Ex Vivo culturing of NK cells and infusion

I guess this is a transition from my recent CAR obsession. NK (natural killer) cells are the other killer lymphocytes than Killer T-cells. They don't have an antigen specific receptor. Instead they have antigen non specific activation receptors (including NKG2C and NKG2D) and, importantly, Fc receptors which bind antibodies then kill the cell to which the antibody sticks (antibody specific cellular toxicity). This means that there is a flexible antigen specific response to any antigen which induces an antibody. That means that they are like natural CAR T-cells too in a way, but with no genetic modification. It is almost odd that growing NK cells outside the patient and infusing them is a fairly small effort reported in a fairly small literature which (as always) I will not responsibly cite (feininger, Mortier, Felicias, Miller sp???). One issue is that the NK cells do not multiply when activated. I think this is not a big problem as they can be stimulated with IL-15 (this requires continuous infusion which is not such a huge hassle). Another problem is that grown for a long time ex vivo with lots of IL15 and also IL12 (too inflamatory to put in patients) and (I think) IL20, they become hyporesponsive, anergic, inactive. (TL:DR version -- I think that it should be checked whether this problem is prevented if the NK cells are grown along with activated macrophages -- the post is definitely TL). There is an article on this (no cite) which says the problem can be solved by one week stimulate with interluekins, then one week pause, then etc. The article also asserts that the problem is that prolonged MTOR activation causes cells to turn off the system for fatty acid oxidation which means that they literally don't have the energy (as in calories) to do their job. I have to mention this, because I want to present a competing hypothesis. I think that NK cells become hyporeactive if they have been too long since touching an activated macrophage. Here I really should cite (all articles include as an author TA (thomas) Waldmann, my late father). This includes "preconditioning ..." Sato, Banfield Tagaya, Waldmann) which notes that naive cells do not respond to IL15 until they have touched a macrophage. "FC IV" Zhang, Anton, Waldmann, al et Ravetch on how NK cells exposed to IL15 and macrophages express an FC receptor which irreversibly binds antibodies. trans endocytosis Anton Waldmann and others The story is that when lymphocytes are in contact with activated macrophages, they engulf little bits of the macrophage forming vescicles with 2 layers with IL15 on the outside of the inner membrane and IL15 beta-gamma receptor on the inside of the outer membrane, so they constantly have IL15 stimulation. This is mainly MTOR not Jak-Stat stimulation (won't define but I think important). If the NK cells divide in pure NK cell culture, they dilute the vescicles which don't divide. I think this may be the problem. In any case (in case anyone has read this far) I think the solution is to add activated macrophages to the solution. Culture a mixture of NK cells and macrophages with IL15, IL12 (and I think IL20). One thing is that, like NK cells, macrophages are ADCC (antibody dependent cellular toxicity) effectors. Last thing (this is due to dad) is that it is important to activate the macrophages with anti-cd40 or Toll like receptor agonists such as lipopolysaccaride (endotoxin) RNA, poly GC DNA (with unmethylated C) c-reactive protein or well there are lots of otpions and a large literature. This is a very simple experiment. I think it is worth doing.

CAR T-Cell III

Unlike my first 2 posts here and here on CAR T-Cells this is an almost serious proposal. It involves more work and expense than current therapy, but does not, as far as I can see add even purely hypothetical risks. The idea is that the problem with CAR T-cell therapy of solid tumors has to do with the original antigen presentation and conversion of the T-Cells from the naive to the memory state. The solution would be to use patient macrophages and incubate the new CAR T-cells with them and lost of added antigen. The macrophages would have to be activated. I think a general Toll like receptor agonist (pattern recognition agonist, inate immunity agonist) such as lipopolysaccharide or RNA or poly GC would do.

It would be best to induce central memory T-cells rather than effector memory T-cells, but I think memory phenotype of either type might do.

convertible CARs

This my second post on CAR T-cells. The first discussed modifying them so that don't have checkpoints and are prepared for nitic oxide. The aim is to make a super CAR T-cell which functions in the solid tumor micro-environment.

This second post is a semi-crazy idea about making off the shelf CAR T-cells rather than modifying cells from the patient. The cost of the patient specific therapy is not prohibitive even now and should go down the learning curve. However, my proposal of multiple modifications would add to the cost and why do them again and again ?

So the idea is to make a CAR T-cell line which will not be rejected by the patient even though the CAR T-cells are made with someone else's T-cells with different surface antigents especially different HLA antigens. Long ago my late father thought of deleting the Beta 2 microglobulin gene so that HLA A B and C would not be expressed on the surface. Here I make a much more radical proposal (which will never be allowed so it is just for a blog post)

The off the shelf CAR can be designed to express the do not kill me signal PDL1. As I already proposed that the receptor PD1 be deleted, these cells will not tell each other not to kill. I think that these cells could be infused into anyone and would function. They would also be dangerous - if some became leukemic dealing with them would have to include anti PDL1. Recall that I propose inserting Herpes TK into the super CAR T-cells so that they can be killed, if necessary, with gangcyclovir. That would be even more clearly needed with the PDL1 expressing super CAR T-cells.

Hot ROd CARs

CARs being chimeric antigen receptors. They are a key contribution to immunotherapy of cancer. The technology is based on genetic modification of the t cell receptor of a CD8 killer T-cell replacing it's variable region with the variable region of a tumor specific monoclonal antibody. Remarkably this creates a cell which specifically kills the cancer cells. A link to a Wikipedia article.

This approach has been very successful in treating Leukemia, but not so successful in treating solid tumors -- the tumor micro environment is not hospitable to killer T-cells. There are a large number of known aspects of the tumor micro-environment which tend to protect tumors from activated killer T-cells

1) Perhaps the most important is myeloid derived suppressor cells -- these are immature granualicytes and macrophages which are attracted to the tumor. Among other things, they produce anti-inflamatory IL-10, and also produce the free radical Nitric Oxide (NO).

2) Tumor inflitrating T-regs which produce and display anti inflammatory TGF beta.

3) Cancer cells display checkpoint "don't kill me signals" including PDL1 and CTLA4 ligand.

4) There are generally low Oxygen, low glucose, low Ph, and high lactic acid levels.

Many of the issues involve specific interaction with specific receptors on the t-cells (eg PD1, CTLA4, IL10 receptor, TGF beta receptor). I think that, since one is already genetically modifying the t-cells, one can also delete those receptors so they do not respond to the anti-inflamatory signals. The NO issue is different -- it is a non specific oxidizing agent. I think here one can make cells which always produce the antioxidant response by deletign KEAP1 which inactivates NRF2 which triggers the anti oxidant response.

So I think it is possible to produce souped up CARs which invade solid tumors.

There is a potential risk of putting killer t-cells which can't be regulated into a patient, so I would also insert the gene for herpes TK so they can be specifically killed by gancyclovir.

This approach makes sense to me. It involves a whole lot of work aiming at a possible future approval of a clinical trial. I can see why it hasn't been done (and will have another post about reducing the cost and effort involved) but I think it makes sense to try.

MMLF Founding Manifesto

With this manifesto I found the Modified Mosquito Liberation Front. Our aim is to liberate mosquitoes which either are resistant to Malaria (and imprisoned on Sao Tome & Principe) or which produce only male spermatazoa (and are imprisoned in Burkino Faso).

The liberation of such mosquitoes is one way to fight malaria. They (and similarly modified members of other species of anopheles mosquitoes) can eliminate malaria.

However they can't do that imprisoned in lab cages. They are not released because of who ? WHO. It is agreed that the important and allegedly for some reason risky decision must be made after careful thorough consideration and that release occur only when all affected countries (which are numerous as mosquitoes don't respect international boundaries) agree.

That is probably roughly never and certainly not until there have been millions more un-necessary deaths.

I quote "'We have got to get going,' Dr. Lanzaro said. 'We can’t just keep saying 10 more years, 10 more years. Six million people have died while we’ve been fiddling around.'"

I think the modified mosquitoes should be liberated using any means necessary.