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Generation of Site‐Specifically Labeled Affinity Reagents via Use of a Self‐Labeling Single Domain Antibody
https://orcid.org/0000-0003-3313-2934
https://orcid.org/0000-0001-9871-8333
AbstractSeveral chemical and enzymatic methods have been used to link antibodies to moieties that facilitate visualization of cognate targets. Emerging evidence suggests that the extent of labeling, dictated by the type of chemistry used, has a substantial impact on performance, especially in the context of antibodies used for the visualization of tumors in vivo. These effects are particularly pronounced in studies using small antibody fragments, such as single‐domain antibodies, or nanobodies. Here, we leverage a new variety of conjugation chemistry, based on a nanobody that forms a crosslink with a specialized high‐affinity epitope analogue, to label target‐specific nanobody constructs with functionalities of choice, including fluorophores, chelators, and click chemistry handles. Using heterodimeric nanobody conjugates, comprised of an antigen recognition module and a self‐labeling module, enables us to attach the desired functional group at a location distal to the site of antigen recognition. Constructs generated using this approach bound to antigens expressed on xenograft murine models of liver cancer and allowed for non‐invasive diagnostic imaging. The modularity of our approach using a self‐labeling nanobody offers a novel method for site‐specific functionalization of biomolecules and can be extended to other applications for which covalent labeling is required.
Generation of Site‐Specifically Labeled Affinity Reagents via Use of a Self‐Labeling Single Domain Antibody
https://orcid.org/0000-0003-3313-2934
https://orcid.org/0000-0001-9871-8333
AbstractSeveral chemical and enzymatic methods have been used to link antibodies to moieties that facilitate visualization of cognate targets. Emerging evidence suggests that the extent of labeling, dictated by the type of chemistry used, has a substantial impact on performance, especially in the context of antibodies used for the visualization of tumors in vivo. These effects are particularly pronounced in studies using small antibody fragments, such as single‐domain antibodies, or nanobodies. Here, we leverage a new variety of conjugation chemistry, based on a nanobody that forms a crosslink with a specialized high‐affinity epitope analogue, to label target‐specific nanobody constructs with functionalities of choice, including fluorophores, chelators, and click chemistry handles. Using heterodimeric nanobody conjugates, comprised of an antigen recognition module and a self‐labeling module, enables us to attach the desired functional group at a location distal to the site of antigen recognition. Constructs generated using this approach bound to antigens expressed on xenograft murine models of liver cancer and allowed for non‐invasive diagnostic imaging. The modularity of our approach using a self‐labeling nanobody offers a novel method for site‐specific functionalization of biomolecules and can be extended to other applications for which covalent labeling is required.
Generation of Site‐Specifically Labeled Affinity Reagents via Use of a Self‐Labeling Single Domain Antibody
https://orcid.org/0000-0003-3313-2934
https://orcid.org/0000-0001-9871-8333
AbstractSeveral chemical and enzymatic methods have been used to link antibodies to moieties that facilitate visualization of cognate targets. Emerging evidence suggests that the extent of labeling, dictated by the type of chemistry used, has a substantial impact on performance, especially in the context of antibodies used for the visualization of tumors in vivo. These effects are particularly pronounced in studies using small antibody fragments, such as single‐domain antibodies, or nanobodies. Here, we leverage a new variety of conjugation chemistry, based on a nanobody that forms a crosslink with a specialized high‐affinity epitope analogue, to label target‐specific nanobody constructs with functionalities of choice, including fluorophores, chelators, and click chemistry handles. Using heterodimeric nanobody conjugates, comprised of an antigen recognition module and a self‐labeling module, enables us to attach the desired functional group at a location distal to the site of antigen recognition. Constructs generated using this approach bound to antigens expressed on xenograft murine models of liver cancer and allowed for non‐invasive diagnostic imaging. The modularity of our approach using a self‐labeling nanobody offers a novel method for site‐specific functionalization of biomolecules and can be extended to other applications for which covalent labeling is required.
Generation of Site‐Specifically Labeled Affinity Reagents via Use of a Self‐Labeling Single Domain Antibody
Advanced Science
Fayn, Stanley (author) / Roy, Swarnali (author) / Cabalteja, Chino C. (author) / Lee, Woonghee (author) / Makala, Hima (author) / Baidoo, Kwamena (author) / Nambiar, Divya (author) / Sheehan‐Klenk, Julia (author) / Chung, Joon‐Yong (author) / Buffington, Jesse (author)
2025-02-18
Article (Journal)
Electronic Resource
English
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