Targeted protein degradation through site-specific antibody conjugation with mannose 6-phosphate glycan

• Published in: mAbs Vol. 16; no. 1; p. 2415333
• Main Authors: Mukai, Kaori, Cost, Robert, Zhang, Xin Sheen, Condiff, Emily, Cotton, Joanne, Liu, Xiaohua, Boudanova, Ekaterina, Niebel, Björn, Piepenhagen, Peter, Cai, Xinming, Park, Anna, Zhou, Qun
• Format: Journal Article
• Language: English
• Published: United States Taylor & Francis 31.12.2024; Taylor & Francis Group
• Subjects: Animals; Antibodies, Monoclonal - chemistry; Antibodies, Monoclonal - immunology; bisM6P-conjugated antibody; CI-MPR; Glycosylation; Humans; Immunoconjugates - chemistry; Immunoconjugates - pharmacology; Lysosomes - metabolism; LYTAC; M6P; Mannosephosphates - chemistry; Mannosephosphates - metabolism; Polysaccharides - chemistry; Polysaccharides - metabolism; protein degradation; Protein Engineering - methods; Proteolysis; Receptor, IGF Type 2 - chemistry; Receptor, IGF Type 2 - metabolism; LYTAC; bisM6P-conjugated antibody; M6P; protein degradation; CI-MPR
• ISSN: 1942-0862; 1942-0870; 1942-0870
• DOI: 10.1080/19420862.2024.2415333

Recent developments in targeted protein degradation have provided great opportunities to eliminating extracellular protein targets using potential therapies with unique mechanisms of action and pharmacology. Among them, Lysosome-Targeting Chimeras (LYTACs) acting through mannose 6-phosphate receptor (M6PR) have been shown to facilitate degradation of several soluble and membrane-associated proteins in lysosomes with high efficiency. Herein we have developed a novel site-specific antibody conjugation approach to generate antibody mannose 6-phosphate (M6P) conjugates. The method uses a high affinity synthetic M6P glycan, bisM6P, that is coupled to an Fc-engineered antibody NNAS. This mutant without any effector function was generated by switching the native glycosylation site from position 297 to 298 converting non-sialylated structures to highly sialylated N-glycans. The sialic acid of the glycans attached to Asn298 in the engineered antibody was selectively conjugated to bisM6P without chemoenzymatic modification, which is often used for site-specific antibody conjugation through glycans. The conjugate is mainly homogeneous by analysis using mass spectrometry, typically with one or two glycans coupled. The M6P-conjugated antibody against a protein of interest (POI) efficiently internalized targeted soluble proteins, such as human tumor necrosis factor (TNF), in both cancer cell lines and human immune cells, through the endo-lysosomal pathway as demonstrated by confocal microscopy and flow cytometry. TNF in cell culture media was significantly depleted after the cells were incubated with the M6P-conjugated antibody. TNF internalization is mediated through M6PR, and it is correlated well with cell surface expression of cation-independent M6PR (CI-MPR) in immune cells. A significant amount of CI-MPR remains on the cell surface, while internalized TNF is degraded in lysosomes. Thus, the antibody-M6P conjugate is highly efficient in inducing internalization and subsequent lysosome-mediated protein degradation. Our platform provides a unique method for producing biologics-based degraders that may be used to treat diseases through event-driven pharmacology, thereby addressing unmet medical needs.