Targeted protein degradation through site-specific antibody conjugation with mannose 6-phosphate glycan
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...
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| Format: | Article |
| Language: | English |
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Taylor & Francis Group
2024-12-01
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| Series: | mAbs |
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| Online Access: | https://www.tandfonline.com/doi/10.1080/19420862.2024.2415333 |
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| author | Kaori Mukai Robert Cost Xin Sheen Zhang Emily Condiff Joanne Cotton Xiaohua Liu Ekaterina Boudanova Björn Niebel Peter Piepenhagen Xinming Cai Anna Park Qun Zhou |
| author_facet | Kaori Mukai Robert Cost Xin Sheen Zhang Emily Condiff Joanne Cotton Xiaohua Liu Ekaterina Boudanova Björn Niebel Peter Piepenhagen Xinming Cai Anna Park Qun Zhou |
| author_sort | Kaori Mukai |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-1dec9122acc045ba9896b198c5b8e79d |
| institution | Kabale University |
| issn | 1942-0862 1942-0870 |
| language | English |
| publishDate | 2024-12-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | mAbs |
| spelling | doaj-art-1dec9122acc045ba9896b198c5b8e79d2025-01-31T04:19:38ZengTaylor & Francis GroupmAbs1942-08621942-08702024-12-0116110.1080/19420862.2024.2415333Targeted protein degradation through site-specific antibody conjugation with mannose 6-phosphate glycanKaori Mukai0Robert Cost1Xin Sheen Zhang2Emily Condiff3Joanne Cotton4Xiaohua Liu5Ekaterina Boudanova6Björn Niebel7Peter Piepenhagen8Xinming Cai9Anna Park10Qun Zhou11Immunology & Inflammation Research, Sanofi, Cambridge, MA, USALarge Molecules Research, Sanofi, Cambridge, MA, USATranslational In Vivo Models Research, Sanofi, Cambridge, MA, USATranslational In Vivo Models Research, Sanofi, Cambridge, MA, USACMC Bioanalytics, Sanofi, Framingham, USALarge Molecules Research, Sanofi, Cambridge, MA, USALarge Molecules Research, Sanofi, Cambridge, MA, USALarge Molecules Research, Sanofi R&D Ghent, Ghent, BelgiumTranslational In Vivo Models Research, Sanofi, Cambridge, MA, USAImmunology & Inflammation Research, Sanofi, Cambridge, MA, USALarge Molecules Research, Sanofi, Cambridge, MA, USALarge Molecules Research, Sanofi, Cambridge, MA, USARecent 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.https://www.tandfonline.com/doi/10.1080/19420862.2024.2415333bisM6P-conjugated antibodyCI-MPRLYTACM6Pprotein degradation |
| spellingShingle | Kaori Mukai Robert Cost Xin Sheen Zhang Emily Condiff Joanne Cotton Xiaohua Liu Ekaterina Boudanova Björn Niebel Peter Piepenhagen Xinming Cai Anna Park Qun Zhou Targeted protein degradation through site-specific antibody conjugation with mannose 6-phosphate glycan mAbs bisM6P-conjugated antibody CI-MPR LYTAC M6P protein degradation |
| title | Targeted protein degradation through site-specific antibody conjugation with mannose 6-phosphate glycan |
| title_full | Targeted protein degradation through site-specific antibody conjugation with mannose 6-phosphate glycan |
| title_fullStr | Targeted protein degradation through site-specific antibody conjugation with mannose 6-phosphate glycan |
| title_full_unstemmed | Targeted protein degradation through site-specific antibody conjugation with mannose 6-phosphate glycan |
| title_short | Targeted protein degradation through site-specific antibody conjugation with mannose 6-phosphate glycan |
| title_sort | targeted protein degradation through site specific antibody conjugation with mannose 6 phosphate glycan |
| topic | bisM6P-conjugated antibody CI-MPR LYTAC M6P protein degradation |
| url | https://www.tandfonline.com/doi/10.1080/19420862.2024.2415333 |
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