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 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 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 pAGMG 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 pAGMG after five days, comparedto close to 90% in films without pAGMG. Computational simulations indicated that the release rate of IgG is governed by positive cooperativity due to pAGMG. 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.

Switching the Immunogenicity of Peptide Assemblies Using Surface Properties

“…For example, peptide and protein self-assemblies are under considerable investigation for many non-immunological applications such as tissue engineering and cell scaffolds, 14, 15, 45 drug delivery, 16, 20, 2146–48 antimicrobials, 49 wound healing, 19, 50 and others. 51, 52 In this broad range of applications, any antibody response against proteins, peptides, or other antigenic components incorporated into the materials could lead to blockage of the factors’ function, early clearance of the material, or at worst, inflammatory rejection responses, although in general peptide assemblies have been found to be non-inflammatory to date.…”

A versatile optical tool for studying synaptic GABAA receptor trafficking

“…This paper lays the groundwork for future investigations of GABA A R trafficking with the γ2 pH FAP sensor. Importantly, this technique can be further extended toward pharmacology-focused efforts in high-throughput screenings (Fisher et al, 2014;Snyder et al, 2015), assay development based on flow cytometry (Saunders et al, 2012;Wu et al, 2014) or high-resolution 96-well plate assays (Larsen et al, 2016) and for in vivo protein labeling (Liu et al, 2016;Zhang et al, 2015). In summary, our γ2 pH FAP is the first protein-FAP conjugate characterized in primary neurons, providing a unique tool to monitor multistage GABA A R trafficking in living cells with relevance both for basic science research and applied pharmacology.…”

Fluorogen-activating proteins: beyond classical fluorescent proteins

“…In combination with polymeric materials, targeted biosensor have been developed to distinct subcellular structures within living cells 56 . More recently, Meng group 57 reported an injectable film by which antibodies can be localized in vivo . Their system builds upon a bifunctional polypeptide consisting of a FAP and a β -fibrillizing peptide ( β FP).…”