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-8877898-B2
Publication Date
2014-11-04
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 present invention provides a polypeptide comprising a fluorescent domain with defined N- and C-termini, a first antibody domain covalently linked to the C-terminus of the fluorescent domain, and a second antibody domain covalently linked to the N-terminus of the fluorescent domain. This design uses the fluorescent domain as a bridge between the antibody domains, thereby stabilizing the molecular geometry for optimal VH/VL pairing and preserving both antigen binding and fluorophore functionality.
The fluorescent domain generally consists of at least a portion of a monomeric fluorescent protein sufficient to emit a fluorescent signal and to maintain steric stability, ensuring the proper distance between antibody domains similar to that found in native Fab fragments. Linkers may be used for covalent connections and to maintain spatial orientation. The antibody domains can specifically bind to the same or different target molecules. The polypeptide can be expressed recombinantly and purified.
The problem addressed by the invention arises from prior approaches that fuse fluorescent proteins directly to single-chain variable fragments (scFv) using long flexible linkers, which often result in aggregation, disassociation, loss of antigen recognition, and non-stoichiometric fluorescent labeling. Additionally, chemical conjugation of fluorophores to antibodies leads to batch variability, partial loss of binding, and photobleaching issues. This invention overcomes these challenges by inserting the fluorescent domain as a stable structural bridge between antibody variable domains, producing a fusion protein that is stable, monomeric, stoichiometric, and retains both antibody binding specificity and fluorescent signal intensity.
Claims Coverage
The patent claims cover a fluorescent fusion polypeptide and compositions thereof, as well as kits including such polypeptides. The claims detail structural features and functional properties of the fusion polypeptides that link antibody domains via a fluorescent domain.
Integration of fluorescent domain as a bridge between antibody domains
A monomeric fluorescent polypeptide domain having a defined C-terminus and N-terminus covalently linked on one end to a first antibody domain and on the other end to a second antibody domain, effectively bridging them while maintaining a specific spatial separation (30-40 Å).
Defined spatial separation between antibody domains via the fluorescent domain
The N-terminus of the first antibody domain and the C-terminus of the second antibody domain are separated by no less than 30 Å and no more than 40 Å, ensuring proper VH and VL interface interactions for functional antigen binding.
Use of short linkers for covalent linkage
At least one covalent link between the fluorescent domain and the antibody domains employs a linker comprising no more than 10 amino acids, supporting structural stability and appropriate spacing.
Antibody domain specificity and composition
The first and second antibody domains can specifically bind to the same target molecule or to different target molecules, and each antibody domain comprises either a variable light chain (VL) or a variable heavy chain (VH).
Stoichiometric and modular design enabling compositions and kits
Compositions comprising two or more such fluorescent fusion polypeptides with differing fluorescent domains and/or antibody specificities, as well as kits containing separate containers with different fluorescent fusion polypeptides binding distinct analytes and exhibiting distinct emission peaks.
The claims protect the novel fusion polypeptide architecture featuring the fluorescent domain positioned between two antibody domains with defined spatial constraints and short linkers, enabling stable, stoichiometric, functionally active fluorescent antibodies useful singly or in compositions and kits for molecular detection and analysis.
Stated Advantages
A single reagent with integrated fluorophore reduces time, cost, and increases reproducibility of binding assays.
The stoichiometric relation between antibody binding site and fluorophore enables quantitative analysis by direct proportional signal generation.
Improved stability and monomeric nature of the fusion proteins compared to conventional scFv fluorescent fusions prone to aggregation.
Robust fluorescent signal retained in bacterial expression and purification, facilitating detection without requiring sophisticated equipment.
The fluorescent domain also serves as a structural bridge to maintain optimal VH/VL pairing and spatial geometry for antigen recognition.
Fusion proteins are genetically encoded and can be expressed in cells in situ, permitting intracellular target detection without cell disruption.
Fluorescent fusion polypeptides can be photoactivated and generate reactive oxygen species under illumination for potential therapeutic applications such as targeted cell ablation.
Documented Applications
Use of fluorescent fusion polypeptides for qualitative and quantitative immunofluorescence analysis and immunodetection assays.
Live cell imaging, immunocytochemical imaging, and flow cytometry.
Use as a nanoprobe for multi-analyte detection in protein arrays and cell surface antigen detection.
In vivo imaging for detection and guidance in surgical removal of tumor cells expressing specific markers.
Development of diagnostic tests for marker-positive cancers such as Her2-positive breast cancer in biopsy samples or circulating cancer cells.
Paratransgenesis applications where recombinant symbiotic bacteria expressing fluorescent antibodies prevent pathogen transmission, for example blocking Xylella fastidiosa transmission by sharpshooters in grapevines.
Monitoring and tracking genetically engineered microbes in environmental or host contexts through intrinsic fluorescence.
Potential use in pest management, disease control, and microbial delivery systems producing antibodies that bind and interfere with pathogens like Clostridium difficile.
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