Research Progress of Extracellular Targeted Protein Degradation Technology
The extracellular protein network composed of secretory and membrane-bound proteins is complex and plays a key role in intercellular communication and physiological regulation. Extracellular protein networks have long been regarded as important therapeutic targets in drug development, and in recent years, the emergence of extracellular targeted protein degradation (eTPD) technology has provided researchers with a novel way to regulate the extracellular proteome. Unlike traditional drugs based on a placeholder mechanism, eTPD technology uses bispecial antibodies, conjugates or small molecules to transfer extracellular protein targets to lysosomes for selective degradation. This unique approach not only expands the range of treatable targets, but also opens up new possibilities for the treatment of a variety of diseases.
What is eTPD and what are the advantages?
eTPD represents a revolutionary drug development strategy focused on the regulation of the extracellular proteome. The extracellular proteome is a complex network of secretory and membrane-bound proteins, such as growth factors, cytokines, hormones, enzymes and receptors, that play key roles in cell signaling, immune response, tissue repair and metabolism. Given that many disease states are closely associated with abnormal levels of extracellular proteins, this collection of proteins is an attractive therapeutic target. In-depth analysis of the function and homeostasis of the extracellular proteome is of core value for the design of precision medicine strategies and interventions.
eTPD, as a cutting-edge technology, cleverly uses bispecial antibodies, coupled molecules or small molecule compounds to achieve selective clearance of extracellular proteins. Unlike the traditional mechanism of action of occupying drugs, these drugs do not simply bind and block the activity of the target protein, but use bispecic molecules to simultaneously lock the target protein and the recycling receptor on the cell surface, induce the endocytosis of the target protein, and then direct it to the lysosome for degradation. This innovative approach not only enhances targeting specificity and reduces the risk of non-specific effects but also renders previously "undruggable" proteins as potential therapeutic targets, opening a new realm in drug discovery, especially for extracellular proteins that are difficult to target with conventional drugs.
Differences between eTPD and iTPD
Although both are designed to interfere with disease processes by promoting protein degradation, eTPD and intracellular targeted protein degradation (iTPD) differ significantly on multiple levels, not only in the location of the target protein, but also in the cellular targeted protein degradation. The diversity of protein degradation mechanisms, kinetic characteristics and types of proteases are also involved.
Protein degradation pathway
iTPD focuses on the use of the proteasome system within the cell, specializing in the degradation of proteins inside the cell. eTPD, on the other hand, is mediated by bispecial biologics or small molecules to promote extracellular or membrane-bound proteins to bind to recycled receptors, and then be internalized and transported to lysosomes for degradation, which is specific to the processing of extracellular proteins.
Kinetics and protease
iTPD responds quickly, often within minutes to hours, thanks to the fact that all essential components are in place in the cytoplasm. In contrast, eTPD processes are slower, ranging from 6 to 48 hours, involving vesicle transport from the cell membrane to the endoplasmic reticulum until fusion with lysosomes to complete protein degradation, a series of steps that contribute to its unique kinetic properties.
Diversity and tissue specificity of E3 ligases
iTPD relies on cereblon (CRBN) and von hippel - lindau protein (VHL), two E3 ligases that are widely expressed in a variety of tissues, setting the threshold for the development of tissee-specific degraders. However, with further research, more than 600 E3 ligases have been identified, and genome-wide screening has revealed a richer degradation mechanism, provided a more diverse degradation pathway for eTPD, enhanced protocol flexibility and tissue specificity, and provided a broader strategic space for the treatment of specific diseases.
Research progress in eTPD
In the field of extracellular targeted protein degradation, the degradation of soluble and membrane-bound protein targets has garnered significant interest. Researchers have developed various protein degradation strategies that leverage different natural recycling and internalization mechanisms to achieve effective degradation of these target proteins.
Sweeping antibodies
"Sweeping antibodies" are engineered to enhance their binding to Fc recycling receptors, neonatal Fc receptors (FcRn), or FcγRIIb. Additionally, they have pH-sensitive complementary determining regions that bind to the target protein (POI) at neutral pH but release the target protein after internalization into acidified endosomes. This process leads to the target protein being transported to the lysosome and degraded. The antibodies bound to Fc receptors are then recycled to the cell surface, where they can continue to capture more target proteins.
Fig. 1 scheme of sweeping antibodies eTPD. (Wells, J. A., 2024)
Targeting glycan receptors
Non-cation-dependent mannose 6-phosphate receptors (CI-M6PR) or asialoglycoprotein receptors (ASGPR) can promote the lysosomal degradation of membrane and soluble proteins. A feasible approach is to conjugate multiple glycan ligands to antibodies targeting specific proteins to create lysosomal targeting chimeras (LYTACs). LYTAC-POI complexes bind to CI-M6PR, get internalized, and are transported to lysosomes, resulting in POI degradation. Researchers have developed similar biologics and bifunctional small molecule constructs to use ASGPR for protein degradation.
Utilizing bispecific antibodies for transmembrane E3 ligase development
This method involves using bispecific antibodies to develop and utilize transmembrane E3 ligases. One arm of these antibodies binds to the extracellular domain of an E3 ligase, while the other binds to the extracellular domain of a transmembrane POI. These constructs are called antibody-based protein degradation targeting chimeras (AbTACs), protein degradation targeting antibodies (PROTABs), or receptor elimination by E3 ubiquitin ligase recruitment (REULR). This approach brings the degrader close to the POI, triggering ubiquitination of the POI's intracellular domain and directing it to the lysosome or proteasome for degradation.
Utilizing bispecific antibody constructs to bind recycling receptors of natural cytokines and growth factors
Cytokine receptor targeting chimeras (KineTACs) are bispecific antibody constructs where one arm binds to a natural cytokine or growth factor and the other specifically binds to the POI. This allows KineTACs to target membrane-bound and soluble proteins for efficient and specific degradation.
Fig. 2 eTPD targeting membrane proteins based on cytokines. (Wells, J. A., 2024)
Using integrin-promoted lysosomal degradation technology
This method involves using bispecific antibodies where one arm specifically binds to integrins and the other arm binds to POI. This approach, called integrin-promoted lysosomal degradation, effectively directs POI to the lysosome for degradation while leveraging integrins' recycling capabilities to enhance target localization and efficiency.
Using small molecule protein degradation chimeras
PROTACs are commonly used for the degradation of intracellular soluble proteins, but some research groups have also utilized PROTACs targeting receptor tyrosine kinases (RTK) containing CRBN or VHL ligand, which are typically transmembrane proteins. It consists of an extracellular domain, a transmembrane region, and an intracellular kinase domain. For example, C4 Therapeutics' EGFR small molecule depressant (L858R) CFT8919 has received investigational new drug approval from FDA. The application of PROTACs to transmembrane proteins broadens the scope of application.
Fig. 3 Six different methods for eTPD. (Wells, J. A., 2024)
Ligand for VHL and CRBN E3 ligases at BOC Sciences
In addition to antibody and small molecule-based eTPD strategies, several biologic-based targeted protein degradation methods have been reported. These methods aim to degrade intracellular and membrane-associated target proteins, including the "Trim-away" system, biologic protein degradation targeting chimeras (bioPROTACs), nanobody-based PROTACs, and protease engineering to target cancer-related mucins.
The rise of eTPD represents a major breakthrough in drug discovery, providing new avenues for selective regulation and therapeutic application of the extracellular proteome. Given its potential applications in cancer therapy, immune modulation, neurodegenerative diseases, and infectious diseases, eTPD is emerging as a versatile therapeutic strategy with broad significance. Looking ahead, eTPD research will focus on achieving multi-targeting, enhancing tissue selectivity, and promoting clinical application development. Addressing resistance mechanisms, reducing off-target effects, and achieving commercialization will be key to realizing the potential of eTPD therapies.
Reference:
- Wells, J. A.; Kumru, K. Extracellular targeted protein degradation: an emerging modality for drug discovery. Nature Reviews Drug Discovery. 2024, 23(2): 126-140.
