CAR V

This post criticizes and presents and alternative to the approach presented in CAR III. update: I am no longer convinced that the approach described here is an improvement, or even as good as the approach presented in CAR III end update In Car III I proposed further genetic manipulation of a CAR T-cell, that is a killer T-cell to which a chimeric antigen receptor which targets cancer cells but not normal cells has been added. The proposed modifications consisted of deleting5 genes. One logistic problem is that the procedure for deleting a gene is different from the procedure for adding a gene. Since the CAR T-cell or further modified CAR T-cell is custom made for each patient, this could be a serious problem.

In any case, this post discussing modifying a CAR T-cell to make it able to function in a tumor micro-environment by adding various genes, one for the CAR receptor, one to make the CAR prepared for reactive oxygen species (ROS think hydrogen peroxide) and 4 to make the cell ignore checkpoints – signals to not kill which have a normal function in healthy people but which can be used by cancer cells to avoid the reaction of the immune system to neo-antigens (new strange proteins basically) which they display.

Our cells handle ROS with various proteins the production of which is stimulated by the transcription facor Nrf2. Nrf2 is bound and inactivated by KEAP1. I proposed deleting the KKEAP1 gene. Amther approach is to add a gene (with a potent promoter and enhancer) which makes lots of Nrf2 so that the normal KEAP1 can’t bind all of it. One issue is that one of the genes stimulated by Nrf2 is the KEAP1 gene (a bit of homeostasis) so the added gene would have to be transcribed more than the normal Nrf2 gene is.

I am not sure that this is easy to manage or strictly necessary. Stimulation of Nrf2 activity in vitro might be enough to give the CAR T-cell a head start. Once the cell is exposed to ROS the natural mechanism (hydrogen peroxide changes the shape of the KEAP-1 protein so it no longer binds Nrf2) might be enough if the CAR T-cell is not quicky killed or inactivated.

Now consider checkpoint inhibitors. Many have been developed and they have been used with dramatic but not wholly satisfying results. They cure a minority of patients (a fact recognized with a Nobel prize) but other patients are not cured. The checkpoints involve receptors on T-cells which tell the cell not to kill (except in one case to kill itself). The receptors are called PD1 CTLA-4 TIGIT and the TGF-beta receptor. The checkpoint inhibitors are monoclonal antibodies which bind to and block the receptor or which bind to and block the molecule which binds to and activates a receptor. As far as I know from my efforts to survey the literature, in clinical trials no more than 2 ckeckpoints are inhibited in any single patient. I assume this is because doctors know that the checkpoints evolved for a reason and fear that blocking too many would lead to autoimmunity (the immune system attacking the normal cells of the patient). I have a crazy idea for inhibition of the checkpoint near the CAR T-cell but not systemically (everywhere in the patient). The idea is for the CAR T-cell to produce blocking antibodies with a short half life, because they are fairly qyuckly removed from the blood and digested by the liver. Most proteins have a short half life for this reason. A key exception is IgG antibodies which have a very long half life because part of the constant region of theantibody signals to the liver to recycle them. The constant region is a tail of the antibody which does not bind the target and which is not highly variable (to make things complicated there are 4 different constant regions of IgG antibodies but nothing like the immense number of variable regions which stick to specific antigens (targets)).

A reduced size molecule called an Fab can bind to and block a receptor even without the constant region. It consists of two proteins stuck together (in genetic engineering they are often one protein which a highly flexible link made of the tiny amino acid glycine which does not get in the way of the two non-glycine parts of the protein aligning and sticking together). These Fab molecules could block receptors of the CAR T-cell which made them and of nearby T-cells without building up to high levels in the blood steam (and lymph) of the patient and making trouble.

An advantage is that nearby T-cells include the patient’s killer T-cells which infiltrate the tumor. This often happens. Also these cells often do not kill the cancer cells because of checkpoints and/or reactive oxygen species. update: This is an advantage of adding genes for anti checkpint Fabs even if some genes, such as part of the normal T-cell receptor have to be deleted. end update. This could be an important added benefit as the tumor would be targeted by different T-cell receptors reacting to different neo antigens, so the cancer cells can’t evade immune surveillance by mutating to lose a specific antigen.

Also the Fab stuck to the receptor on the CAR T-cell or other nearby T-cells will not send the signals that full antibodies send to other immune cells which include “kill this cell” sent to natural killer cells, swallow and kill this cell sent to macrophages and will not fix complement which kills cells. I think this should be an approach which works as well as the checkpoint inhibitors which are currently being used.

update: It is possible that the CAR itself will attack normal cells – CAR T-cells have the added chimeric antigen receptor but they also have the original T-cell receptors. Self reacting killer T-cells should be eliminated in the fetal thymus, but Killer T-cells do sometimes cause autoimmune problems (hence the use of short half live Fab’s) update: this makes it necessary to delete a gene for part of the normal T-cell receptor to prevent attack on patient. That elimiates the original logic of this approach vs that presented in CAR III and explains the update that I am no longer convinced that the approach preseented here is a better appraoch end update. Also one of the cells or all of the cells might become a leukemia (this can and has happened with addition of genes with a retrovirus). For both reasons it would be wise to add the gene for herpes thymidine kinase which makes cells vulnerable to Ganciclovir. end update.

In any case it is an idea which I find interesting.