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Updated: Feb 28, 2026
Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation
Published on: August 21, 2017
Targeted shedding of extracellular membrane proteins by induced protease recruitment
Zi Yao1, Fangzhu Zhao1, Kun Miao1
1Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA, USA.
Abstract:
Extracellular targeted protein degradation has emerged as a promising therapeutic modality to eliminate proteins of interest (POIs) at the cell surface, by using bifunctional molecules to recruit natural recycling receptors or membrane-bound E3 ligases that redirect POIs to the lysosome. Another natural mechanism involves extracellular proteases that cleave and shed extracellular domains. Here, we exploit this endogenous mechanism by engineering bispecific antibody Shedders, that recruit a classic sheddase ADAM10 to POIs, inducing selective ectodomain shedding. We first targeted the immune checkpoint receptor LAG-3 and observed robust depletion of surface LAG-3 accompanied by accumulation of soluble LAG-3 fragments in both engineered cell lines and primary human T cells. Using biochemical and imaging assays, we confirmed that this antibody-induced shedding is restricted to extracellular protease activity and occurs independently of lysosomal trafficking. Notably, induced shedding of LAG-3 on activated primary T cells partially alleviated inhibitory signaling and reinvigorated IFNγ secretion. We extended the scope of induced shedding by developing Shedders that recognize synthetic epitope-tags that enabling rapid assessment of substrate compatibility across diverse targets. Using this platform, we identified multiple immune modulatory cell-surface receptors, including IL6Rα, CD62L and MIC-A that can be targeted for shedding. In summary, this work establishes a new paradigm for targeted extracellular proteolysis and expands the toolkit for studying extracellular proteolysis with potential therapeutic benefit.
Insights
Researchers developed "Shedders," bispecific antibodies that use natural proteases to remove cell surface proteins like LAG-3. This approach offers a new way to target extracellular proteins for therapeutic benefit.
Area of Science:
- Biochemistry
- Immunology
- Proteolysis
Background:
- Extracellular protein degradation is a therapeutic strategy targeting cell surface proteins.
- Existing methods often rely on lysosomal pathways.
- Natural extracellular proteases offer an alternative degradation mechanism.
Purpose of the Study:
- To engineer bispecific antibodies, termed "Shedders," that hijack endogenous sheddases to induce targeted extracellular protein degradation.
- To investigate the mechanism and efficacy of antibody-induced ectodomain shedding.
- To explore the therapeutic potential of this approach for immune modulation.
Main Methods:
- Engineering bispecific antibodies ("Shedders") to recruit ADAM10 sheddase to target proteins.
- Targeting the immune checkpoint receptor LAG-3 on engineered cell lines and primary human T cells.
- Utilizing biochemical assays, imaging, and functional assays to assess protein depletion, shedding, and T cell activity.
Main Results:
- Demonstrated robust depletion of surface LAG-3 and accumulation of soluble fragments.
- Confirmed antibody-induced shedding relies on extracellular protease activity, independent of lysosomal trafficking.
- Showed that shedding of LAG-3 on T cells partially reduced inhibitory signaling and enhanced IFN-γ secretion.
- Developed a platform for rapid assessment of substrate compatibility, identifying IL6Rα, CD62L, and MIC-A as targetable receptors.
Conclusions:
- Established a novel paradigm for targeted extracellular proteolysis using engineered "Shedders."
- Expanded the toolkit for studying extracellular proteolysis with potential therapeutic applications.
- Demonstrated the feasibility of targeting immune modulatory receptors for shedding.
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