Protein degradation, isdAbs and screening: We explore a key presentation by Nicolas Bery, Ph.D, Senior Postdoctoral Scientist at Cancer Research Centre of Toulouse CRCT
Silvia Hnatova highlights a presentation by Nicolas Bery from the Cancer Research Centre of Toulouse, outlining the intracellular single domain approaches for targeted protein degradation.
In his presentation, Dr. Nicolas Bery introduced single domain antibodies (sdAbs), explaining that the characteristics that make them particularly desirable as antibody candidates.
sdAbs have similar specificity and affinity as natural antibodies, and all sdAbs can be expressed in cells to bind targets intracellularly (intracellular single domain antibodies, isdAbs). isdAbs can be fused with GFP proteins to produce chromobodies.
Another application of isdAbs is to inhibit the target activity of an enzyme or protein-protein interactions.
Some targeted protein degradation strategies also utilize isdAbs, by fusing the isdAb to E3 ligase, leading to targeting the protein for degradation by the proteasome (Caussinus et al., 2011; Moutel et al., 2016; Fulcher et al., 2016).
isdAbs can also be used for GFP fusion degradation or endogenous target degradation through a similar strategy.
The focus of Dr. Bery’s talk was on the induction of protein degradation, highlighting the potential for using isdAbs for this purpose.
The main advantage of using isdAbs for inducing targeted protein degradation is the specificity, including isoform specificity, which is traditionally difficult to obtain.
Another advantage is the quick generation using IACs or phage displays combined with cell-based screens.
isdAbs bind to a large variety of targets, able to target active or oxidized conformations of a protein, or specific post-translational modifications.
They are easy to manipulate by modifying E3 ligases/linkers for degrader optimization.
Dr. Bery cautioned that targeted protein degradation mediated by macromolecules is not always straightforward, by using an example from his laboratory (Moutel et al., 2016).
He overexpressed isdAb fused to an FBOX domain in HeLa cells labeled with GFP. Immunofluorescence imaging revealed that using two different isdAbs, there was a variable degree of targeted degradation in the mitochondria.
Dr. Bery concluded that not all isdAbs can be directly converted into efficient degraders.
To overcome this problem, Dr. Bery demonstrated that the choice and the position of the E3 ligase on the isdAb are of utmost importance (Bery et al., 2020).
He ligated the E3 ligase on the N or C-terminal of the isdAb, leading to variability in the degradation of the target protein.
He concluded that each specific application needs to be tested to ensure that the optimal E3 ligase type and its position are used to obtain the best degrader.
Dr. Bery then outlined a screening approach towards identifying the best degraders.
He developed a stable cell line expressing the target protein fused to H2B-mCherry-RHOB protein to enable the visualization of the protein in the nucleus during the screening.
Next, he created a sub-library of sdAb-based degraders using phage display followed by cloning and transfection into the stable cell line established in the first step. This enabled the screening of sdAbs that lead to endogenous target degradation (Bery et al., 2021).
A fluorescent GFP reporter was added to visualize the transfected cells, in which the mCherry fluorescence of the target protein was measured.
Using this strategy, Dr. Bery was able to directly select an antibody working to induce target degradation.
Dr. Bery further optimized the protocol to find the degraders of the active conformation of the small GTPase RHOB, that is involved in the resistance to targeted therapies.
He explained that it was particularly challenging to inhibit specifically one member of the RHOB GTP family, aiming to target RHOB-GTP, as the RHOB-GTPase cycles between active and inactive form.
Using the H2B-mCherry-RHOB reporter assay, several FBOX-isdAba were tested to elicit H2B-mCherry-RHOB-GTP construct degradation (Bery et al., 2019).
He transfected the different antibodies into HeLa cells, finding that some isdAbs were more efficient than others, selecting the F-B6 degrader that achieved specific degradation of the active conformation of RHOB-GTP.
The efficiency of degradation was validated in vitro, confirming that RHOB-GTP degradation induced migration and invasion of bronchial epithelial cells.
The next GTPase targeted by Dr. Bery was KRAS, a GTPase often mutated in human cancer. For this, he developed a DARPin that bound to KRAS-GDP and KRAS-GTP only, creating a KRAS-specific degrader by fusing VHL to DP KRAS binder.
To test the efficiency of degradation, the KRAS degrader was expressed in cancer cell lines, and the KRAS degradation was induced by doxycycline (Bery et al., 2020).
The findings were surprising - KRAS degradation was proteasome-dependent and KRAS depletion was sustained to almost three days following induction of targeted degradation.
Dr. Bery observed that KRAS degrader only inhibits RAS signaling pathways in mutant KRAS cells, confirming that the degrader can be used to specifically target mutant KRAS GTPases for degradation – a finding pivotal to human applications.
Dr. Bery tested whether the KRAS degrader could affect the development of tumors in vivo, expressing KRAS H358 tumors in mice. After the mice were treated with the degrader, tumor volume was reduced, highlighting the potency of the degrader.
To conclude his talk, Dr. Bery outlined his current research into targeted degradation in pancreatic cancer, which could target proteins that confer resistance to chemotherapies, blocking cellular migration and invasion.
To target specific isoforms of proteins, he is using the aforementioned isdAb strategy to identify whether some of the isdAbs could induce tumor regression in vivo.
For this, Dr. Bery is using an optimized cell-based screening assay for sdAbs-based degraders using an automatic screening system (Operetta), allowing screening of a higher number of potential degraders.
