The "SWITCH" Project

Proteins in medical treatments

In the past decade, proteins have gained increasing attention as therapeutic agents. Among them, monoclonal antibodies have gained a particular importance, since they present several advantages compared to standard drugs. Reduced risk of non-specific action, longer therapeutic effect and smaller risk of toxicity upon elimination are just several advantages monoclonal antibodies possess when used as drugs.

Problems with synthesis

Despite all of their advantages, monoclonal antibodies are sometimes challenging to produce. The majority of them are manufactured by recombinant cells that are cultured in huge volumes (called “bioreactors”). In order to achieve a reasonable price for the final product, the antibody needs to be produced at a high concentration. Unfortunately, many antibodies are toxic to the producing cells at high concentrations, which means they get killed before a sufficient concentration of final product is reached.

So far, this problem has been solved by using cells that are designed to produce the desired protein only when they uptake and sense a particular small molecule. With this technique, cells begin by growing normally and start to produce the desired protein only when the small molecule signal is present in sufficient concentration to trigger the production of the desired product at an optimal concentration, maintaining a balance between protein production and cell health. Unfortunately, this method has a couple of disadvantages. First, the “switch” molecule is often difficult and costly to remove. Second, redesigning cells so that they react to the “switch” molecule can perturb many biological pathways, which in turn decreases the efficiency of antibody production by disrupting normal cell growth and proliferation.

Cool solution

We thought that the best solution for this problem would be a simple switch, that would activate the synthesis of a therapeutic protein in cells just by shining a specific wavelength of light on the cell culture. Our hope was that this method would have a minimal effect on cellular pathways and reduce the effect on cell health. This way the cells would grow happily in darkness and start producing the toxic protein only when light is supplied. With such a switch, there would be nothing to remove from the cell culture, allowing recombinant proteins to be produced more economically than by supplying large amounts of a small molecule trigger.

How are we going to do it: LovTAP protein

A really nice way to realize our idea would be to use the LovTAP protein. When illuminated with blue light, LovTAP changes configuration and acts as a negative regulator of gene expression in bacteria, enabling the ability to switch on and off gene activity. Unfortunately, LovTAP does nothing in the mammalian cells, which are the chemical factories used for the synthesis of antibodies. We've decided to overcome this problem by attaching a VP16 viral promoter domain to the LovTAP protein, so that it will become a powerful positive regulator of gene expression in mammalian cells. If we transect the LovTAP-VP16 construct along with the gene of the protein we would like to synthesize (monoclonal antibodies) under the control of LovTAP, we could use it as the perfect light “switch” to control gene expression. It will activate protein production when mammalian cells are exposed to blue light and minimally disturb other cellular pathways.

Another approach: Melanopsin

An another nice tool to realize a light-induced switch would be to use a light receptor that already exists in mammalian cells, such as melanopsin. This way, the receptor will sense the light and transmit a chemical signal to the rest of the cell in order to activate the production of the desired protein. Even though this switch is more complex then the previous one and will affect several pathways, it still eliminates the need for an activating small molecule. In addition, it might be more efficient then the LovTAP-VP16 based switch, since it uses a protein that already exists in mammalian cells, and not a fusion protein.

CHO cells & Co

In order to check the validity of our ideas, we attempted to implement both light-induced switches in CHO cells. For the LovTAP-VP16 switch, we've transfected CHO cells with LovTAP-VP16 encoding plasmid along with a reporter plasmid containing a protein that fluoresces in red (dsRed). We can tell that the switch functions correctly if after illumination with blue light we can detect a substantial increase of red fluorescence in the cells. For the melanopsin-based switch, we will transfect a plasmid expressing a natural light receptor (melanopsin), along with a reporter that would synthesize green fluorescent protein in response to the signaling pathway that is naturally triggered by melanopsin. We will know the switch functions correctly if we can detect a substantial increase in green fluorescence in cell culture after illumination with blue light.