It is important to be able to identify and validate new therapeutic strategies for proteins that are not currently targeted by inhibitors in cancers.
Therapeutic antibodies are molecules of choice for the standard treatment of many forms of cancer. Their application is currently restricted to the extracellular compartment due to their excessive size, which prevents them from crossing the cell membrane. As most therapeutic cancer targets appear to be located in the intracellular medium. With the development of genetic engineering of antibodies, it has been possible to use recombinant antibody fragments produced in cells by integrating into the cell the DNA molecule necessary for its production by the cellular machinery. These antibodies can be functionalised with degradation domains for example. Once produced by the cell, they degrade (known as degrading antibodies) their target and cause cell death and thus the eventual regression of the tumour.
In this study, the team has developed a detailed protocol for the selection of intracellular “degrading” antibodies using the cell screening technique which aims to identify those that have an interesting activity at the cell level, either by modifying a particular cell function or by acting on the cell as a whole.
Previously, this team has used this method to select degrading antibodies targeting GFP fusions (Moutel S, et al. Elife, 2016) and more recently the active conformation of the small RHOB GTPase (RHB-GTP) using a modified cell line (Bery N, et al. Cell Chem Biol).
In this new publication, antibodies selected by an in vitro technique (called phage display) are directly fused to a domain of an E3 ubiquitin ligase. This domain allows the antibody target protein to be sent to the proteasome, which is a cellular machinery that eliminates proteins in cells. In order to visualize the ability of these antibodies to degrade their intracellular antigen, we fused the target protein of the antibodies to a fluorescent protein that is expressed in a cell line. During cell screening, an antibody is considered to be degrading if it decreases the fluorescent signal of the fluorescent target protein. We used this protocol as a starting point to select a RHOB-GTP degrading antibody and study its function in various cellular processes. This protocol can be applied to custom select intracellular “degrading” antibodies that could be particularly useful in the fight against pancreatic cancer.
Discover the published article :
STAR Protoc. 2020 Dec 30;2(1):100249.doi: 10.1016/j.xpro.2020.100249. eCollection 2021 Mar 19.
Protocol to select conformation-specific intracellular antibodies for targeted protein degradation in an engineered cell line
Nicolas Bery, Gilles Favre , Aurélien Olichon
Key words :
- Intracellular antibodies,
- targeted degradation,
- antibody-based degraders,
- cell-based screening
Collaborations and thanks
This work was carried out between team 3 (Pr Gilles Favre, Dr Aurélien Olichon) and team 10 (Dr Nicolas Béry).
This work is supported by a grant from Fondation pour la Recherche Médicale (FRM) (Equipe labellisée FRM [DEQ20170839117]) G.F. and A.O.
N.B. is supported by a fellowship from the Fondation de France (n°00097692).