SignificanceTumors are often surrounded and invaded by bone marrow-derived cells. Imaging the infiltration of such immune cells into tumors may therefore be an attractive means of detecting tumors or of tracking the response to anticancer therapy. We show that it is possible to detect these cells noninvasively by positron emission tomography (PET) via the surface markers displayed by them. The ability to monitor the immune response in the course of therapy will enable early determination of the efficacy of treatment and will inform decisions as to whether treatment should be stopped or continued. Noninvasive monitoring could therefore change how therapies are applied and assessed, to the benefit of many patients.
mAbs specific for surface proteins on APCs can serve as Ag-delivery vehicles that enhance immunogenicity. The practical use of such constructs is limited by the challenge of expressing and modifying full-sized mAbs. We generated single-domain Ab fragments (VHHs) specific for class II MHC (MHCII), CD11b, and CD36. VHH sequences were modified by inclusion of a C-terminal sortase motif to allow site-specific conjugation with various Ag payloads. We tested T cell activation using VHHs that target distinct APC populations; anti-MHCII adducts elicited strong activation of CD4+ T cells, whereas anti-CD11b showed CD8+ T cell activation superior to targeting via MHCII and CD36. Differences in Ag presentation among constructs were unrelated to dendritic cell subtype or routing to acidic compartments. When coupled to antigenic payloads, anti-MHCII VHH primed Ab responses against GFP, ubiquitin, an OVA peptide, and the α-helix of influenza hemagglutinin’s stem; the last afforded protection against influenza infection. The versatility of the VHH scaffold and sortase-mediated covalent attachment of Ags suggests their broader application to generate desirable immune responses.
Background: Nanobodies represent a specific means to modulate enzyme function. Results: HypE-specific nanobodies modulate AMPylation activity and identify HypE as localized to the nuclear envelope. Conclusion: HypE function can be interfered with and probed for with new tools. Significance: HypE nanobodies are the first known HypE activators and inhibitors.
Antibody-drug conjugates (ADCs) of defined structure hold great promise for cancer therapies, but further advances are constrained by the complex structures of full-sized antibodies. Camelid-derived single domain antibody fragments (VHHs or nanobodies) offer possible solutions to this challenge by providing expedited target screening/validation through shuttling between imaging and therapy. Here, we used a nanobody (VHH7) specific for murine MHC-II and rendered sortase-ready for introduction of oligoglycine-modified cytotoxic payloads or NIR fluorophores. The VHH7 conjugates outcompeted commercial mAbs for internalization and exhibited high specificity and cytotoxicity against the A20 murine B-cell lymphoma. Non-invasive NIR imaging with a VHH7-fluorophore conjugate showed rapid tumor targeting on both localized and metastatic lymphoma models. Subsequent treatment with the nanobody-drug conjugate efficiently controlled tumor growth and metastasis without obvious systemic toxicity.
The exact mechanism of retinal ganglion cell loss in the pathogenesis of glaucoma is yet to be understood. Mitochondrial damage-associated molecular patterns (DAMPs) resulting from mitochondrial dysfunction have been linked to Leber’s hereditary optic neuropathy and autosomal dominant optic atrophy, as well as to brain neurodegenerative diseases. Recent evidence shows that, in conditions where mitochondria are damaged, a sustained inflammatory response and downstream pathological inflammation may ensue. Mitochondrial damage has been linked to the accumulation of age-related mitochondrial DNA mutations and mitochondrial dysfunction, possibly through aberrant reactive oxygen species production and defective mitophagy. The present review focuses on how mitochondrial dysfunction may overwhelm the ability of neurons and glial cells to adequately maintain homeostasis and how mitochondria-derived DAMPs trigger the immune system and induce neurodegeneration.
Peripheral blood can provide valuable information on an individual’s immune status. Cell-based assays typically target leukocytes and their products. Characterization of leukocytes from whole blood requires their separation from the far more numerous red blood cells.[1] Current methods to classify leukocytes, such as recovery on antibody-coated beads or fluorescence-activated cell sorting require long sample preparation times and relatively large sample volumes.[2] A simple method that enables the characterization of cells from a small peripheral whole blood sample could overcome limitations of current analytical techniques. We describe the development of a simple graphene oxide surface coated with single domain antibody fragments. This format allows quick and efficient capture of distinct WBC subpopulations from small samples (~30 μL) of whole blood in a geometry that does not require any specialized equipment such as cell sorters or microfluidic devices.
Site-specific modification of proteins with fluorophores can render a protein fluorescent without compromising its function. To avoid self-quenching of multiple fluorophores installed in close proximity, we used Holliday junctions to label proteins site-specifically. Holliday junctions enable modification with multiple fluorophores at reasonably precise spacing. We designed a Holliday junction with three of its four arms modified with a fluorophore of choice and the remaining arm equipped with a dibenzocyclooctyne substituent to render it reactive with an azide-modified, fluorescent single domain antibody fragment or an intact immunoglobulin, produced in a sortase-catalyzed reaction. We conclude that fluorescent Holliday junctions improve fluorescence yields for both single domain and full-sized antibodies, without deleterious effects on antigen binding.
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