The Second Generation LYTAC Technology

Targeted protein degradation is a very promising treatment, and it is also an important tool for discovering cell biology pathways and apoptosis mechanisms. Many methods for targeted protein degradation have been established, such as targeted induced protein degradation technology (PROTAC), autophagy targeted chimera (AUTAC), etc., but most of these methods target proteins in the cytoplasm. Extracellular proteins and membrane proteins are also key factors for cancer, aging, autoimmune diseases, etc. In order to target degradation of these proteins, Carolyn R. Bertozzi  from Stanford University established a lysosome targeting chimeras lytacs in 2020 (LYTACs), chimeras include small molecules or antibodies that bind to the target, and mannose-6-containing mannose-6-containing molecules that bind to the CI-M6PR receptor in the cell surface lysosomal targeted receptor family (LTRs). Phosphate branched glycopeptide ligands. Although CI-M6PR is widely expressed in most tissues, some LTR family proteins are expressed tissue-specifically, which gives the LYTAC technology the possibility of tissue-specific treatment.

Recently, Bertozzi ’s research group has developed the LYTAC technology that uses ASGPR, a transmembrane glycoprotein specifically expressed in the liver, as a lysosomal targeting receptor. This study was published in the journal Nature Chemical Biology with the title “LYTACs that engage the asialoglycoprotein receptor for targeted protein degradation”.

Previous studies have shown that the tri-acetylglucosamine ligand (tri-GalNAc) ligand can bind to the ASGPR protein and is safer. Therefore, the author designed the tri-GalNAc-DBCO ligand (Figure 2) for one end Targeting the ASGPR protein, the alkynyl group at the other end can be connected to the azide-labeled small molecule or antibody through a click chemistry reaction. The authors proved that the ligand can enter liver cells through a fluorescent confocal microscope.

After that, the author chose to use the membrane protein EGFR, which is highly expressed in liver cancer cells, to detect the degradation effect of the method. The EGFR antibody Ctx was coupled to tri-GalNAc to treat the cells and found that it can degrade 70% of the EGFR on the cell surface. The first generation of LYTAC probes are similar, and knock down ASGPR to verify that its degradation is mediated by ASGPR.

In order to verify the specificity of the GalNAc-LYTAC method to degrade cells, the author co-cultured hepatocarcinoma cells HEP3B cells and Hela-GFP cells, and treated the cells with the first and second generation ligands connected to Ctx (Figure 3). It is found that Ctx-GalNAc can selectively degrade EGFR on the surface of HEP3B cells, while EGFR maintains high expression in Hela-GFP cells, while Ctx-M6Pn (first-generation ligand) degrades EGFR in both cells.

In addition to the EGFR protein, the authors also used HER2 protein for verification, indicating that ASFPR-mediated LYTAC has the ability to target other proteins. Although LYTAC mediates the transport of membrane proteins to lysosomes, it has been tested that this method will not disrupt the normal function of lysosomes.

Finally, the author used Ctx-GalNAc to validate the method in vivo. This method is less frequently administered than non-specific chimeras, so it is more advantageous in vivo, and the treatment method does not cause liver toxicity.

In summary, on the basis of the first generation of LYTAC technology, the author has developed a liver-specific protein degradation technology based on ASGPR protein, which is expected to serve as a new generation of targeted therapy.