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Tickling cancers into committing suicide

Pembrolizumab and sebelipase alfa. What do these two odd sounding words have in common? They are both life saving proteins, one for treating cancer and one for treating an enzyme deficiency disease. Both incredible medicines are the result of years of painstaking research and development work. At the same time, both medicines fall under the umbrella of protein therapeutics, with the most famous protein therapeutic being insulin, a lifesaver for diabetic patients.

Protein therapeutics are of course, made by protein engineers. These scientific engineers design proteins, engineer them into cells and test and optimize them in countless experiments. This simple reiterative cycle of design, test and repeat serves as the foundation of nearly all protein therapeutics today. But this post is not about past successes, but rather it is about future ones. Our team at Ranomics, along with our VariantFind technology platform, has been fortunate to work with leading healthcare companies in engineering next generation therapies. While we cannot share our partners’ brilliant work with you, we want to highlight some inspiring innovation in the public domain.

With cancer being one of the leading causes of death in the United States, it comes as no surprise that pharmaceutical companies spend endless resources in developing the next market-changing cancer therapy. These companies have incredible programs to identify traditional medicines such as small molecules and antibodies, but also next generation medicines such as CAR-T cells (which we will cover in another post).

While these efforts are great, they are not nearly enough. What most folks don’t realize is that cancer cells are highly evasive and find ways to escape treatment. This means that every time a cancer becomes non-responsive to treatment, we need to throw another weapon at it and hope the new treatment eradicates the cancer faster than it can escape. The bad news here is that the pace of developing new therapies has slowed and as a community, scientists need to be creative in making medicines faster.

Luckily, researchers at Johns Hopkins University recently came up with a new weapon idea using a synthetic biology approach. They generated a synthetic protein that only functions inside a cancer cell. The function of this protein? To “TICKLE” cancer cells into committing suicide.

“TICKLE”, more scientifically known as Trigger-Induced Cell-Killing Lethal-Enzyme, started off as a suicide-gene from the Herpes Simplex Virus (HSV), in this case the gene encoding for Thymidine Kinase (TK). The label for this gene pretty much says it all – cellular suicide. But of course, in order for a cellular suicide gene to have therapeutic effect, the gene needs to be ON in a cancer cell and OFF everywhere else. Thus, the research team needed to engineer an activity switch for HSV-TK that is responsive to a cancer specific signal.

Fortunately, cancer cells have a number of notable differences compared to normal cells. One particular difference is a protein call HIF1-alpha, which is highly abundant in hypoxic environments such as tumors. H1F1-alpha was a good choice because it is known that a second protein called p300, specifically the CH1 domain of p300, senses levels of HIF1-alpha and changes shape depending on how much HIF1-alpha is around. The two proteins theoretically can function as a responsive switch to detecting cancer versus non-cancer cells.

The next piece of the puzzle was to figure out a way to fuse HSV-TK and CH1 into a new protein that would only be suicidal when HIF1-alpha is around. This is a difficult task for any protein engineer as it is commonly known that there are a millions of ways to disrupt function, but only a few finite ways to create new functions, which is exactly what the researchers were trying to do here.

Brilliantly, instead of guessing how to fuse the two proteins together, the researchers took a systematic and unbiased approach by making variant libraries (always a great idea by the way). They made a diverse insertion library by inserting CH1 at every possible position within HSV-TK. In addition, at each insertion point, they tested three different flavors of fusion linkers: (a) no linker, (b) Gly/Ser-rich linkers and (c) site-saturation linkers. Using this diverse library of approximately 140 million possibilities, the group screened for HSV-TK-CH1 fusion proteins that were active only in the presence of HIF1-alpha.

The group deployed a 2-stage bacteria screen for finding the perfect HSK-TK-CH1 suicide protein. The first stage isolated HSV-TK-CH1 fusion proteins that did not cause suicide in the absence of HIF1-alpha, a really important task for keeping patients alive (by not killing normal cells). The second stage went on to isolate HSV-TK-CH1 fusion proteins that turned on when HIF1-alpha was overexpressed, also a really important task for keeping patients alive (by specifically killing cancer cells). All of this work was done in the E. coli strain KY895, where the group engineered growth-based assays for the biochemical activity of HSV-TK-CH1.

At the end of the day, the 140 million possibilities boiled down to a single HSV-TK-CH1 fusion protein that was only active when HIF1-alpha was overexpressed. This lucky fusion protein, now known as TICKLE, had CH1 inserted between amino acids 150 and 151 of HSV-TK. Multiple growth based experiments show that TICKLE was only effective at promoting suicide when HIF1-alpha was present. Further analysis showed that this effect was due in part, by HIF1-alpha’s ability to keep TICKLE around. When HIF1-alpha is not around at high levels, the bacteria cell destroys TICKLE.

As impressive as this protein-engineering project was, it will still be a while before we see TICKLE in a clinic near you. This new weapon has miles to travel before reaching any patients. First, all of the work so far has been done in a bacteria system, which in no way, shape, or form resembles a tumor. Second, HIF1-alpha is also highly abundant in the gallbladder, appendix and bone marrow, how will this affect TICKLE-mediated toxicity? Lastly, how would we even get TICKLE into tumors? That itself will be a tricky task. Nonetheless, this is surely a promising start towards making a new protein therapeutic and filling up our arsenal of cancer targeting weapons.



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