As described previously, the first generation of CAR's that were constructed some 25 years ago consisted of a receptor composed of three parts from different proteins fused together: a modified antibody, a transmembrane section, and an intracellular domain. These showed promise but weren't particularly effective and gave rise to succeeding generations that added one or two costimulatory domains to the intracellular portion. It is these latter versions that have proven successful in combating a variety of blood cancers. However, since non-blood cell tumors haven't responded particularly well to either the first or later generations, numerous laboratories around the world have begun devising new CAR's with enhanced ability aimed at eradicating solid tumors.
These new CAR's have been designed to address several issues.
A very recent article by Nicholas Tokarew and colleagues ("Teaching an old dog new tricks: next-generation CAR T cells", British Journal of Cancer, Volume 119 - Available behind a paywall) describes how researchers have begun to deal with these points.
Upon biopsy, many tumors are found to be surrounded by T cells. For patients, that's generally good news. The presence of T cell infiltration is an indicator that the tumor may well respond to checkpoint inhibitors and other immune therapy. In order to improve the recruitment of T cells to the cancer, scientists have made use of chemokines, kinds of cytokines that are chemoattractants. Cancer cells "learn" to use these small proteins to further tumor progression. They secrete them into their immediate environment in an effort to recruit cells that inhibit the immune response. Scientists have tried to turn these molecules against the tumor. They have added chemokine receptors to the intracellular domain of the CAR so that the CAR-T cells will move to the cancer site. Such fourth generation CAR's have been called "TRUCKs", for "T cell Redirected for Universal Cytokine-mediated Killing". It isn't yet clear whether such vehicles are effective or not in humans,. However, because different tumors often express distinct populations of chemokines, the intriguing possibility exists that CAR's might be designed to respond to particular combinations and thereby specifically target specific cancers.
Ordinary killer T cells will die off it they do not encounter molecules to which their receptors bind. Moreover, they also require the presence of co-stimulants and cytokine signals. Second generation CAR's carry both an external binding site that is likely to be engaged and co-stimulatory domains. Newer CAR's have been created that also carry a cytokine signalling domain that will hopefully enhance the survival and killing ability of the CAR-T cell. Results are not yet in.
As I've already mentioned, determining the appropriate target for a CAR is a challenge. The tumor antigen must be located on the cell surface and it must be specific to the cancer being attacked - not found on normal tissues. In practice, these two objectives are rarely achieved. What's more, even if they can be, tumors mutate. By selecting for the appropriate mutations, they are able to evolve and move antigens internally or eliminate them entirely. One strategy to get around the selectivity problem is to create a T cell that carries more than one CAR (so called "dual CARs"), each with a different antibody recognition site. The idea is that only when a cancer cell is detected that bears both antigens will the full killing response occur. Alternatively, some very clever researchers have developed what are called "split CARs". Again, T cells are made to carry two different CARs, one of which lacks a stimulatory domain, the other, directed at a different antigen, has a costimulatory domain but is missing an intracellular signalling domain. Only when these two CARs recognize a cell carrying the two antigens that they are directed against, does the T cell become active (see the figure at the right). Both dual and split CARs are being evaluated in ongoing clinical trials.
Besides being hypoxic (low in oxygen) and lacking a reasonable blood supply, tumors often express inhibitory molecules such as PD-L1 that interact with PD-1 receptors on T-cells in order to prevent the immune system from attacking them. A few years ago, with the advent of CRISPR/Cas9 (a potent DNA editing system), it became possible to knock out the gene for the PD-1 receptor on CAR-T cells. This proved effective when tested in animal models. However, in 2017 the process was taken a step further. A group of scientists used CRISPR/Cas9 to eliminate three genes from a CAR-T cell. They removed the PD-1 receptor gene and the endogenous T cell receptor as well as the MHC genes. This created what is called a "universal CAR". It's universal because CAR-T cells can be collected from healthy individuals and deployed in anyone. That's because it's lacking both a T cell receptor and a MHC protein. If it and other techniques prove successful, having an "off the shelf" CAR-T will bring down the time needed to treat a patient as well as the costs, an exciting development.
Because several patients have lost their lives due to CAR-T therapy, scientist have looked for ways to control CAR-T cells after they have been added to the bloodstream. These controls have taken several forms. I'll onlly discuss one - the NOT-gate. A NOT-gate circuit consists of a CAR T cell that carries two CARs. One is traditional, bearing a tumor seeking antigen and CD3 activation domain and costimulatory domains. The other, carries an antibody directed against normal cell surface antigens. Instead of being linked to an activation domain, when it finds a normal surface antigen it activates an inhibitory protein. When this dual CAR-T encounters a tumor, it will be activated and hopefully carry out its assigned task. However, in the presence of normal cells, its activity will be inhibited and it will not function. The idea is that this will increase the specificity of CAR-T therapy.
It's clear from this and previous posts that the CAR-T technique has caught the attention of the scientific community. As further evidence, I offer the following statistics that come from clinicaltrials.gov, the website that the US government runs that tracks clinical trials worldwide. When I entered "CAR-T OR chimeric antigen receptor" into their search box a week ago I got 735 hits. They included trials that were recruiting (367), completed (150), not yet recruiting (69), and active, but not recruiting (55) (the status of the remaining trials were either unknown, terminated, suspended or withdrawn). The fact that there's so much attention being given to CAR-T therapy offers hope for the future. But the number of clinical trials also demonstrates that we're got a long way to go. While I've described many ingenious new CAR-T variants, I've only told you about the tip of the elephant's tail. And it's highly probable that some combination of techniques may be optimal. Ultimately, new procedures and combinations will have to be tried in humans. So, it's likely that many more clinical trials will be added to the government's database in the near future.
Meanwhile, scientists will have to make use of mouse models of cancer to develop promising approaches. One brand new technique that may allow researchers to more quickly manipulate new combinations of CARs in both mice and humans was recently published. Called "SUPRA", for split, universal, and programmable, it divides a CAR into two parts. As shown in the figure, one part carries an external antibody (three different ones are shown); the other a CAR's intracellular signalling domain (it's carrying a red squiggly thing on its end). The two sections can be tethered together by means of a "zipper", a binding site on the intracellular piece that can match with one on the antibody (the blue and red squigglies bind to one another). Without going into too much detail, this allows researchers to add different antibodies to the CAR much more easily. One can imagine that a SUPRA CAR can be quickly switched from one antibody to another in a patient that hasn't responded to a given therapy.
Well, that's all folks. I've decided to put these posts together into an electronic book. That's my next project. Look for it in the near future. Thanks for reading.