Team:Montpellier/Safety
BIOSAFETY
Although our project was mainly focused on the realization of a solid proof of concept around the activity of our new KARMA tool, we thought further about its application in therapeutics and in vivo fields. For an application against beta-lactamase or exotoxins, administration of KARMA would be intravenous. As soon as this tool is in the blood, biosafety issues arise and it should obviously be ensured that the KARMA tool is not toxic or harmful to the patient's health.
As our tools consist of a fusion between a VHH against the protein to be degraded and a specific protease we have identified two types of problems. The first would be the non-specific degradation of other proteins present in the blood and the second would be about the body's immune response against the recombinant protein that KARMA would be.
We have considered several ways to reduce or even counter these problems below.
How to decrease the risk of non specific degradation?
Protease library: The idea is to mutate the active site of the protease of interest to decrease its catalytic activity and avoid non specific degradation of other proteins that will be on the close environment of the KARMA tool.
With the creation of a library of mutated proteases by directed mutagenesis with different levels of activity for different applications of KARMA it will lead to an interesting platform of protease choice for different applications
Other strategy? Protease dimerization.
Another way to prevent non-specific degradation is to produce the device in two parts using a dimeric protease. This KARMA in two parts will only be active if the two monomers are close enough to reassemble and form a complete protease. For this reason, it would be easier to use a KARMA with a whole antibody that can recognize a first epitope of the target, this KARMA carries a first monomer of the protease. A second KARMA would recognize the constant part of the first antibody, providing the second monomer of the protease. Both monomers being very close, there could be a dimerization of the protease in order to make it functional.
Is unspecific degradation this bad?
VWhen we think about it, non-specific degradation would not necessarily be so serious. Indeed, in an organism, the turn over of proteins is extremely fast, the cycle of a protein is to be produced and constantly degraded. Our tool is designed to target extracellular proteins, which does not seem extremely dangerous in the event of non-specific degradation of an unwanted target. In addition, the tool would be delivered in very low concentrations and the half-life would be controlled.
How to avoid immune response?
VHH are humanized VH fragments, they are designed to avoid/escape immune response in humans.
We could use non immunogenic proteases such as human proteases. We had thought of trypsin, for example, which keeps its activity in the blood [1].
The tool we used for our proof of concept was designed with a VHH, but we could build it with more human immunoglobulin fragments such as ScFv, Fab or complete immunoglobulin... to be able to approach a near-total humanization of the tool. And thus avoid immunogenicity in the host.
Safety in the lab
In order to achieve our proof of concept, we had to work with tools that are widely known, mastered and characterized in the world of biology. For the protease, we used the TEV protease (Tabacco Ech Virus). This virus is not pathogenic for humans. We have also worked only with the protein sequence and have never manipulated the virus itself. All laboratory transformations were performed in E. coli strain NEB10B, which is a BioSafety Level 1 commercial strain. Manipulation and waste management were carried out following all safety rules.
[1] Lefkowitz, Roy B., Geert W. Schmid-Schönbein, et Michael J. Heller. 2010. « Whole Blood Assay for Trypsin Activity Using Polyanionic Focusing Gel Electrophoresis ». ELECTROPHORESIS 31(14): 2442‑51.