Joao N. Duarte

HypE-specific Nanobodies as Tools to modulate HypE-mediated Target AMPylation

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. The covalent addition of mono-AMP to target proteins (AMPylation) by Fic domain-containing proteins is a poorly understood, yet highly conserved post-translational modification. Here, we describe the generation, evaluation, and application of four HypE-specific nanobodies: three that inhibit HypE-mediated target AMPylation in vitro and one that acts as an activator. All heavy chain-only antibody variable domains bind HypE when expressed as GFP fusions in intact cells. We observed localization of HypE at the nuclear envelope and further identified histones H2–H4, but not H1, as novel in vitro targets of the human Fic protein. Its role in histone modification provides a possible link between AMPylation and regulation of gene expression.

Fluorophore‐Conjugated Holliday Junctions for Generating Super‐Bright Antibodies and Antibody Fragments

The site-specific modification of proteins with fluorophores can render a protein fluorescent without compromising its function. To avoid self-quenching from 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. These fluorescent Holliday junctions improve fluorescence yields for both single-domain and full-sized antibodies without deleterious effects on antigen binding.

Graphene Oxide Nanosheets Modified with Single-Domain Antibodies for Rapid and Efficient Capture of Cells

Peripheral blood can provide valuable information on an individuals 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.

Generation of Immunity against Pathogens via Single-Domain Antibody–Antigen Constructs

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.

Rapid capture and labeling of cells on single domain antibodies-functionalized flow cell

Current techniques to characterize leukocyte subgroups in blood require long sample preparation times and sizable sample volumes. A simplified method for leukocyte characterization using smaller blood volumes would thus be useful in diagnostic settings. Here we describe a flow system comprised of two functionalized graphene oxide (GO) surfaces that allow the capture of distinct leukocyte populations from small volumes blood using camelid single-domain antibodyfragments (VHHs) as capture agents. We used site-specifically labeled leukocytes to detect and identify cells exposed to fungal challenge. Combining the chemical and optical properties of GO with the versatility of the VHH scaffold in the context of a flow system provides a quick and efficient method for the capture and characterization of functional leukocytes.

Nanobody–Antigen Conjugates Elicit HPV-Specific Antitumor Immune Responses

A targeted purely protein-based therapeutic vaccine elicits CD8+ T-cell responses in an HPV model of cancer, resulting in tumor regression. High-risk human papillomavirus-associated cancers express viral oncoproteins (e.g., E6 and E7) that induce and maintain the malignant phenotype. The viral origin of these proteins makes them attractive targets for development of a therapeutic vaccine. Camelid-derived single-domain antibody fragments (nanobodies or VHHs) that recognize cell surface proteins on antigen-presenting cells (APC) can serve as targeted delivery vehicles for antigens attached to them. Such VHHs were shown to induce CD4+ and CD8+ T-cell responses against model antigens conjugated to them via sortase, but antitumor responses had not yet been investigated. Here, we tested the ability of an anti-CD11b VHH (VHHCD11b) to target APCs and serve as the basis for a therapeutic vaccine to induce CD8+ T-cell responses against HPV+ tumors. Mice immunized with VHHCD11b conjugated to an H-2Db-restricted immunodominant E7 epitope (E749-57) had more E7-specific CD8+ T cells compared with those immunized with E749-57 peptide alone. These CD8+ T cells acted prophylactically and conferred protection against a subsequent challenge with HPV E7-expressing tumor cells. In a therapeutic setting, VHHCD11b-E749-57 vaccination resulted in greater numbers of CD8+ tumor–infiltrating lymphocytes compared with mice receiving E749-57 peptide alone in HPV+ tumor-bearing mice, as measured by in vivo noninvasive VHH-based immune-positron emission tomography (immunoPET), which correlated with tumor regression and survival outcome. Together, these results demonstrate that VHHs can serve as a therapeutic cancer vaccine platform for HPV-induced cancers. Cancer Immunol Res; 6(7); 870–80. ©2018 AACR.

Targeted delivery of cyclotides via conjugation to a nanobody

Many naturally occurring peptides have poor proteolytic stability, which limits their therapeutic applications. Cyclotides are plant-derived cyclic peptides that resist proteolysis due to their highly constrained structure, comprising a head-to-tail cyclic backbone and three disulfide bonds that form a cystine-knotted core. This structure makes them useful as scaffolds onto which peptide sequences (epitopes) can be grafted. In this study, VHH7, an alpaca-derived nanobody that targets murine class II MHC molecules, was used for the targeted delivery of cyclotides to antigen-presenting cells (APCs). The cyclotides MCoTI-I, and MCoTI-I with a HA-tag (YPYDVPDYA) grafted into loop 6 (MCoTI-HA), were tested for immunogenic properties. To produce the requisite VHH7-peptide conjugates, a site-specific sortase A-catalyzed reaction in combination with a copper-free strain-promoted cycloaddition reaction was used. MCoTI-I alone did not display any obvious antibody response, thus showing the capacity of cyclotides as immunologically silent scaffolds. By contrast, MCoTI-I conjugated to VHH7 elicited antibodies against cyclic or linear MCoTI-I, thus suggesting a simple and robust approach for targeting cyclotides to APCs, and potentially to other cell types. A similar antibody response was observed when MCoTI-HA was conjugated to VHH7, but there was no reactivity toward a linear HA-tag itself, suggesting differences in conformational constraint between cyclotide-presented and linear epitopes. Studies of commercially available HA antibodies applied to MCoTI-HA confirmed that the conformation of peptide immunogens affects their reactivity. Thus, the production of antibodies that recognize constrained epitopes may benefit from engraftment onto scaffolds such as cyclotides. More broadly, this study validates that a prototypic cyclotide, a member of a peptide family that has proven to be useful as drug design scaffolds in many other studies, can efficiently reach a specific target in vivo.