Fluorescent fusion polypeptides and methods of use
Inventors
MARKIV, ANATOLIY • Durvasula, Ravi Venkata • Kang, Angray Singh
Assignees
US Department of Veterans Affairs • UNM Rainforest Innovations
Publication Number
US-9566353-B2
Publication Date
2017-02-14
Expiration Date
2031-04-28
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Abstract
Embodiments of the present invention provide for the facile generation of a stable recombinant fusion polypeptides with intrinsic fluorescent properties. The recombinant antibodies may be suitable for qualitative and/or quantitative immunofluorescence analysis. Generally, the fluorescent polypeptides include a fluorescent domain comprising a C-terminus and an N-terminus; a first antibody domain covalently linked to the C-terminus; and a second antibody domain covalently linked to the N-terminus.
Core Innovation
The invention provides a novel fusion polypeptide comprising a fluorescent domain with a C-terminus and an N-terminus, a first antibody domain covalently linked to the C-terminus, and a second antibody domain covalently linked to the N-terminus. This design uses a β-barrel fluorescent protein domain as a structural bridge between the variable heavy (VH) and variable light (VL) chains of antibodies, stabilizing their spatial orientation and preserving their antigen-binding functionality.
The problem addressed is the instability and aggregation of conventional single-chain fragment variable (scFv) antibodies when fused directly to fluorescent proteins via long flexible linkers. Conventional scFv domains linked by such linkers are prone to dissociation, leading to reduced antigen recognition and decreased stability. Prior antibody-fluorophore conjugates also suffer from issues including photobleaching, batch variation, Fc receptor binding, aggregation, and partial loss of binding.
The invention overcomes these limitations by replacing the flexible linker of scFvs with a fluorescent domain (e.g., monomeric red fluorescent protein mRFP1) that maintains the optimal spacing (about 30 to 40 Å) between the VH and VL chains. This enables recombinant fluorescent antibodies with both functional fluorescence and high-affinity antigen binding. The polypeptides can be genetically encoded for in situ expression inside cells, enabling detection of intracellular targets without cell disruption.
Claims Coverage
The patent includes one independent method claim focused on a method of detecting an analyte using a fluorescent fusion polypeptide.
Fluorescent fusion polypeptide structure
A polypeptide comprising a monomeric fluorescent domain with a C-terminus and an N-terminus, where a first antibody domain (VL or VH) is covalently linked to the C-terminus and a second antibody domain (VL or VH) is covalently linked to the N-terminus, maintaining a spatial distance of 30 to 40 Å between the N-terminus of the first antibody domain and the C-terminus of the second antibody domain.
Specific binding of antibody domains to analyte
At least one of the first or second antibody domains specifically binds to the analyte in the sample, enabling specific detection.
Sequential detection of multiple analytes
The method optionally includes contacting the sample with a second similar polypeptide with distinct antibody domains and fluorescent domains to detect a second analyte, allowing multiplex detection through distinct fluorescent signals.
Quantitative fluorescence detection
The method includes removing unbound polypeptides and detecting (and optionally quantifying) the fluorescent signal produced by the specific binding, enabling qualitative and quantitative analyte detection.
The claims cover a method of analyte detection using genetically encoded fluorescent fusion polypeptides with structurally defined antibody-fluorescent protein configurations that allow specific, stable, and potentially multiplexed fluorescent detection of target analytes.
Stated Advantages
Each antibody binding site carries a single, stoichiometric fluorescent molecule, enabling quantitative detection proportional to antigen binding.
The fusion polypeptides exhibit enhanced stability and maintain optimal VH/VL spatial orientation, reducing aggregation compared to conventional scFv linkers.
The intrinsic fluorescence allows monitoring protein expression and purification by visual color without complex equipment.
The fluorescent domain can also produce reactive oxygen species upon light exposure, providing potential therapeutic photo-ablation capabilities.
The modular platform allows exchange of fluorescent domains to generate multicolor antibody reagents for multiplexed analysis.
Documented Applications
Qualitative and quantitative immunofluorescence analysis and detection of antigens in samples using fluorescent fusion polypeptides.
Flow cytometry and immunocytochemical imaging for cell surface antigens, including cancer biomarkers.
In vivo imaging and molecular imaging to detect and guide removal of tumor cells, including improved surgical navigation.
Paratransgenesis in pest management by engineering symbiotic bacteria to express fluorescent antibodies that interfere with pathogen transmission (e.g., Xylella fastidiosa in grapevine sharpshooters).
Non-invasive imaging of inflammation markers in subjects via administration of fluorescent fusion polypeptides that bind inflammatory targets.
Use as nanoprobes in multiplex immunoassays and diagnostic kits containing multiple fluorescent antibody constructs.
Detection of analytes by applying fluorescent fusion polypeptides directly to samples, immobilizing samples, and measuring specific fluorescent signals.
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