Synthetic Antibody‐Rhamnose Cluster Conjugates Show Potent Complement‐Dependent Cell Killing by Recruiting Natural Antibodies

Ou, Chong; Prabhu, Sunaina Kiran; Zhang, Xiao; Zong, Guanghui; Yang, Qiang; Wang, Lai-Xi

doi:10.1002/chem.202200146

Cited by 15 publications

(8 citation statements)

References 36 publications

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“…We have previously described the use of a chemoenzymatic Fc glycan remodeling approach to install drugs and mannose-6-phosphate glycan ligands site-specifically on monoclonal antibodies. ,− For example, we have reported that M6P-modified LYTACs prepared through this chemoenzymatic method can degrade transmembrane proteins effectively through the CI-M6PR receptor . We sought to use this chemoenzymatic Fc glycan remodeling method to site-specifically install natural and synthetic glycan ligands of ASGPR to the Fc domain of an anti-PCSK9 antibody, which has not been explored before.…”

Section: Resultsmentioning

confidence: 99%

Synthetic Site-Specific Antibody–Ligand Conjugates Promote Asialoglycoprotein Receptor-Mediated Degradation of Extracellular Human PCSK9

Donahue

Yang

et al. 2023

ACS Chem. Biol.

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Targeted degradation using cell-specific lysosome targeting receptors is emerging as a new therapeutic strategy for the elimination of disease-associated proteins. The liver-specific human asialoglycoprotein receptor (ASGPR) is a particularly attractive lysosome targeting receptor leveraged for targeted protein degradation (TPD). However, the efficiency of different glycan ligands for ASGPR-mediated lysosomal delivery remains to be further characterized. In this study, we applied a chemoenzymatic Fc glycan remodeling method to construct an array of site-specific antibody–ligand conjugates carrying natural bi- and tri-antennary N-glycans as well as synthetic tri-GalNAc ligands. Alirocumab, an anti-PCSK9 (proprotein convertase subtilisin/kexin type 9) antibody, and cetuximab (an anti-EGFR antibody) were chosen to demonstrate the ASGPR-mediated degradation of extracellular and membrane-associated proteins, respectively. It was found that the nature of the glycan ligands and the length of the spacer in the conjugates are critical for the receptor binding and the receptor-mediated degradation of PCSK9, which blocks low-density lipoprotein receptor (LDLR) function and adversely affects clearance of low-density lipoprotein cholesterol. Interestingly, the antibody–tri-GalNAc conjugates showed a clear hook effect for its binding to ASGPR, while antibody conjugates carrying the natural N-glycans did not. Both the antibody–tri-antennary N-glycan conjugate and the antibody–tri-GalNAc conjugate could significantly decrease extracellular PCSK9, as shown in the cell-based assays. However, the tri-GalNAc conjugate showed a clear hook effect in the receptor-mediated degradation of PCSK9, while the antibody conjugate carrying the natural N-glycans did not. The cetuximab–tri-GalNAc conjugates also showed a similar hook effect on degradation of the membrane-associated protein, epidermal growth factor receptor (EGFR). These results suggest that the two types of ligands may involve a distinct mode of interactions in the receptor binding and target-degradation processes. Interestingly, the alirocumab–tri-GalNAc conjugate was also found to upregulate LDLR levels in comparison with the antibody alone. This study showcases the potential of the targeted degradation strategy against PCSK9 for reducing low-density lipoprotein cholesterol, a risk factor for heart disease and stroke.

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Section: Resultsmentioning

confidence: 99%

Synthetic Site-Specific Antibody–Ligand Conjugates Promote Asialoglycoprotein Receptor-Mediated Degradation of Extracellular Human PCSK9

Donahue

Yang

et al. 2023

ACS Chem. Biol.

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“…18,19 Once cancer cells are rhamnose-tagged, anti-Rha Abs from human serum recognize the cells and activate downstream immune pathways, such as the complement cascade, leading to cancer cell death in vitro and in vivo. 18,19 Here we describe CB2, a Nb discovered by serendipity that specifically binds to lymphoma and breast cancer via a novel thiol-dependent binding mode, followed by internalization of the Nb. We show that CB2 binding and internalization correlate with higher surface levels of reduced cysteines on lymphoma cells compared to healthy lymphocytes and that CB2 can be easily functionalized for different applications.…”

Section: ■ Introductionmentioning

confidence: 99%

“…To exploit these naturally occurring anti-Rha Abs for cancer therapy, cancer cells have been labeled with rhamnose using rhamnose-functionalized liposomes or antibodies. 18,19 Once cancer cells are rhamnose-tagged, anti-Rha Abs from human serum recognize the cells and activate downstream immune pathways, such as the complement cascade, leading to cancer cell death in vitro and in vivo. 18,19 Here we describe CB2, a Nb discovered by serendipity that specifically binds to lymphoma and breast cancer via a novel thiol-dependent binding mode, followed by internalization of the Nb.…”

Section: ■ Introductionmentioning

confidence: 99%

“…In human blood, endogenous Abs against a variety of small molecules and glycans are highly abundant because these molecules are recognized as nonself by the immune system. , For example, rhamnose (Rha) is found in many bacterial polysaccharides, and humans produce anti-Rha Abs due to constant exposure to bacteria in their environment. To exploit these naturally occurring anti-Rha Abs for cancer therapy, cancer cells have been labeled with rhamnose using rhamnose-functionalized liposomes or antibodies. , Once cancer cells are rhamnose-tagged, anti-Rha Abs from human serum recognize the cells and activate downstream immune pathways, such as the complement cascade, leading to cancer cell death in vitro and in vivo. , …”

Section: Introductionmentioning

confidence: 99%

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Thiol-Mediated Uptake of a Cysteine-Containing Nanobody for Anticancer Drug Delivery

et al. 2023

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The identification of tumor-specific biomarkers is one of the bottlenecks in the development of cancer therapies. Previous work revealed altered surface levels of reduced/oxidized cysteines in many cancers due to overexpression of redox-controlling proteins such as protein disulfide isomerases on the cell surface. Alterations in surface thiols can promote cell adhesion and metastasis, making thiols attractive targets for treatment. Few tools are available to study surface thiols on cancer cells and exploit them for theranostics. Here, we describe a nanobody (CB2) that specifically recognizes B cell lymphoma and breast cancer in a thiol-dependent manner. CB2 binding strictly requires the presence of a nonconserved cysteine in the antigen-binding region and correlates with elevated surface levels of free thiols on B cell lymphoma compared to healthy lymphocytes. Nanobody CB2 can induce complement-dependent cytotoxicity against lymphoma cells when functionalized with synthetic rhamnose trimers. Lymphoma cells internalize CB2 via thiol-mediated endocytosis which can be exploited to deliver cytotoxic agents. CB2 internalization combined with functionalization forms the basis for a wide range of diagnostic and therapeutic applications, rendering thiol-reactive nanobodies promising tools for targeting cancer.

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“…For example, rhamnose (Rha) is found in many bacterial polysaccharides, and humans produce anti-Rha Abs due to constant exposure to bacteria in their environment. To exploit these naturally occurring anti-Rha Abs for cancer therapy, cancer cells have been labeled with rhamnose using rhamnose-functionalized liposomes or antibodies 18,19 . Once cancer cells are rhamnose-tagged, anti-Rha Abs from human serum recognize the cells and activate downstream immune pathways, such as the complement cascade, leading to cancer cell death in vitro and in vivo 18,19 .…”

Section: Introductionmentioning

confidence: 99%

Thiol-mediated Uptake of a Cysteine-containing Nanobody for Anti-Cancer Drug Delivery

Goerdeler

Reuber

Lühle

et al. 2022

Preprint

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The identification of tumor-specific biomarkers is one of the bottlenecks in the development of cancer therapies. Previous work revealed altered surface levels of reduced/oxidized cysteines in many cancers due to overexpression of redox-controlling proteins such as protein disulfide isomerases on the cell surface. Alterations in surface thiols can promote cell adhesion and metastasis, making thiols attractive targets for treatment. Only a few tools are available to study surface thiols on cancer cells and exploit them for theranostics. Here, we describe a nanobody (CB2) that recognizes B cell lymphoma in a thiol-dependent manner. CB2 binding strictly requires the presence of a non-conserved cysteine in the antigen-binding region and correlates with elevated surface levels of free thiols on B cell lymphoma compared to healthy lymphocytes. Nanobody CB2 can induce complement-dependent cytotoxicity against lymphoma cells when functionalized with synthetic rhamnose trimers. Lymphoma cells internalize CB2 in a thiol-mediated manner such that the nanobody can be used to deliver cytotoxic agents. Hence, surface thiols can be used as lymphoma biomarkers and targeted by thiol-binding nanobodies. Functionalization of internalizable CB2 is the basis for a range of diagnostic and therapeutic applications of this thiol-binding nanobody.TOC GraphicSynopsisNanobody CB2 specifically binds and internalizes into B cell lymphoma via thiol-based interactions. Functionalized CB2 can be used for complement recruitment or drug delivery to lymphoma cells.

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Synthetic Antibody‐Rhamnose Cluster Conjugates Show Potent Complement‐Dependent Cell Killing by Recruiting Natural Antibodies

Cited by 15 publications

References 36 publications

Synthetic Site-Specific Antibody–Ligand Conjugates Promote Asialoglycoprotein Receptor-Mediated Degradation of Extracellular Human PCSK9

Synthetic Site-Specific Antibody–Ligand Conjugates Promote Asialoglycoprotein Receptor-Mediated Degradation of Extracellular Human PCSK9

Thiol-Mediated Uptake of a Cysteine-Containing Nanobody for Anticancer Drug Delivery

Thiol-mediated Uptake of a Cysteine-containing Nanobody for Anti-Cancer Drug Delivery

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