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Msg  17 of 24  at  1/22/2022 3:18:29 PM  by


The following message was updated on 1/23/2022 7:52:36 AM.

Cancer Immunology

Immune Surveillance 
The immune system is the primary surveillance system for preventing cancer. We have mutations occur all the time, but it only evolves into cancer once it learns to escape the immune system.
The tumor begins when a cell gains a mutation that allows it to grow and replicate faster then normal. Otherwise, these cells are completely normal cells in every way. We call this hyperplasia or a benign growth.
Natural Killer (NK) cells are the primary cells for detecting damaged or defective cells. They have pattern recognition receptors on their surface for Damage Associated Molecular Patterns (DAMPs).
As cancer cells grow and mutate, the mutated self proteins can become antigens which the immune system can recognize and use to mount a response. These are called Tumor Associated Antigens (TAA).
The T cells can recognize these new mutated self proteins and mount a cell mediated response against these tumor cells. As the T cell kill tumor cells, the Antigen Presenting Cells (APCs) will clean up the dead cells.
This allows the APC to present new antigens from the tumor cells to the T cells and activate a further response. This called Epitope Spreading. As the tumor cells die, they release more tumor antigens for the immune cells to use to continue to kill more tumor cells.
The tumor can only survive in this hostile environment if it develops mutations that allow it to escape this process of immune surveillance. 
Tumor Micro Environment (TME) 
There is a complex set of interactions that must go on between a tumor and its surroundings. It has to interact with the tissue cells, it needs nutrients, it needs oxygen, it needs to survive and proliferate in a hostile environment.
One of the many things it has to achieve to exist and thrive is to overcome the immune system and its natural ability to find, target and destroy tumors. There are 3 key cells in the immune system that are designed to find and kill cells that are infected or defective.
They are the Natural Killer (NK) cell, the Cytotoxic T cells, and the Macrophage. The NK cells deploy a set of damage receptors called Damage Associated Molecular Patterns (DAMPs) that recognize stressed cells. This can be things like Mic-a or Mic-b.
Normal cells will express MHC I on their surface that tells the immune system they are healthy. These MHC I receptors are made up of an alpha chain and a beta chain.
Defective cells will often display just 1 chain called MIC-a for the MHC I Chain related alpha chain or MIC-b for the MHC I Chain related beta chain. This is just one of the many ways NK cells can recognize an unhealthy cancer cell.
The Cytotoxic T cells can detect Tumor Associated Antigens (TAA) which are just mutated versions of the normal self proteins. This can cause T cell activation and a cell mediated response. The Cytotoxic T cells will bind to and kill tumor cells that display their antigen.
The last cells that are key are the Antigen Presenting Cells (APCs) like the Dendritic cells and Macrophage. The tumor not only competes with the healthy tissue for survival, but the cancer cells will battle each other in a survival of the fittest.
There is a lot of cell death going on in the tumor. This naturally allows APCs like the Macrophage to clean up these dead cells and process TAA antigens. They can present these antigens to the T cells to activate a response.
One of the hallmarks of tumor progression is the tumor evolves mechanism to evade the immune response. There are multiple ways the tumor will evade or redirect the immune system.
The most common is the tumor cells will release signals that will recruit T regulatory cells and Myeloid Derived Suppressor Cells (MDSC) to the tumor which will promote tolerance by releasing signals that inactivate the immune cells like T cells and APCs.
This is one of these hardest effects to overcome in the tumor microenvironment. One effective way to clear all these cells is the use of a lymphodepletion regiment like Flu/Cy. This works in cell therapies to clear the battle field.
The next mechanism the tumor cells will deploy is the expression of immune cell suppressing receptors like PD-1, CTLA-4 and CD-47. These will engage with T cells and Macrophages and inhibit them from cell killing. We call them checkpoints as they stop the immune response.
There is a huge amount of development going on to understand all the interactions between the immune cells and the tumor. There has been some great success with PD-1 and CTLA-4 and a lot of disappointments along the way trying to develop checkpoint inhibitors.
Some of the early developing checkpoints are LAG-3, TIM-3, TIGIT and CD47. All of these are natural receptors used by healthy cells to block the immune system from killing any healthy tissues like with PD-1.
The problem is the Tumor cells mutate to exploit this system to block the immune system and protect the tumor cells.
The critical role of the immune system in protecting us from cancer has been well established by following patients who are immune suppressed from cancer treatments or organ transplants.
Immune compromised patients have a significant risk for cancer. Even HIV patients will have higher risk of cancer related to the immune suppression of the virus. Some cancer patients get secondary cancers while their immune systems are suppressed from chemotherapy.
The role of the immune system in oncology is now well established. The many interactions that play a role in cancer surviving in a healthy immune system is still developing.
T cell engineering 
The T cell, specifically the CD8+ cytotoxic T cell, is the perfect cell killing machine. Its natural function is to find and destroy any cells that are infected or defective in the body so they can do no harm.
There are a few attributes about the cytotoxic T cell that need to be understood when using it as a therapy to target and kill cancer.
The first is a T cell has a T cell receptor. This functions off MHC I. We covered the HLA and MHC I in the immunology threads. Each T cell only recognizes the right antigen for its receptor and only when its presented by the exactly right HLA type. This is called restriction.
Even if you insert a CAR receptor into a T cell, it will still express its original TCR that can react to antigens and other MHC I classes in the form of rejection.
The best way to fix this issue is to insert the CAR receptor directly into the DNA location for the TCR and just replace the original TCR. This location is called the T cell Receptor Alpha Chain (TRAC) locus.
This method solves 2 problems with one edit by replacing (knocking out the TCR) with the CAR receptor itself. The original TCR is no longer there to interact with any antigens or reject any HLA types.
What is a CAR anyway? It stands for Chimeric Antigen Receptor. It take the binding region of an antibody and inserts it into the T cell. Why? because antibodies have more diverse ability to bind antigens without the need of HLA or MHC I.
It takes that binding region of the antibody and binds it together with the activity domains of the TCR receptor complex like CD3 and Zeta. This creates a chimeric receptor for activation.
The main thing for a T cell is it is a cell. All cells display MHC I of the host they came from. This means they can be attacked and killed if put into another patient that has a different HLA type.
This type of potential rejection is fixed by knocking out the B2 microglobulin (B2M) of the MHC I which blocks it from being presented on the T cells surface.
Knocking out the B2M protect these CAR-T or TCR T cells from being attacked by the recipients T cells and rejected before they can do their job of killing the cancer. The problem with just knocking out the MHC I is the NK cells.
Knocking out the B2M protect these CAR-T or TCR T cells from being attacked by the recipients T cells and rejected before they can do their job of killing the cancer. The problem with just knocking out the MHC I is the NK cells.
NK cells measure the MHC I level on a cell as part of their damage checks. If any cell lacks any MHC I, the NK cell will kill it. By just knocking out B2M, you save these T cells from rejection by other T cells, but you open them up to killing my NK cells.
The fix is to insert into that B2M locus another HLA type that won't be rejected by T cells, but will be accepted by NK cells. This can be HLA-E or other equivalent. This then gives you protection by both T and NK cells in the recipient.
The next key edit needed for TCR or CAR-T cells will be the knockout of the PD-1 receptor. Many tumors will display large amounts of PD-L1 on their surface. This is a checkpoint designed to protect healthy cells form being killed by T cells.
Its a way tumor cells block killing by T cells. This can be over come by just knocking out the gene for the PD-1 receptor. This prevents the need for a PD-1 inhibitor drug which typically are more toxic as they work systemically vs localized for a CAR-T cell.
The last big thing about CAR-T and TCR cell engineering is co-stimulation. Much study has been put into what kind of co-stimulation is necessary. Some use CD28 and others use 4-1BB.
From my years of looking at the data for these CAR-T programs its my summary that CD28 has shorter, but more robust responses while 4-1BB has longer more slowly ramped responses. Newer generation CAR-T therapies use both now.
The key to remember is the co-stimulatory signals on a normal T cells are separate from the receptor and act like a second safety signal to activation. With a CAR-T, they are hardwired right into the CAR receptor complex.
This can add additional risk to these therapies as they are always active.
NK cell Engineering
NK cells are a close cousin to the cytotoxic T cell so it is only logical that scientists would look at them for use in cell therapies to treat cancer. They use the same cytotoxic mechanisms to kill infected or defective cells.
There are some major difference between the CD8+ T cells and the NK cell. The first is the NK cell does not use MHC I the same as the T cell. The cytotoxic T cell reads the MHC I on the cell along with any antigens it is presenting. It works with the MHC I.
NK cells read the level of MHC I on the surface using sets of activating and inhibitors receptors on its surface like NKG2D and KIR. The NK cell activates when MHC I is missing.
Many infected or tumor cells will stop producing MHC I to protect themselves from the T cell. The NK cell is designed to detect this and kill those cells that lack MHC I expression.
The combination of the Cytotoxic T cell and the NK cell are like the dynamic duo that regulate the 2 aspects of MHC I expression on cells. One activates in its presence while the other activates in its absence.
When using a NK cell to do CAR-NK, you can easily leave these receptors active as they can still do their jobs without any problems unlike the original TCR receptor for the T cells.
One of the major benefits of using NK cells over T cells is they produce much lower levels of pro inflammatory cytokines which lead to a lot of the toxicities of the CAR-T therapies like CRS and neurotoxicity.
NK cells do tend to be less robust and don't last as long as the T cells. The data so far has show to be lower on responses and durability. It is still early though.
Another major advantage is the NK cell has the CD16 receptor with allow them to work with antibodies in a process called Antibody Dependent Cellular Cytotoxicity (ADCC). This allows for great combination treatments with already existing and newly developed cancer antibodies.
With a NK cell, you don't have to worry about knocking out PD-1 as it does not effect them. That saves you an edit.
They still display MHC I of their donor as they are still cells. This can be fixed the same as with T cells by doing the knock out and replacement of the MHC I on the NK cell. They can replace it with HLA-E or equivalent.
induced Pluripotent Stem Cells 
This has been a huge love of mine for years. The engineering of cells to create cancer therapies. The induced Pluripotent Stem Cells (iPSC) can revolutionize the way we create CAR-T or CAR-NK cells for cancer treatments.
The process of the iPSC can be automated, duplicated and consistent as a source of low cost cell therapies. So how does this work?
They start by taking any cell. They mix it with a combination of transcription factors like Klf4, Sox2, Oct4 and Myc. This will take those cells back into a stem cell.
Its believed this is done by unpackaging the DNA which has been turned off as the cells specialize during development. The colonies of these iPSC cells can then be developed into new cell lines using a combination of cytokines and DNA editing.
The DNA of the cell is its programming. Just like with any computer, if you reprogram the DNA with editing, you change the cells behavior. Studies have shown that transplanting 1 cells DNA into another cell with cause the new cell to assume the behavior of the original cell.
This showed us that DNA is the master programming for any cell. If we change that program with gene editing, we can create any cell with any behavior we wish.
When I covered T cells, I explained how a T cell could develop millions of random receptors by randomly selecting from a set of genes called Variable, Diversity, and Junctional. This creates the random T cell receptor.
But what if we took out all those genes for the TCR using CRISPR editing and replaced them with just one CAR receptor? We could use cytokines to develop those stem cells down that pathway of T cell lineage.
Once you have a stem cell with all the edits you desire to the DNA, you can create a master bank of those stem cells. This creates a renewable resource of cells that can be used to develop cell therapies.
There are a lot of benefits for using iPSC manufacturing for cell therapies. The first is their consistency to create high quality cells at a low cost.
The second is expansion of the T cells. Each T cell can replicate about 50 times before they run into the Hayflick limit. Since you have to have room in the body for them to expand to respond, you can only expand them so far in the lab.
Taking the T cells from a donor and editing them after they are fully grown T cells will end up with about 60% to 70% having all the desired edits when you do multiple edits like insertion of a CAR into the T cell locus, PD-1 knockout, HLA-E insertion into the MHC I locus.
With iPSC every cell is an identical copy of the DNA that is editing during the stem cell phase. They might end up with 1 in every 1,000 stem cell that has every edit they want, but they can collect those specific stem cells and build a master bank.
That master bank can go on and make 15,000 doses per year at a cost of less than $2,000 per. 

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