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Based on Antibody Targeted Protein Degradation

In recent years, PROteolysis TArgeting Chimeras (PROTAC), which utilizes the cell’s own degradation mechanisms to eliminate specific disease-related proteins, has emerged as one of the most promising methods. Apart from PROTAC, various targeted protein degradation (TPD) strategies are emerging, involving molecular glue, Autophagy-Targeting Chimera (AUTEC), Autophagosome Tethering Compound (ATTEC), and Autophagy Targeting Chimera (AUTOTAC). These compounds are generally bifunctional small molecules, consisting of two parts: one part contains an E3 recruitment ligand to activate the ubiquitination cascade, and the other part contains a head that targets the protein of interest (POI), assembled using an appropriate linker. Once inside the body, a POI-PROTAC-E3 ternary complex is formed, inducing POI ubiquitination and subsequent degradation through the ubiquitin-proteasome system (UPS). In many cases, their relatively large molecular weight (usually > 1000 Da) may affect oral bioavailability, solubility, and/or in vivo pharmacokinetic properties (not conforming to the Lipinski’s Rule of Five). Additionally, certain intracellular proteins may be resistant to such molecules because they might not be substrates for proteasomal clearance. To address these issues and complement PROTAC, antibodies, with their high selectivity and affinity for any immunogenic target (in principle) and their successful commercialization, have been extensively applied to lysosome-based degradation strategies. Depending on their composition, this class of molecules includes antibody-based PROTAC (AbTAC/PROTAB), Lysosome-Targeting Chimeras (LYTAC), GlueBody Targeting Chimera (GlueTAC), Cytokine Receptor-Targeting Chimeras (KineTACs), and Signal-Mediated Lysosome-Targeting Chimeras (SignalTACs). These technologies not only significantly expand the scope of TPD but also offer new insights into antibody drug discovery.


Signal-Mediated Lysosome-Targeting Chimera (SignalTAC)

Membrane protein degradation technology relies on specific cell surface lysosome-targeting receptors to exert its effects. However, the expression of these internalizing receptors varies depending on tissue and cell type, which poses significant limitations to their further application. The internalization of lysosome-targeting receptors is a complex process mediated by signals within the receptor’s cytoplasmic domain. Most signals are based on double leucine or tyrosine-based peptide sequences, which are recognized by components of the clathrin coat to ensure the precise transport of proteins to lysosomes through clathrin-mediated endocytosis. Among these, the tyrosine-based lysosome sorting signal NPXY (Asn-Pro-X-Tyr, where X represents any amino acid) in the low-density lipoprotein receptor (LDLR) can induce the internalization and lysosomal transport of anti-EGFR nanobodies. Therefore, based on this research, Professor Xiaoqing Cai’s research group at Sun Yat-sen University proposed that membrane protein complexes with lysosome sorting signals could form SignalTACs for protein degradation. After binding to membrane proteins, SignalTACs dissociate from the membrane protein in the acidic endosomal compartment and enter lysosomes for degradation.


The Cation-Independent Mannose-6-Phosphate Receptor (CI-M6PR) is a single transmembrane glycoprotein that mediates the internalization of extracellular ligands and the intracellular sorting of newly synthesized lysosomal enzymes. Its efficacy does not depend on any lysosome shuttle receptor. Therefore, SignalTAC technology was developed using CI-M6PR’s inherent lysosome sorting signal. The internalization and sorting of CI-M6PR are guided by a signal based on double leucine (composed of a cluster of acidic amino acids and subsequent double leucine). Based on this information, a series of IgG/Nb-based SignalTACs was constructed, where the CI-M6PR sorting signal was fused to the C-terminus of IgG antibodies using heavy chains, light chains, or both. Among these, Nb1 showed significantly enhanced cell fluorescence compared to the parental 5F7. Additionally, SignalTACs containing the RRRRK sequence, based on Trastuzumab (Tz) modification (Ab6), exhibited the highest internalization capability. Structure-activity relationship studies indicated that the double leucine-based sequence DEDLL (Asp7-Ile11) is critical for its function. Moreover, truncation of 13 residues at P1 of the sequence was found to synergistically enhance Nb1 internalization.


The use of IgG-type SignalTACs to treat HER2+ cell lines (SKBR3, BT474, and SKOV3) showed that SignalTACs Ab1-Ab6 induced degradation in all three cell lines. Further investigation of the degradation effects of Ab6 revealed that the maximum protein degradation occurred after treating SKBR3 cells with a concentration of 100 nM for 48 hours. Mechanistic studies indicated that clathrin-mediated endocytosis is a key pathway for SignalTAC-induced degradation. However, further research is needed to fully understand the molecular mechanism of SignalTAC-induced degradation. In addition to causing degradation of the target protein HER2, SignalTAC also downregulates downstream signaling (PI3K/AKT/mTOR) and promotes apoptosis and inhibits the proliferation of HER2-driven cancer cells.


Cytokine Receptor-Targeting Chimeras (KineTACs)

Due to limitations in lysosome delivery targeting chimeras, such as low modularity, high development difficulty, poor practicality, and limited tissue specificity, Professor James A. Wells’ team at the University of California, San Francisco (UCSF) developed KineTACs. KineTACs are fully genetically encoded bispecific antibodies composed of a cell factor arm that binds to its homologous cytokine receptor and a binding arm that targets the desired protein. The construction of bispecific antibodies in the KineTAC class is not complicated by light-heavy chain mismatches, a common problem in traditional bispecific antibodies. Furthermore, the KineTAC platform only requires the design of the antigen-binding arm for the target protein, as natural cytokines can recruit the relevant degradation receptors.


To validate the KineTAC platform design concept, the authors developed KineTAC bispecific antibodies targeting different target proteins (PD-L1, HER2, PD-1, EGFR, CDCP1, and TROP2). The N-terminus of the human CXCL12 chemokine was fused to the Fc domain of the bispecific antibody to form the cytokine-binding arm, and the antigen-binding arm contained antigen-binding fragment (Fab) antibody sequences fused to the Fc domain, consistent with a regular antibody. The six KineTACs demonstrated maximum degradation efficiencies (Dmax) of approximately 70%, 51%, 84%, 82%, 93%, and 51%, respectively, demonstrating that the KineTAC platform can degrade a variety of cell surface proteins, offering good technical versatility.


Pretreatment with Bafilomycin (a lysosome inhibitor) reduced PD-L1 degradation, while MG132 (a proteasome inhibitor) treatment did not affect PD-L1 degradation. This suggests that KineTAC mediates degradation by delivering the target protein to lysosomes. Based on the previous research, CXCR7 is the primary receptor responsible for KineTAC-mediated degradation, and alternative cytokines (such as CXCL11 and vMIPII) were also shown to degrade target proteins. Compared to traditional antibody therapies, KineTAC-mediated degradation offers functional advantages as traditional antibodies only bind and inhibit without causing degradation. Pharmacokinetic experiments in vivo showed that KineTAC remained in the plasma for up to 10 days after injection, with a half-life of 8.7 days, similar to the reported half-life of mouse IgG.

While KineTAC demonstrated excellent results in in vitro experiments, its in vivo experiments have not been extensively explored. Only pharmacokinetic studies have been presented, leaving room for further animal experiments to validate whether the in vitro effective dose might lead to potential “cytokine storm” side effects in vivo due to the intensity of cytokine action in vivo.


Proteolysis-Targeting Antibodies (PROTABs)

In 2021, Professor James A. Wells and his team developed AbTACs, and published their findings in JACS. They utilized fully recombinant bispecific antibody AC-1 to recruit the membrane-binding E3 ligase RNF43 for the degradation of the cell surface protein PD-L1, achieving a Dmax of 63%. Although AbTAC represents a novel type in the PROTAC field, using fully recombinant biomolecules for targeting the degradation of cell surface proteins, its industry impact is far less than that of Genentech’s PROTAB.


The Genentech-developed PROTAB platform technology constructs bispecific antibodies remarkably similar to those developed by Professor James A. Wells’ team. One end targets the E3 ligase N-terminal glycoprotein D site, while the other end binds to the target protein, facilitating degradation. The Genentech team delved into its mechanism of action and found that PROTAB can participate in two degradation pathways: proteasomal and lysosomal. MG132 and Baf can both influence the degradation of the target protein, unlike several other TPD technologies discussed earlier. It also expanded the application scope of the PROTAB technology platform, demonstrating the replicability of PROTAB for different target proteins. Moreover, they optimized the assembly forms of PROTAB to investigate variations in protein degradation efficiency. Although PROTAB exhibits a Hook effect similar to PROTAC, it is believed that combining modular antibody engineering technology can expedite its translation, making more significant contributions to drug development.


Lysosome Targeting Chimeras (LYTACs)

LYTACs use IgG-polysaccharide bioconjugates to effectively clear target proteins. This involves the chemical synthesis and in vitro conjugation of polysaccharides to enable efficient target protein clearance. The mechanism of action is illustrated in Fig. 6, where the target protein ligand portion binds to the extracellular domain of the target protein. Simultaneously, polysaccharides bind to lysosome-targeting receptors (LTRs) on the cell surface, forming a ternary complex. This complex enters cells via clathrin-mediated endocytosis and is transported to endolysosomes. As the endolysosomes become acidified, the ternary complex continues to be transported to lysosomes for degradation. Similar to SignalTAC, the authors targeted CI-M6PR as the receptor and conjugated M6Pn polysaccharide peptides to antibodies that specifically recognize CI-M6PR to create LYTACs with specificity for the degradation of membrane proteins like EGFR and CD71. Constructing LYTACs using this method is intricate, requiring in vitro conjugation reactions. The introduced polysaccharide peptides may have certain immunogenicity, leading to accelerated clearance and reduced degradation efficiency.


GlueBody Targeting Chimeras (GlueTACs)

GlueTAC technology was developed by Professor Peng Chen’s team at Peking University and is based on covalent Nb-PROTAC technology. The covalently modified single-domain antibody, GlueBody (modified with proximity-enabled non-natural amino acids PrUAAs), can form a covalent bond with membrane protein antigens like PD-L1 through a proximity-enabled reaction, reducing the dissociation and escape of the target protein during endocytosis and degradation. Additionally, cell-penetrating peptides (CPPs) and lysosome-sorting sequences (LSS) covalently linked to the single-domain antibody facilitate the internalization and lysosomal transport of complexes without the need for specific cell surface proteins, ultimately achieving the targeted degradation of membrane proteins in lysosomes.


The GlueTACs constructed using this method were assessed for their degradation effect on target proteins using a non-small-cell lung cancer cell line, H460. Western blot analysis demonstrated a significantly higher rate of PD-L1 degradation induced by GlueTAC (remaining 11%) compared to NbTAC (remaining 68%), highlighting the crucial role of covalent bonds in target protein degradation. Atezolizumab, Nb-PD-L1, and GlueBody showed lower degradation rates, indicating that individual antibodies or Nb might not be sufficient for target protein degradation, and PD-L1 might be reinternalized and recirculated to the cell membrane after endocytosis.

Studies into the degradation mechanism of GlueTACs revealed that treatment with Chloroquine/Bafilomycin significantly reduced PD-L1 degradation, whereas MG132 treatment did not, suggesting that the degradation process depends on the endolysosome pathway. In vivo experiments showed that both GlueBody and GlueTAC treatment significantly inhibited tumor growth, with tumors treated with GlueTAC having a lower average weight compared to those treated with GlueBody. Based on in vivo results, GlueBody performed slightly better than drug treatment, and the difference between GlueBody and GlueTAC groups was minimal. This might be related to the efficiency of the proximity-enabled covalent binding used in GlueBody. Changing the conjugation method might help enhance the anti-tumor activity of GlueTAC. Replacing GlueBody with antibodies might also improve its anticancer activity, as Fc and FcR interactions contribute to its efficacy. However, this approach might result in some non-specific conjugation, potentially causing the degradation of non-target proteins and side effects. Additionally, the introduction of PrUAA might induce some immunogenicity, leading to faster metabolism in in vivo experiments, masking its therapeutic effect. Overall, GlueTAC technology opens up an important new direction for antibody drug development.


Challenges in the Development of TPD

The development of PROTAC-like technologies still faces numerous challenges. Firstly, there are issues related to their drug-like properties, often including poor cell permeability and low oral bioavailability. Secondly, while the human genome encodes over 600 E3 ubiquitin ligases, only a few (such as VHL, CRBN, IAP, and MDM2) are used for degrading target proteins, and the scope of applications needs further expansion. Additionally, it’s worth noting the toxicity concern. PROTACs might potentially exhibit greater toxicity than small molecule inhibitors because they lead to the degradation of the entire target protein rather than merely inhibiting it. Controlling the degree of degradation is an urgent issue in the TPD field.

The development of TPD technologies based on lysosomes has significantly broadened the range of target proteins compared to PROTACs and molecular glues, leading to a surge in research interest in this field. However, it is still in its early stages. Lysosomes, as essential organelles, regulate many important physiological functions aside from protein degradation. It remains unclear whether the “hijacking” of lysosomal degradation pathways may affect the organism’s physiological functions. Additionally, the degradation of target proteins may lead to negative feedback regulation in cascading signaling pathways or overactivation of bypass signals, potentially causing more severe side effects, an area where research has yet to be conducted.

Furthermore, it is uncertain whether the excessive activation of specific receptor internalization functions may impair their original biological functions and lead to acquired drug resistance. Despite these challenges, the development of TPD offers a powerful tool for biomedical research and holds great promise for the future of drug development.



  1. Han, Y., et al., Protein labeling approach to improve lysosomal targeting and efficacy of antibody–drug conjugates, Biomol. Chem., 2020, 18, 3229-3233.
  2. Yu, J., et al., Harnessing the Lysosomal Sorting Signals of the Cation-Independent Mannose-6-Phosphate Receptor for Targeted Degradation of Membrane Proteins, Am. Chem. Soc., 2023, 145, 34, 19107–19119.
  3. Pance, K., et al., Modular cytokine receptor-targeting chimeras for targeted degradation of cell surface and extracellular proteins, Nature Biotechnology, 2023. 41, 273-281.
  4. Cotton, A.D., et al., Development of Antibody-Based PROTACs for the Degradation of the Cell-Surface Immune Checkpoint Protein PD-L1, Am. Chem. Soc., 2021, 143, 593-598.
  5. Banik, S.M., et al., Lysosome-targeting chimaeras for degradation of extracellular proteins. Nature, 2020. 584(7820): 291-297.
  6. Zhang, H., et al., Covalently Engineered Nanobody Chimeras for Targeted Membrane Protein Degradation, Am. Chem. Soc.,2021. 143(40),16377-16382.



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