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Local retention of antibodies in vivo with an injectable film embedded with a fluorogen-activating protein.

ABSTRACT: Herein we report an injectable film by which antibodies can be localized in vivo. The system builds upon a bifunctional polypeptide consisting of a fluorogen-activating protein (FAP) and a ?-fibrillizing peptide (?FP). The FAP domain generates fluorescence that reflects IgG binding sites conferred by Protein A/G (pAG) conjugated with the fluorogen malachite green (MG). A film is generated by mixing these proteins with molar excess of EAK16-II, a ?FP that forms ?-sheet fibrils at high salt concentrations. The IgG-binding, fluorogenic film can be injected in vivo through conventional needled syringes. Confocal microscopic images and dose-response titration experiments showed that loading of IgG into the film was mediated by pAG(MG) bound to the FAP. Release of IgG in vitro was significantly delayed by the bioaffinity mechanism; 26% of the IgG were released from films embedded with pAG(MG) after five days, compared to close to 90% in films without pAG(MG). Computational simulations indicated that the release rate of IgG is governed by positive cooperativity due to pAG(MG). When injected into the subcutaneous space of mouse footpads, film-embedded IgG were retained locally, with distribution through the lymphatics impeded. The ability to track IgG binding sites and distribution simultaneously will aid the optimization of local antibody delivery systems.

SUBMITTER: Liu W

PROVIDER: S-EPMC5716469 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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Local retention of antibodies in vivo with an injectable film embedded with a fluorogen-activating protein.

Liu Wen W Saunders Matthew J MJ Bagia Christina C Freeman Eric C EC Fan Yong Y Gawalt Ellen S ES Waggoner Alan S AS Meng Wilson S WS

Journal of controlled release : official journal of the Controlled Release Society 20160331

Herein we report an injectable film by which antibodies can be localized in vivo. The system builds upon a bifunctional polypeptide consisting of a fluorogen-activating protein (FAP) and a β-fibrillizing peptide (βFP). The FAP domain generates fluorescence that reflects IgG binding sites conferred by Protein A/G (pAG) conjugated with the fluorogen malachite green (MG). A film is generated by mixing these proteins with molar excess of EAK16-II, a βFP that forms β-sheet fibrils at high salt concen ...[more]

PMID: 27038493

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Project description:The development and function of tissues, blood, and the immune system is dependent upon proximity for cellular recognition and communication. However, the detection of cell-to-cell contacts is limited due to a lack of reversible, quantitative probes that can function at these dynamic sites of irregular geometry. Described here is a novel chemo-genetic tool developed for fluorescent detection of protein-protein proximity and cell apposition that utilizes the Fluorogen Activating Protein (FAP) in combination with a Dye Activated by Proximal Anchoring (DAPA). The FAP-DAPA system has two protein components, the HaloTag and FAP, expressed on separate protein targets or in separate cells. The proteins function to bind and activate a compound that has the hexyl chloride (HexCl) ligand connected to malachite green (MG), the FAP fluorogen, via a poly(ethylene glycol) spacer spanning up to 28 nm. The dehalogenase protein, HaloTag, covalently binds the HexCl ligand, locally concentrating the attached MG. If the FAP is within range of the anchored fluorogen, it will bind and activate MG specifically when the bath concentration is too low to saturate the FAP receptor. A new FAP variant was isolated with a 1000-fold reduced KD of ∼10-100 nM so that the fluorogen activation reports proximity without artificially enhancing it. The system was characterized using purified FRB and FKBP fusion proteins and showed a doubling of fluorescence upon rapamycin induced complex formation. In cocultured HEK293 cells (HaloTag and FAP-expressing) fluorescence increased at contact sites across a broad range of labeling conditions, more reliably providing contact-specific fluorescence activation with the lower-affinity FAP variant. When combined with suitable targeting and expression constructs, this labeling system may offer significant improvements in on-demand detection of intercellular contacts, potentially applicable in neurological and immunological synapse measurements and other transient, dynamic biological appositions that can be perturbed using other labeling methods that stabilize these interactions.

Dataset Information

Local retention of antibodies in vivo with an injectable film embedded with a fluorogen-activating protein.

Publications

Local retention of antibodies in vivo with an injectable film embedded with a fluorogen-activating protein.

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