Lysosome-Targeting Chimera Using Mannose-6-Phosphate Glycans Derived from Glyco-Engineered Yeast.

Lysosome-targeting chimeras (LYTACs) harness the cell's lysosomal degradation machinery to break down extracellular and membrane proteins. Previous methods used a synthetic glycopeptide containing multiple serine-O-mannose-6-phosphate (poly-M6Pn), which presented challenges such as synthetic complexity and potential immunogenicity associated with poly-M6Pn. This study introduced a LYTAC formulation, LYTAC^gyM6pG, which uses glyco-engineered yeast-derived mannose-6-phosphate glycans (gyM6pGs) for lysosomal transport, overcoming synthetic complexities and immunogenic risks. The gyM6pGs used in LYTAC^gyM6pG are human-compatible (identical to the structures found in humans) and are efficiently produced through yeast fermentation, followed by the preparation of cell wall glycans and their in vitro modifications. We employed copper-free click chemistry (azide and dibenzocyclooctyne reactions) for the robust conjugation of gyM6pGs to a nanobody targeting the immune checkpoint protein PD-L1, thereby streamlining the assembly of LYTAC^gyM6pG. We demonstrated that LYTAC^gyM6pG effectively degraded endogenous and recombinant PD-L1 proteins on the cell surface by directing them to the lysosome via the cation-independent mannose-6-phosphate receptor pathway. Furthermore, LYTAC^gyM6pG significantly enhanced T cell-mediated cytotoxicity against cancer cells, surpassing the efficacy of nanobodies alone. The successful application of gyM6pGs in the development of LYTAC^gyM6pG highlights the potential for a more viable and scalable therapeutic production of LYTACs, paving the way for broader therapeutic applications, including cancer treatment.