. The car T cells. So his T cells will attack me. I will try to attack his Tcells. If these cells will try to attack me it's gonna be a mess.
So that's why we use allogenic cells, allogenic means from the same person that is going to receive them.
Of course, there are limitations with this. Sometimes these patients have undergone chemotherapy. So they're already immunocompromised. So trying to get some cells is hard. There are actually many reasons we cannot get this many. There's only so much we can amplify them in culture and so on and so forth. So people have been developing, we, we'll come back to this later.
FDA-approved CAR T cell therapies
You don't have to know the tables by heart.
so the video also mentioned limited efficacy of CAR T cells against solid tumors
that is because unfortunately, we have for the hematologic tumors which are the tumors of the blood. We have this brilliant, especially for the ones that are derived from B cells. We have this brilliant antigen CD19 that can be targeted with car T cells.
And why is a brilliant antigen because CD19 is only expressed in developing B cells or in cancers that are derived from B cells. So the toxicity and that's it.
These are the only cells that express it in the body. So when you kill the cells that express that antigen, you're killing a few developing B cells but it's not the end of the world.
You're not completely immunocompromised the person, but you're killing the tumor.
Or this BCMA which is expressed almost exclusively in multiple myeloma.
Multiple myeloma is a tumor of the bone marrow.
CAR T cell therapy for solid tumors
The problem with the solid tumors, lung, colorectal liver, pancreatic gastric breast, you name it hepatocellular.
The problem is that if you look at what the antigens are it's pretty sad because the majority of them are things like EGFR, HER2 , these are the receptors that are expressed in almost every cell of the body.
Yes, they are mutated in these tumors, and one can design an FV that targets specifically the mutated version, the FV, that targets specifically that mutated region.
But still they will cross react a little bit with the non mutated is expressed in every cell.
So they have high toxicity because of the low selectivity, they work.
But you see that we're still at the level of clinical trial, most of them are between phase one and phase 1/2.
Definitely they've not been approved by now all of this
GPC3. Also, it's very commonly expressed.
There are even CAR T cells that target PD-L1. This is interesting. So this is a fusion of both concepts. But again, these are in clinical trial in phase one
On-Target, off-tumor toxicity of CAR T cell therapy
So we call it on target off tumor toxicity of car T cell therapy
it targets what it is supposed to target, for example, the EGF receptor, but it's off tumor because this receptor is also expressed in a bunch of other good cells that are not necessarily transformed.
So we say on target, yes, it's targeting GFR but off tumor.
Ok. This is the most common type of toxicity of Car T cells. Well, not just car T cells but we saw that also immune check points have the same kind of toxicity on target off tumor.
CAR-NK cells may be more efficient and less toxic than CAR-T cells
People have been developing other strategies to avoid graft versus host disease and have been using NK cells instead of T cells
because NK cells remember they're not specific for a specific antigen. So they're not gonna recognize another person just because they have an HLA MHC that is slightly different from us.
No, as long as there isn't a pathogen with the normal pattern of molecular patterns of pathogens, they're fine.
So NK cells don't cause graft versus host disease and we can get them from many people.
We can expand them a million times and bank them.
We don't have to take them from the patient.
So they could be ready to go when the patient needs them instead of waiting weeks and weeks and weeks to get them from the patient transduce them, select them, expand them in vitro and reinfuse them.
So car T cells, car NK cells are basically the same concept, the cells are so two differences.
A the cells are different and B the car is also slightly different
because for car T we use co-stimulatory elements from co-stimulatory molecules such as CD 28, 41BB and things like that, which are molecules that stimulate T cells.
Concept still pretty much the same with uh NK cells. But we use slightly different molecules that simulate NK cells.
NK cells 41BB is still the same. Um 2B4, we don't use it for T cells. We use it for NKD. DAP10, DAP12. These are also bits and pieces that activate muscle. They activate better NK cells than T cells.
CAR-T Limitations
Short video.
Chimeric Antigen receptor T cell therapy which involves the engineering of a patient's own T cells to eliminate cancer.
T cells has revolutionized cancer treatment.
This highly personalized immunotherapy typically starts by collecting T cells from the patient's peripheral blood.
These T cells are then genetically modified, often using viral vectors to express CARS on their surface before being expanded into millions in the lab.
After quality control testing, the final step is the reinfusion of the car T cells back into the patient to attack cancer cells.
Cars are modular synthetic molecules comprising four components, an extracellular antigen binding domain that is most commonly a single chain variable fragment derived from a specific monoclonal antibody, a hinge region, a transmembrane portion and an intracellular signaling domain that triggers cell activation.
Cars, redirect T cell cytotoxicity toward tumor cells expressing a specific antigen.
When the extracellular domain binds to the target antigen on tumor cells. The intracellular domain mediates T cell activation and anti tumor responses.
Despite their considerable success, car T therapy still have limitations including limited efficacy against solid tumors.
Car T cell associated cytokine release syndrome or CRS and neurotoxicity on target off tumor effects and antigen escape.
Additionally, using autologous T cells as a source material to prevent graft versus host disease or gvhd requires individualized collection modification and expansion processes that limit the scalability and accessibility of autologous car T therapy.
Given these limitations, there is growing interest in exploring car natural killer cells as an alternative to car T cells due to their promising pre clinical and clinical results.
Car NK cells offer unique advantages.
They can be generated from various sources as allogeneic Car NK cells don't cause GVHD, making them attractive candidates for universal off the shelf, immunotherapy.
CAR NK cell therapies have demonstrated superior safety profiles with low risk for CRS and neurotoxicity.
Furthermore, car NK cells identify and eliminate cancer cells through both car dependent and car independent mechanisms, boosting anti tumor efficacy.
Nevertheless, there are still hurdles to overcome in car NK cell therapy.
Major roadblocks include their limited expansion, ex vivo, lack of durable persistence and survival in vivo, inefficient trafficking and infiltration into tumor sites and susceptibility to the immunosuppressive tumor microenvironment.
While there are obstacles, potential strategies continue to evolve and may pave the way for more accessible, more effective and safer car NK and T therapies.