Sortase-Mediated Site-Specific Conjugation to Prepare Fluorine-18-Labeled Nanobodies.

Single-domain antibodies, or nanobodies (Nbs), are promising biomolecules for use in molecular imaging due to their excellent affinity, specificity, and fast clearance from the blood. Given their short blood half-life, pairing Nbs with short-lived imaging radioisotopes is desirable. Because fluorine-18 (F) is routinely used for clinical imaging, it is an attractive radioisotope for Nbs.

Highly biased agonism for GPCR ligands via nanobody tethering.

Ligand-induced activation of G protein-coupled receptors (GPCRs) can initiate signaling through multiple distinct pathways with differing biological and physiological outcomes. There is intense interest in understanding how variation in GPCR ligand structure can be used to promote pathway selective signaling ("biased agonism") with the goal of promoting desirable responses and avoiding deleterious side effects. Here we present an approach in which a conventional peptide ligand for the type 1 parathyroid hormone receptor (PTHR1) is converted from an agonist which induces signaling through all relevant pathways to a compound that is highly selective for a single pathway.

Sortase-mediated labeling: Expanding frontiers in site-specific protein functionalization opens new research avenues.

New applications for biomolecules demand novel approaches for their synthesis and modification. Traditional methods for modifying proteins and cells using non-specific labeling chemistry are insufficiently precise to rigorously interrogate the mechanistic biological and physiological questions at the forefront of biomedical science. Site-specific catalytic modification of proteins promises to meet these challenges.

Nanobody-Mediated Dualsteric Engagement of the Angiotensin Receptor Broadens Biased Ligand Pharmacology.

Dualsteric G protein-coupled receptor (GPCR) ligands are a class of bitopic ligands that consist of an orthosteric pharmacophore, which binds to the pocket occupied by the receptor's endogenous agonist, and an allosteric pharmacophore, which binds to a distinct site. These ligands have the potential to display characteristics of both orthosteric and allosteric ligands. To explore the signaling profiles that dualsteric ligands of the angiotensin II type 1 receptor (AT1R) can access, we ligated a 6e epitope tag-specific nanobody (single-domain antibody fragment) to angiotensin II (AngII) and analogs that show preferential allosteric coupling to Gq (TRV055, TRV056) or -arrestin (TRV027).

Semi-synthetic nanobody-ligand conjugates exhibit tunable signaling properties and enhanced transcriptional outputs at neurokinin receptor-1.

Antibodies have proven highly valuable for therapeutic development; however, they are typically poor candidates for applications that require activation of G protein-coupled receptors (GPCRs), the largest collection of targets for clinically approved drugs. Nanobodies (Nbs), the smallest antibody fragments retaining full antigen-binding capacity, have emerged as promising tools for pharmacologic applications, including GPCR modulation. Past work has shown that conjugation of Nbs with ligands can provide GPCR agonists that exhibit improved activity and selectivity compared to their parent ligands.

Semi-synthetic nanobody-ligand conjugates exhibit tunable signaling properties and enhanced transcriptional outputs at neurokinin receptor-1.

Antibodies have proven highly valuable for therapeutic development; however, they are typically poor candidates for applications that require activation of G protein-coupled receptors (GPCRs), the largest collection of targets for clinically approved drugs. Nanobodies (Nbs), the smallest antibody fragments retaining full antigen-binding capacity, have emerged as promising tools for pharmacologic applications, including GPCR modulation. Past work has shown that conjugation of Nbs with ligands can provide GPCR agonists that exhibit improved activity and selectivity compared to their parent ligands.

Highly biased agonism for GPCR ligands via nanobody tethering.

Ligand-induced activation of G protein-coupled receptors (GPCRs) can initiate signaling through multiple distinct pathways with differing biological and physiological outcomes. There is intense interest in understanding how variation in GPCR ligand structure can be used to promote pathway selective signaling ("biased agonism") with the goal of promoting desirable responses and avoiding deleterious side effects. Here we present a new approach in which a conventional peptide ligand for the type 1 parathyroid hormone receptor (PTHR1) is converted from an agonist which induces signaling through all relevant pathways to a compound that is highly selective for a single pathway.

Altered Signaling and Desensitization Responses in PTH1R Mutants Associated with Eiken Syndrome.

The parathyroid hormone receptor type 1 (PTH1R) is a G protein-coupled receptor that plays key roles in regulating calcium homeostasis and skeletal development via binding the ligands, PTH and PTH-related protein (PTHrP), respectively. Eiken syndrome is a rare disease of delayed bone mineralization caused by homozygous PTH1R mutations. Of the three mutations identified so far, R485X, truncates the PTH1R C-terminal tail, while E35K and Y134S alter residues in the receptor's amino-terminal extracellular domain.

Site-Specifically Conjugated Single-Domain Antibody Successfully Identifies Glypican-3-Expressing Liver Cancer by Immuno-PET.

Primary liver cancer is the third leading cause of cancer-related deaths, and its incidence and mortality are increasing worldwide. Hepatocellular carcinoma (HCC) accounts for 80% of primary liver cancer cases. Glypican-3 (GPC3) is a heparan sulfate proteoglycan that histopathologically defines HCC and represents an attractive tumor-selective marker for radiopharmaceutical imaging and therapy for this disease.

Rapid Covalent Labeling of Membrane Proteins on Living Cells Using a Nanobody-Epitope Tag Pair.

Synthetic molecules that form a covalent bond upon binding to a targeted biomolecule (proximity-induced reactivity) are the subject of intense biomedical interest for the unique pharmacological properties imparted by irreversible binding. However, off-target covalent labeling and the lack of molecules with sufficient specificity limit more widespread applications. We describe the first example of a cross-linking platform that uses a synthetic peptide epitope and a single domain antibody (or nanobody) pair to form a covalent linkage rapidly and specifically.

Characterization of a Nanobody-Epitope Tag Interaction and Its Application for Receptor Engineering.

Peptide epitope tags offer a valuable means for detection and manipulation of protein targets for which high quality detection reagents are not available. Most commonly used epitope tags are bound by conventional, full-size antibodies (Abs). The complex architecture of Abs complicates their application in protein engineering and intracellular applications.

A conserved Bacteroidetes antigen induces anti-inflammatory intestinal T lymphocytes.

The microbiome contributes to the development and maturation of the immune system. In response to commensal bacteria, intestinal CD4 T lymphocytes differentiate into functional subtypes with regulatory or effector functions. The development of small intestine intraepithelial lymphocytes that coexpress CD4 and CD8αα homodimers (CD4IELs) depends on the microbiota.

Protein visualization and manipulation in through the use of epitope tags recognized by nanobodies.

Expansion of the available repertoire of reagents for visualization and manipulation of proteins will help understand their function. Short epitope tags linked to proteins of interest and recognized by existing binders such as nanobodies facilitate protein studies by obviating the need to isolate new antibodies directed against them. Nanobodies have several advantages over conventional antibodies, as they can be expressed and used as tools for visualization and manipulation of proteins in vivo.

Activation of a G protein-coupled receptor through indirect antibody-mediated tethering of ligands.

Antibodies raised against many cell surface proteins, including G protein-coupled receptors, remain important tools for their functional characterization. By linking antibodies to ligands for cell surface proteins, such adducts can be targeted to the surface of a cell type of choice. Site-specific functionalization of full-size antibodies with synthetic moieties remains challenging.

Strategies for targeting cell surface proteins using multivalent conjugates and chemical biology.

Proper function of receptors on the cell surface is essential for homeostasis. Compounds that target cell surface receptors to address dysregulation have proven exceptionally successful as therapeutic agents; however, the development of compounds with the desired specificity for receptors, cells, and tissues of choice has proven difficult in some cases. The use of compounds that can engage more than one binding site at the cell surface offers a path toward improving biological specificity or pharmacological properties.

Converting an Anti-Mouse CD4 Monoclonal Antibody into an scFv Positron Emission Tomography Imaging Agent for Longitudinal Monitoring of CD4 T Cells.

Immuno-positron emission tomography (PET), a noninvasive imaging modality, can provide a dynamic approach for longitudinal assessment of cell populations of interest. Transformation of mAbs into single-chain variable fragment (scFv)-based PET imaging agents would allow noninvasive tracking in vivo of a wide range of possible targets. We used sortase-mediated enzymatic labeling in combination with PEGylation to develop an anti-mouse CD4 scFv-based PET imaging agent constructed from an anti-mouse CD4 mAb.

Activity-based, bioorthogonal imaging of phospholipase D reveals spatiotemporal dynamics of GPCR-Gq signaling.

Canonically, G-protein-coupled receptor (GPCR) signaling is transient and confined to the plasma membrane (PM). Deviating from this paradigm, the parathyroid hormone receptor (PTHR1) stimulates sustained G signaling at endosomes. In addition to G, PTHR1 activates G signaling; yet, in contrast to the PTHR1-G pathway, the spatiotemporal dynamics of the G branch of PTHR1 signaling and its relationship to G signaling remain largely ill defined.

Engineering Protease-Resistant Peptides to Inhibit Human Parainfluenza Viral Respiratory Infection.

The lower respiratory tract infections affecting children worldwide are in large part caused by the parainfluenza viruses (HPIVs), particularly HPIV3, along with human metapneumovirus and respiratory syncytial virus, enveloped negative-strand RNA viruses. There are no vaccines for these important human pathogens, and existing treatments have limited or no efficacy. Infection by HPIV is initiated by viral glycoprotein-mediated fusion between viral and host cell membranes.

In vivo detection of antigen-specific CD8 T cells by immuno-positron emission tomography.

The immune system's ability to recognize peptides on major histocompatibility molecules contributes to the eradication of cancers and pathogens. Tracking these responses in vivo could help evaluate the efficacy of immune interventions and improve mechanistic understanding of immune responses. For this purpose, we employ synTacs, which are dimeric major histocompatibility molecule scaffolds of defined composition.

Exploring cellular biochemistry with nanobodies.

Reagents that bind tightly and specifically to biomolecules of interest remain essential in the exploration of biology and in their ultimate application to medicine. Besides ligands for receptors of known specificity, agents commonly used for this purpose are monoclonal antibodies derived from mice, rabbits, and other animals. However, such antibodies can be expensive to produce, challenging to engineer, and are not necessarily stable in the context of the cellular cytoplasm, a reducing environment.

Improved GPCR ligands from nanobody tethering.

Antibodies conjugated to bioactive compounds allow targeted delivery of therapeutics to cell types of choice based on that antibody's specificity. Here we develop a new type of conjugate that consists of a nanobody and a peptidic ligand for a G protein-coupled receptor (GPCR), fused via their C-termini. We address activation of parathyroid hormone receptor-1 (PTHR1) and improve the signaling activity and specificity of otherwise poorly active N-terminal peptide fragments of PTH by conjugating them to nanobodies (VHHs) that recognize PTHR1.

A nanobody that recognizes a 14-residue peptide epitope in the E2 ubiquitin-conjugating enzyme UBC6e modulates its activity.

A substantial fraction of eukaryotic proteins is folded and modified in the endoplasmic reticulum (ER) prior to export and secretion. Proteins that enter the ER but fail to fold correctly must be degraded, mostly in a process termed ER-associated degradation (ERAD). Both protein folding in the ER and ERAD are essential for proper immune function.

Recognition of Class II MHC Peptide Ligands That Contain β-Amino Acids.

Proteins are composed of α-amino acid residues. This consistency in backbone structure likely serves an important role in the display of an enormous diversity of peptides by class II MHC (MHC-II) products, which make contacts with main chain atoms of their peptide cargo. Peptides that contain residues with an extra carbon in the backbone (derived from β-amino acids) have biological properties that differ starkly from those of their conventional counterparts.

Internalization of Influenza Virus and Cell Surface Proteins Monitored by Site-Specific Conjugation of Protease-Sensitive Probes.

Commonly used methods to monitor internalization of cell surface structures involve application of fluorescently or otherwise labeled antibodies against the target of interest. Genetic modification of the protein of interest, for example through creation of fusions with fluorescent or enzymatically active protein domains, is another approach to follow trafficking behavior. The former approach requires indirect methods, such as multiple rounds of cell staining, to distinguish between a target that remains surface-disposed and an internalized and/or recycled species.

Receptor selectivity from minimal backbone modification of a polypeptide agonist.

Human parathyroid hormone (PTH) and N-terminal fragments thereof activate two receptors, hPTHR1 and hPTHR2, which share ∼51% sequence similarity. A peptide comprising the first 34 residues of PTH is fully active at both receptors and is used to treat osteoporosis. We have used this system to explore the hypothesis that backbone modification of a promiscuous peptidic agonist can provide novel receptor-selective agonists.