T Cell Therapies

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 that a T cell has a T cell receptor which functions with MHC I. We covered the HLA and MHC I in immunology. Each T cell only recognizes the right antigen for its receptor and only when it is presented by the exactly right MHC I type. This is called T cell 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. Some CAR-T therapies now insert the CAR into the T Cell Alpha chain (TRAC) locus which replaces the T cell receptor with the CAR receptor. This is designed to get around possible rejection caused by the original T cell receptor.

What is a CAR receptor? It stands for Chimeric Antigen Receptor. It takes the binding region of an antibody and inserts it into the T cell. Why? because antibodies have a more diverse ability to bind antigens without the need of HLA or MHC I. It takes the 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 person they came from. This means they can be attacked and killed when put into another patient that has a different HLA type. This type of potential rejection is what many companies are trying to fix now. They are using approaches like 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 protects these CAR-T or TCR T cells from being attacked by the recipient's 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 enough 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. Some of the attempts to fix this are to insert into that B2M locus something that will pacify the NK cells. There are other HLA types that won't be rejected by T cells, but will be accepted by NK cells. This can be HLA-E, HLA-G. Some companies are testing out checkpoint inhibitors like C47. This then gives you protection by both T and NK cells in the recipient. Most of the data in these immune evasion therapies is still early.

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 from being killed by T cells. It is a way tumor cells block killing by T cells. This can be overcome 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. Some now use both. From my years of looking at the data for these CAR-T programs it is my summary that CD28 has shorter, but more robust responses while 4-1BB has longer and more slowly ramped responses. The key to remember is the co-stimulatory signals on 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. This leads to some of the side effects of CAR-T cell therapies.

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NK Cell Therapies

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 differences between the CD8+ T cells and the NK cell. The first is that 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 of the cell using sets of activating and inhibitory receptors on its surface like NKG2D and KIR. The NK cell activates when MHC I is low or missing. Many infected or tumor cells will stop producing MHC I, or they produce only fragments of the MHC I. The NK cell is designed to detect this and kill those cells that lack proper MHC I expression. NK cells also have other receptors which allow them to detect stress ligands from cells. The combination of the Cytotoxic T cell and the NK cell are like the dynamic duo of the immune system as they 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 leads to a lot of the toxicities of the CAR-T therapies like CRS and neurotoxicity. That also tends to be their drawback too. The lower level of cytokines usually leads to lower responses with CAR-NK cell therapies.

Some other advantages of the NK cell is it has the CD16 receptor which allows 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. Another advantage of NK cells is they are tissue oriented immune cells. Where the T cells and B cells typically remain in the lymph nodes and circulation, the NK cells normally occupy the tissues where many tumors arise. In the same way as T cells, NK cells still express MHC I like all cells. This means they can still be rejected by a healthy immune system. They tend to work fine in patients with immune suppression, but they will need immune evasion editing to work better and have long term durability.

NK cells are still in early stages of development. Data from some of the phase 1 programs have shown similar efficacy to CAR-T programs in this space. With additional edits, there is the potential to close the gap between autologous CAR-T therapies and some of these newer allogeneic CAR-T and CAR-NK therapies.

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Delta/Gamma T Cells

In all my years of taking Immunology courses, I have yet to find one that gets into this variant of the T cell called the Delta/Gamma T cell. I think most of that has to do with how little we know about them. It wasn't until cell therapies like CAR-T came onto the scene before an interest in this subset of T cells got some interest. Here we will look at the differences they have with T cells, and the similarities they have with innate cells.

We know about T cells and how they are developed in the thymus. All precursor T cells go into the thymus where they mature into Alpha/Beta T cells with the CD4+ and CD8+ types. About 5% of the precursor Thymocytes that enter the thymus will become the Delta/Gamma T cell subset. These variants of T cells have very different receptors than their siblings the Alpha/Beta T cells. The first thing that happens is the T cell will attempt to make a good Beta chain. If it is successful, it will make the Alpha chain which removes the Delta genes. Notice the Delta chain genes sit inside the Alpha chain gene between the variable and joining regions. It is believed that thymocytes that fail to do a Beta chain rearrangement will attempt to make a Gamma chain as a rescue mechanism. If they succeed at the Gamma chain, then they will develop the Delta chain and become Delta/Gamma T cells.

There are a few differences between these two types of T cells. The first is the Alpha/Beta T cells have many Variable, Diversity and Junction genes to choose from. Their diversity has millions of possibilities. The Delta/Gamma T cells only have 4 Variable and a few Diversity and Junction options. They have extremely limited options for which to arrange their receptors. Once they become a Delta/Gamma T cell, they leave the thymus immediately and undergo no further selection. Their receptors do not work with MHC at all. They work more like pattern recognition receptors. This means they can't participate in rejection based on MHC I mismatch. This is what brought them interest as cell therapies. They actually have a ton of other receptors like CD1, CD16, NKG2D and Toll Like Receptors (TLRs). They are like a Swiss Army Knife of cells. They have some of the receptors of NK cells and some of the receptors of Antigen Presenting Cells (APCs). It is believed they can process and present antigens to Alpha/Beta T cells.

They even release many of the same cytokines to initiate inflammation like Macrophages such as Interferon and TNF-a. Beside having their own limited Delta/Gamma T cell receptor, they have many other receptors. It is also believed that these Delta/Gamma T cells can differentiate like the helper T cells into a Delta/Gamma T17 cell. The first of the many receptors for the Delta/Gamma T cell is the NKG2D receptors which measures the level of MHC I on cells to see if they are healthy. They also have the receptors to detect Mic-a and Mic-b variants of MHC I that are often stress ligands. They have a CD1 receptor which works as a limited ability of the immune system to present lipids as antigens by T cells. They also have some Toll Like Receptors. I found no reference yet to exactly which ones, but some references are toward viral TLRs which would be 3, 7, 8 and 9. They also have the CD16 receptor which makes them able to bind to antibodies and participate in Antibody Dependent Cellular Cytotoxicity ADCC.

Since the Delta/Gamma T cell is a cell, they will express the MHC I and MHC II of the person who created them. This means they could be targeted and rejected by a healthy immune system. They would still need edits for immune evasion to be used as therapies. The Delta/Gamma T cells are in two subtypes with the Vd1+ and Vd2+ variants. They have different roles within the body. They tend to home into different tissues. There has been a lot of new research into the subtypes of Delta/Gamma T cells. This has been to study which subtypes do best with specific tissues. This could lead to helping target these therapies to specific tissue types of cancer.

The Delta/Gamma T cells have so many of the functions used by many of the different immune cells. It is only natural that they would have their time in development for cell therapies. What we don't know is how they will perform. Some of the early data suggests they are on par with other allogeneic therapies. It is going to take time and data to differentiate these cell types as therapies. We might end up finding there is no difference between any of them. They might all work equally well.

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iPSC Cells

This technology is about taking a mature cell and backward engineering it back into a stem cell. It was developed by Shinya Yakamana in 2006. Alone, this technology had limited applications and didn't do much for years. 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 state. This is called an induced Pluripotent Stem Cells.

Once these cells are engineered back into stem cells they can be combined with gene editing to allow for engineering new cells. Then along came CRISPR gene editing and everything changed with a cheap, fast and easy to use editor. The DNA of the cell is its programming. Just like with any computer, if you reprogram the DNA with editing, you change the cell's behavior. Studies have shown that transplanting 1 cell's DNA into another cell will cause the new cell to assume the behavior of the original cell. The combination of iPSC and CRISPR brought together the two key components for complex cell engineering. You can engineer a stem cell with edits such as those for immune evasion in cell therapies.

Since stem cells are immortal, they can make master banks from these cells by running them through the cell cycle and expanding them. They can cryopreserve these cells. They build these master banks as a starting source for therapies. They can take some cells from the master bank and walk them down the path to development using growth factors and transcription factors. A single batch run can produce a trillion cells with 99% efficiency. Traditional ex-vivo cell therapy editing takes time, has high costs and can have low yields for efficiency. The development of iPSC cells cuts costs to as low as a few thousand dollars per therapy. It can produce hundreds or even thousands of doses per batch run. Typically it takes a few weeks to run a batch of these cells. The development of engineer iPSC banks has changed the way we can manufacture cell therapies.

It has the benefits of being able to work with any cell. The combination of iPSC with CRISPR has opened up a whole new world of highly engineered cell therapies. Some companies are developing master banks of cells that have upward of 12 edits. They create master banks of common progenitor cells which then just need the CAR inserted before running the batch. Due to stem cells having immortality, they can do many edits and only need 1 of the stem cells to have all the edits to become an entire master bank. I see a lot of people get hung up about one cell type or one therapy for the iPSC cell companies. The real technology in iPSC is how it changes the manufacturing of cell therapies. It can be used for any cells from T cells, NK cells, liver cells, heart cells, islet cells and even glial cells. The companies that wield iPSC as a tool will be able to do complex cell engineering that other companies will never be able to do.

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