Appetite for destruction: ‘Plug and play’ targeted protein degradation using synthetic VHH antibodies to take out tricky targets

Much of modern pharmacy is built on the concept of inhibition: identify a target that plays a role in a particular disease, then find a molecule that binds and blocks it.  

Yet while this approach has led to game-changing therapies for many conditions, from cancer to infections to auto-immune diseases, it has its limitations. Many physiologically important targets lack suitable functional binding sites or are otherwise incompatible with conventional drug development approaches.  

Targeted protein degradation: a fresh therapeutic paradigm

One promising alternative for these ‘undruggable’ targets is to skip over the idea of inhibition and just get rid of them altogether. Over the past two decades, researchers have been exploring ways of manipulating targeted protein degradation (TPD) pathways within cells to treat disease¹. 

Of the variety of approaches being investigated², the leaders of the protein degradation pack are proteolysis-targeting chimeras (PROTACs). These are bifunctional molecules that attach to the target of interest and bring it into contact with the cell’s degradation machinery via ubiquitin modification by E3 ligase³.  

However, the first PROTACs coming through development rely on typical small molecule inhibitors for the protein binding function (smPROTACs), limiting their use for more challenging targets. They are also dependent on the presence of a limited suite of endogenous E3 ligases and their associated components, which may not be expressed in the tissue of interest. 

 Researchers are now applying sophisticated protein engineering techniques to build on these foundations and create a new generation of PROTACs.  

Biological PROTACs (bioPROTACs) combine highly specific antibody-based target binding domains with an E3 ligase to direct antibody-bound targets for proteasomal degradation – when the antibody is coupled to the E3 ligase TRIM21 this approach is known as ‘Trim-Away’⁴. 

A bioPROTAC breakthrough

In an important step forward for the field, the ability of TRIM21-based bioPROTACs to target an endogenous protein for destruction has now been demonstrated for the first time in a recent paper published in Nature Communications⁵.  

This study focused on developing a bioPROTAC against human antigen R (HuR) – a ubiquitous but enigmatic RNA-binding protein that is overexpressed in high grade tumours and associated with poor prognosis, making it an attractive target for cancer therapy⁶˒⁷.

The TRIM21 component was fused to a targeting domain created from a highly specific VHH single domain antibody. These small but highly versatile molecules were first identified in species such as camels and llamas⁸, and are becoming a modality of choice in multiple biopharma applications such as bi-specific antibodies and CAR-T targeting. 

In this case, the anti-HuR VHH antibody was identified from one of Isogenica’s vast synthetic libraries, which contains the equivalent number of unique antibodies that would be found in one million llamas. 

In vitro colony formation and proteomic studies showed that the engineered bioPROTAC could effectively bind and degrade HuR in cell cultures. These effects were associated with altered cellular processes including splicing and transcription, confirming that depleting HuR was impacting RNA-mediated pathways as expected.  

Further experiments with mouse xenograft models established from cell lines carrying a doxycycline-inducible construct expressing the bioPROTAC demonstrated in vivo depletion of endogenous HuR, along with a rapid and sustained negative impact on tumour growth starting just two days after induction. 

A ‘plug and play’ platform for targeted protein degradation 

While more work needs to be done to establish the most effective ways of delivering bioPROTACs into tumours, these results are the first demonstration that VHH-targeted bioPROTACs can degrade endogenous proteins and impact cancer growth.  

Importantly, the VHH and TRIM21 components alone did not cause any significant phenotypic or proteomic disruptions, suggesting that these bioPROTAC building blocks may be suitable for use against a wider range of disease targets.  

Finally, the study is a powerful proof of a ‘plug and play’ platform for the rapid discovery and development of similar approaches to investigate other intractable targets, as well as the more general utility of VHH antibodies for targeting other cellular components for the creation of next-generation biotherapeutics. 

References:

1 Lai, A., Crews, C. Induced protein degradation: an emerging drug discovery paradigm. Nat Rev Drug Discov 16, 101–114 (2017). https://doi.org/10.1038/nrd.2016.211 

2 Cresser-Brown, JO., Marsh,GP. And Maple, H.J. Reviewing the toolbox for degrader development in oncology. Curr Op Pharmacology 59: 43-51 (2021) https://doi.org/10.1016/j.coph.2021.04.009  

3 Békés, M., Langley, D.R. & Crews, C.M. PROTAC targeted protein degraders: the past is prologue. Nat Rev Drug Discov 21, 181–200 (2022). https://doi.org/10.1038/s41573-021-00371-6 

4 Clift, D., So, C., McEwan, W.A. et al. Acute and rapid degradation of endogenous proteins by Trim-Away. Nat Protoc 13, 2149–2175 (2018). https://doi.org/10.1038/s41596-018-0028-3 

5 Fletcher, A., et al. A TRIM21-based bioPROTAC highlights the therapeutic benefit of HuR degradation. Nat. Commun. 14, 7093 (2023). 

6 Mukherjee, N., et al. Integrative regulatory mapping indicates that the RNA-binding protein HuR couples pre-mRNA processing and mRNA stability. Molecular Cell 43, 327-339 (2011). 

7 Lebedeva, S., et al. Transcriptome-wide Analysis of Regulatory Interactions of the RNA-Binding Protein HuR. Molecular Cell 42, 340-352 (2011).  

8 Wesolowski J, Alzogaray V, Reyelt J, et al. Single domain antibodies: promising experimental and therapeutic tools in infection and immunity. Med Microbiol Immunol. 2009;198(3):157-174. doi:10.1007/s00430-009-0116-7