Ube2R1 (Cdc34) is a K48-ubiquitin chain-specific ubiquitin-conjugating (E2) enzyme central to proteasomal degradation, yet the regulatory potential of its unique structural elements remains underexplored. Here, we report the isolation and biochemical characterization of a nanobody, VHH12R1, that binds selectively to the N-terminal extension of Ube2R1 with no detectable cross-reactivity to its paralog Ube2R2 or other E2s. Engagement of this N-terminal region by VHH12R1 transiently delays ubiquitin charging, promotes accumulation of stable mono-ubiquitin Ube2R1 conjugates, and markedly reduces self-directed polyubiquitination chain formation by Ube2R1 without impairing di-ubiquitin synthesis. Although Ube2R1 can catalyze ubiquitination of VHH12R1 in the absence of an E3 ligase, this modification occurs independently of stable nanobody binding and is not required for modulation of Ube2R1 activity. Together, these findings support a model in which VHH12R1 selectively restricts processive self-elongation by Ube2R1 through engagement of its N-terminal extension, without broadly inhibiting ubiquitin transfer reactions. Our results reveal an unappreciates regulatory role for the Ube2R1 N-terminus in controlling catalytic outcomes and highlight nanobody-based approaches as precise tools to dissect E2 enzyme function.

Sortase A, originating from Staphylococcus Aureus, has become the gold standard for site-specific transpeptidation of protein substrates. Wild-type and engineered variants of sortase A catalyze the recognition and cleavage of a five-amino acid substrate motif (LPxTG), followed by transpeptidation with oligoglycine nucleophiles (peptides that contain one or more glycines at their N-terminus). This LPxTG sortagging motif can be introduced by genetic engineering of recombinantly expressed proteins of interest. Oligoglycine nucleophiles can be readily synthesized on solid-phase resin and allow the introduction of a wide range of functional groups. Here, we describe the transpeptidation of two nanobodies (VHHs, variable region of the heavy chain of heavy-chain only antibodies) with various GGG-nucleophiles.

Through screening of a VHH phage display library constructed from an alpaca immunized with the recombinantly expressed murine myelin oligodendrocyte (MOG)-specific 2D2 T cell receptor (TCR), we identified nanobodies that reduced antigen-driven 2D2 T cell activation in vitro. Immunoblotting and staining confirmed TCRβ chain reactivity and Vβ11-associated recognition in polyclonal T cell populations. Site-specific sortase-mediated conjugation with desferrioxamine and 89Zr enabled nanobody-based immuno-PET/CT. In vivo imaging showed tracer accumulation in secondary lymphoid organs in settings enriched for Vβ11-expressing 2D2 T cells. In mice symptomatic for experimental autoimmune encephalomyelitis that had received MOG-specific 2D2 T cells, we observed a spinal cord-associated signal. This corresponded to accumulation of transferred Vβ11-expressing 2D2 T cells within inflamed spinal cord tissue. Compared with full-length immunoglobulins and multimeric peptide-MHC reagents that primarily support ex vivo detection of antigen-specific T cells, TCR-specific nanobodies show rapid clearance and improved tissue penetration that favor high-contrast immuno-PET/CT imaging of defined T cell populations in vivo.

Ubiquitin conjugating E2 enzymes are a set of ~40 proteins that play a central role in the ubiquitination cascade. They transfer ubiquitin from an E1 enzyme to substrates with the help of an E3 enzyme. The members of the E2 family share structural similarity in their conserved UBC fold. This complicates an assessment of the specificity of E2-E3 interactions. We identified a nanobody that binds to the 'backside' region of Ube2G2, an E2 involved in ER protein quality control. This binding does not affect ubiquitin loading but shows varying degrees of inhibition on E3-mediated ubiquitination, in the order HRD1 > CHIP >> TRC8. A naturally occurring segment that binds Ube2G2's backside, referred to as G2BR (Ube2G2 Binding Region), shows a similar inhibitory effect depending on the identity of the interacting E3. The G2BR in the Ube2G2-cognate E3 Gp78 enhances Ube2G2's activity, but its deletion results in a similar inhibition upon addition of the nanobody. Occupation of a single binding site on an E2 can thus affect its interactions with different E3s.

T cell tolerance to self can prevent self-reactive B cells from mounting effective autoimmune responses by limiting their available help. However, tolerance may be compromised during infection if small amounts of soluble cross-reactive pathogen antigens containing functional T cell epitopes are released for capture by activated B cells. Here, we assess this scenario and show that naïve B cells engaged and activated by membrane-bound antigens lacking T helper epitopes are impaired in their ability to capture and present additional soluble antigens containing T helper epitopes. This limits their ability to acquire help from cognate CD4+ T cells required for effective antibody responses. Failure to capture and present soluble antigen is due to IgM and IgD downregulation when engaged with membrane-bound antigen. Interestingly, IgG+ B cells are not similarly constrained and effectively capture soluble antigens. Our findings suggest control of naïve B cell tolerance partially depends on efficient receptor downregulation upon antigen engagement.

Four nanobodies (VHH1-4SMT3) that target the yeast SUMO protein Smt3p were isolated and characterized. VHH1-4SMT3 bind to Smt3p and Smt3p-tagged proteins with high affinity (Kd: low nM). NMR analysis shows that the four nanobodies all bind near the C-terminus of Smt3p, partially overlapping with the binding site for the SUMO protease Ulp1p. Binding of Smt3p-specific nanobodies impairs Ulp1-mediated cleavage of Smt3p-tagged proteins, with VHH1SMT3 showing complete inhibition. The use of immobilized VHH2SMT3 enabled efficient purification of Smt3p-tagged proteins, while VHH1SMT3 can be used for immunoblotting and detects both Smt3p-tagged and free Smt3p. When expressed in yeast, VHH1SMT3 causes significant growth defects, particularly when targeted to the nucleus or fused with GFP, indicative of interference with essential SUMOylation-dependent processes.

Influenza remains a significant public health threat. Both monoclonal antibodies and small-molecule inhibitors can target the influenza surface glycoproteins hemagglutinin (HA) or neuraminidase (NA) for prevention and treatment of influenza. Here, we combine the strengths of anti-influenza antibodies and small molecules by site-specific conjugation of the NA inhibitor zanamivir to MEDI8852, an HA-specific fully human monoclonal antibody. MEDI8852 targets the conserved stem region of HA and inhibits HA-mediated fusion of the viral and host cell membranes. Elimination of virus-infected cells involves Fcγ receptor–mediated effector functions. The efficacy of MEDI8852 is limited to influenza A viruses. Zanamivir, on the other hand, binds to the active site of NA in both influenza A and B viruses to inhibit NA activity and virus release. However, because of its small size, zanamivir has a short half-life and requires repeated dosing at high concentrations. We produced a MEDI8852–zanamivir antibody–drug conjugate (ADC) that engages Fc-mediated effector functions and benefits from neonatal Fc receptor (FcRn)-mediated recycling. The MEDI8852–zanamivir conjugate extends the circulatory half-life of zanamivir, targets both influenza HA and NA, and shows enhanced antibody-dependent cellular cytotoxicity (ADCC) compared to MEDI8852 alone. The MEDI8852–zanamivir conjugate protected mice from a lethal (10 × LD 50 ) challenge with influenza A and B viruses at a dose similar to that required for broadly neutralizing anti-NA antibodies, with the added advantage of simultaneously targeting NA (influenza A and B) and HA (influenza A).

Oncogenic mutations in the epidermal growth factor receptor (EGFR) promote tumorigenesis by stabilizing active or pre-active receptor conformations. Most EGFR-driven cancers are characterized by kinase domain mutations that directly activate the receptor. However, brain cancers such as glioblastoma multiforme (GBM) uniquely harbor mutations in the EGFR ectodomain that allosterically activate the kinase domain. Despite significant advances in understanding the physiological and pathogenic roles of EGFR, the conformational characteristics that define ligand-independent EGFR activation in GBM remain poorly understood. In this study, we use naive and post-immune yeast-displayed nanobody libraries to discover four nanobody groups that with benchmark nanobodies define a total of five groups with unique binding signatures and specificities for GBM mutation-stabilized conformational states. Nanobodies in Groups 1 and 2 block ligand, selectively bind the inactive, tethered conformation, and favor wild-type EGFR over GBM-stabilized conformations. In contrast, nanobodies in Groups 4 and 5 do not block ligand, target active or pre-active conformations, and selectively bind GBM-stabilized conformations. Additionally, nanobodies in Group 3 block ligand and appear to be conformation agnostic. We observed domain-specific bias in the nanobodies' selectivity for GBM mutations, suggesting that mutations across different ectodomain regions stabilize distinct conformations. This work advances our understanding of EGFR conformational equilibria in the context of GBM. The observed cooperativity and mutation-dependent binding of nanobodies emphasize their utility in dissecting EGFR activation mechanisms and in developing targeted therapies for EGFR-driven cancers, including GBM.

As immune checkpoint blockade induces durable responses in only a subset of patients, more effective immunotherapies are needed. Here we present bispecific antibody engagers, fusion proteins composed of a nanobody that recognizes immunoglobulin kappa light chains (VHHkappa) and a nanobody that recognizes either CTLA-4 or PD-L1. These fusions show strong antitumour activity in mice through recruitment of polyclonal immunoglobulins independently of specificity or isotype. The anti-CTLA-4 VHH-VHHkappa conjugate demonstrates superior antitumour activity compared with the conventional monoclonal anti-CTLA-4 antibody and reduces the number of intratumoural regulatory T cells in a mouse model of colorectal carcinoma. The anti-PD-L1 VHH-VHHkappa conjugate is less effective in the colorectal carcinoma model while still outperforming a conventional antibody of similar specificity. The potency of the anti-PD-L1 VHH-VHHkappa conjugate was enhanced by installation of the cytotoxic drug maytansine or a STING agonist. The ability of such fusions to engage the Fc-mediated functions of all immunoglobulin isotypes is an appealing strategy to further improve on the efficacy of immune checkpoint blockade, commonly delivered as a monoclonal immunoglobulin of a single defined isotype. Nanobody-based immune checkpoint inhibitors conjugated to an immunoglobulin kappa light chain-binding nanobody showed antitumour efficacy in colorectal carcinoma and melanoma models.

Interferon-induced ubiquitin (Ub)-like modifier Interferon Stimulated Gene 15 (ISG15) functions both intracellularly and as a secreted protein with cytokine-like properties. The ISG15 pathway is implicated in various diseases, including cancer and inflammatory disorders, but understanding its precise roles has been challenging because of limited availability of tools to study ISG15 biology. Here, we report the development of two novel nanobodies that target human ISG15, obtained through alpaca immunization and phage display. These nanobodies, VHHISG15-A and VHHISG15-B, exhibit nanomolar binding affinities and recognize distinct epitopes on ISG15's C- and N-terminal domains, respectively, as demonstrated by NMR and X-ray structural analyses. Both nanobodies enable the immunoprecipitation and proteomic identification of ISGylated substrates with minimal background contamination. VHHISG15-A is compatible with immunoblotting and recognizes unconjugated ISG15 under denaturing conditions. Functional assays showed that VHHISG15-A, but not VHHISG15-B, inhibits ubiquitin-specific peptidase 16-mediated deISGylation, likely by steric hindrance at the ISG15-binding interface. These results underscore the utility of VHHISG15-A and VHHISG15-B as tools to study ISG15 biology.

Two anti-transferrin receptor (TfR) nanobodies, V H H123 specific for mouse TfR and V H H188 specific for human TfR, were used to track transplants non-invasively by PET/CT in mouse models, without the need for genetic modification of the transferred cells. We provide a comparison of the specificity and kinetics of the PET signals acquired when using nanobodies radiolabeled with 89 Zr, 64 Cu, and 18 F, and find that the chelation of the 89 Zr and 64 Cu radioisotopes to anti-TfR nanobodies results in radioisotope release upon endocytosis of the radiolabeled nanobodies. We used a knock-in mouse that expresses a TfR with a human ectodomain (Tfrc hu/hu ) as a source of bone marrow for transplants into C57BL/6 recipients and show that V H H188 detects such transplants by PET/CT. Conversely, C57BL/6 bone marrow and B16.F10 melanoma cell line transplanted into Tfrc hu/hu recipients can be imaged with V H H123. In C57BL/6 mice impregnated by Tfrc hu/hu males, we saw an intense V H H188 signal in the placenta, showing that TfR-specific V H Hs accumulate at the placental barrier but do not enter the fetal tissue. We were unable to observe accumulation of the anti-TfR radiotracers in the central nervous system (CNS) by PET/CT but showed evidence of CNS accumulation by radiospectrometry. The model presented here can be used to track many transplanted cell types by PET/CT, provided cells express TfR, as is typically the case for proliferating cells such as tumor lines.

Conditional therapeutics that rely on disease microenvironment-specific triggers for activation are a promising strategy to improve therapeutic cargos. Among the investigated triggers, protease activity is used most often because of its dysregulation in several diseases. How to optimally fine-tune protease activation for different therapeutic cargos remains a challenge. Here, we designed nanobody-targeted conditional antimicrobial therapeutics to deliver a model therapeutic peptide and protein to the site of bacterial infection. We explored several parameters that influence proteolytic activation. We report the use of targeting nanobodies to enhance the activation of therapeutics that are otherwise activated inefficiently despite extensive optimization of the cleavable linker. Specifically, the pairing of Ly6G/C or ADAM10-targeting nanobodies with ADAM10-cleavable linkers improved activation via proximity-enabled reactivity. This study demonstrates a distinct role of active targeting in conditional therapeutic activation. More broadly, this optimization framework provides a guideline for the development of conditional therapeutics to treat various diseases in which protease activity is dysregulated.

Expression of the BCR is essential for survival, development, and effector functions of B cells. Naive B cells express surface IgM and IgD, while surface IgG1 is expressed by class-switched (memory) B cells. Despite similar overall structures, the different BCR isotypes show differences in distribution and expression levels. The dynamics of BCR behavior have been difficult to explore owing to a lack of appropriate tools that can track the BCR without causing concomitant activation. Using CRISPR-Cas9, we inserted a sortase recognition motif (LPETG [LeuProGluThrGly]) at the C-terminus of the OB1 transnuclear ovalbumin-specific Cκ chain (Igκ-LPETG mice). The surface BCR from Igκ-LPETG mice is fully functional and can be labeled site-specifically with biotin or fluorophores. Igκ-LPETG mice show near-normal B-cell development, with an increase in Igλ-producing cells, presumably due to massive contraction of the κ locus V-region cluster upon V-J recombination to generate the OB1 light chain. Using the Igκ-LPETG mice, we compared organization and density of BCRs on the surface of IgM/IgD+ B cells bearing a wild-type (WT) heavy chain locus and IgG1 B cells in the OB1 model. The density of IgG1 BCRs is much reduced compared to IgM/IgD BCRs on primary B cells. Upon activation, IgM/IgD BCRs are found in detergent-insoluble domains, whereas IgG1 BCRs are not. The isotype of the Ig heavy chain thus contributes to surface expression and nanoscale organization of the BCR.

Post-translational modifications modulate protein function, causing a variety of downstream consequences. Best known for its role in proteasomal degradation, ubiquitination is a post-translational modification also responsible for changing protein-protein interactions, the modification of protein localization, and the control of protein activity, to name a few. Ubiquitination involves addition on substrate lysine residues of a 76-residue ubiquitin (Ub) polypeptide, executed by a cascade of 3 enzymes. Ub itself has 7 lysine residues and can form poly-Ub chains of different topologies, recognized by appropriately specific Ub-binding proteins. The family of E2-type ubiquitin conjugating enzymes transfers an activated Ub from an Ub-activating enzyme (E1) to the substrate, facilitated by a member of the E3 family of Ub ligases. Humans have ∼40 E2s (including E2s for ubiquitin-like proteins) that share a highly conserved ubiquitin conjugating domain. How these structurally similar E2s compete for E1∼Ub and engage with a multitude of specific E3s is not well understood and hampers our understanding of how thousands of substrates are targeted. We address this gap in knowledge by using nanobodies as tools. Nanobodies are the recombinantly expressed variable regions sourced from camelid heavy-chain-only antibodies. We obtained a set of E2-specific nanobodies by screening of several nanobody libraries constructed from alpacas immunized with purified recombinant E2s. We explored in detail the biochemical properties of a nanobody specific for Ube2G2, an E2 involved in endoplasmic reticulum-associated protein degradation (ERAD). As nanobodies can be expressed in a cytoplasmic environment with retention of binding properties, they offer a unique possibility to probe the importance Ube2G2 in a cellular context without the need for genetic alterations of Ube2G2.

The Class I MHC molecule (MHC-I) HLA-E presents peptides that are derived from the signal sequences, either those of other MHC-I products, or of viral type I membrane glycoproteins. Monoclonal antibodies with proven specificity for HLA-E, and with no cross-reactions with other MHC-I products, have yet to be described. To obtain anti-HLA-E-specific antibodies suitable for a range of applications, we generated monoclonal antibodies against a unique feature of HLA-E: its cytoplasmic tail. We created an immunogen by performing an enzymatically catalyzed transpeptidation reaction to obtain a fusion of the cytoplasmic tail of HLA-E with a nanobody that recognizes murine Class II MHC (MHC-II) products. We obtained a mouse monoclonal antibody that recognizes a 13-residue stretch in the HLA-E cytoplasmic tail. We cloned the genes that encode this antibody in expression vectors to place an LPETG sortase recognition motif at the C-terminus of the heavy and light chains. This arrangement allows the site-specific installation of fluorophores or biotin at these C-termini. The resulting immunoglobulin preparations, labeled with 4 equivalents of a fluorescent or biotinylated payload of choice, can then be used for direct immunofluorescence or detection of the tag by fluorescence or by streptavidin-based methods. We also show that the 13-residue sequence can serve as an epitope tag, independent of the site of its placement within a protein's sequence. The antibody can be used diagnostically to stain for HLA-E on patient tumor samples, it can be used as an antibody-epitope tag for extracellular proteins, and it enables research into the unique role of the cytoplasmic tail of HLA-E.

The glycoproteins MICA and MICB are upregulated on the surface of cells undergoing stress, for instance due to (viral) infection or malignant transformation. MICA/B are the ligands for the activating receptor NKG2D, found on cytotoxic immune cells like NK cells, CD8+ T cells, and gamma delta T cells. Upon engagement of NKG2D, these cells are activated to eradicate the MICA/B-positive targets, assisted by the secretion of cytokines. Nanobodies, or VHHs, are derived from the variable regions of camelid heavy-chain only immunoglobulins. Nanobodies are characterized by their small size, ease of production, stability, and specificity of recognition. We generated nanobodies that recognize membrane-bound MICA with high affinity. Here, we use these nanobodies as building blocks for a chimeric antigen receptor (CAR) to establish VHH-based CAR NK cells. These anti-MICA nanobody-based CAR NK cells recognize and selectively kill MICA-positive tumor cells in vitro and in vivo. We track localization of the VHH-based CAR NK cells to MICA-positive lung metastases by immuno-positron emission tomography imaging.

Mycobacterium tuberculosis (Mtb) is known to survive within macrophages by compromising the integrity of the phagosomal compartment in which it resides. This activity primarily relies on the ESX-1 secretion system, predominantly involving the protein duo ESAT-6 and CFP-10. CFP-10 likely acts as a chaperone, while ESAT-6 likely disrupts phagosomal membrane stability via a largely unknown mechanism. we employ a series of biochemical analyses, protein modeling techniques, and a novel ESAT-6-specific nanobody to gain insight into the ESAT-6’s mode of action. First, we measure the binding kinetics of the tight 1:1 complex formed by ESAT-6 and CFP-10 at neutral pH. Subsequently, we demonstrate a rapid self-association of ESAT-6 into large complexes under acidic conditions, leading to the identification of a stable tetrameric ESAT-6 species. Using molecular dynamics simulations, we pinpoint the most probable interaction interface. Furthermore, we show that cytoplasmic expression of an anti-ESAT-6 nanobody blocks Mtb replication, thereby underlining the pivotal role of ESAT-6 in intracellular survival. Together, these data suggest that ESAT-6 acts by a pH dependent mechanism to establish two-way communication between the cytoplasm and the Mtb-containing phagosome.

Bispecific antibody engagers are fusion proteins composed of a nanobody that recognizes immunoglobulin kappa light chains ( VHH kappa ) and a nanobody that recognizes either CTLA-4 or PD-L1. These fusions show strong antitumor activity in mice through recruitment of polyclonal immunoglobulins independently of specificity or isotype. In the MC38 mouse model of colorectal carcinoma, the anti-CTLA-4 VHH-VHH kappa conjugate eradicates tumors and reduces the number of intratumoral regulatory T cells. The anti-PD-L1 VHH-VHH kappa conjugate is less effective in the MC38 model, whilst still outperforming an antibody of similar specificity. The potency of the anti-PD-L1 VHH-VHH kappa conjugate was strongly enhanced by installation of the cytotoxic drug maytansine or a STING agonist. The ability of such fusions to engage the Fc-mediated functions of all immunoglobulin isotypes is an appealing strategy to further improve on the efficacy of immune checkpoint blockade, commonly delivered as a monoclonal immunoglobulin of a single defined isotype.

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) impacts multiple anatomical sites. Whether this is due to the virus itself or is a secondary effect caused by the influx and activation of immune cells is not known. Positron emission tomography (PET) with immunoglobulins can provide insights into which sites and cells are activated in a living animal. Our aim is to use two nanobodies as tools to monitor (1) the distribution of antigen presenting cells (APC) by virtue of their Mafa-DR expression profile, (2) virus-infected cells and viral particles using a nanobody against the SARS-CoV-2 spike protein. Two [89Zr]-labeled nanobodies that target the SARS-CoV-2 spike protein and major histocompatability complex (MHC) class II antigens (Mafa-DR), respectively, are used to monitor their distribution during an experimental SARS-CoV-2 infection in a nonhuman primate model. Scans are obtained before infection and on Day 3 and 10 post infection (pi) in two macaques each. The [89Zr]anti-SARS-CoV-2 spike nanobody localized to SARS-CoV-2-associated lung lesions and the nasal mucosa, while the [89Zr]anti-human leukocyte antigen (HLA)-DR nanobody was predominantly found in non-affected lung tissue after infection. We also detected, pi, upregulation of the Mafa-DR signal, indicative of recruitment of professional APCs, in the superior sagittal sinus. [89Zr]-labeled nanobodies show recruitment of macrophages/monocytes in non-lesional lung tissue in cynomolgus macaques after experimental infection with SARS-CoV-2, as well as accumulation of the spike protein in both lung lesions and the nasal mucosa during infection. These results show the possibility of in vivo monitoring the quality and quantity of immune responses during the initial stages of an infection.

Inflammasome activation results in the cleavage of gasdermin D (GSDMD) by pro-inflammatory caspases. The N-terminal domains (GSDMDNT) oligomerize and assemble pores penetrating the target membrane. As methods to study pore formation in living cells are insufficient, the order of conformational changes, oligomerization, and membrane insertion remained unclear. We have raised nanobodies (VHHs) against human GSDMD and find that cytosolic expression of VHHGSDMD-1 and VHHGSDMD-2 prevents oligomerization of GSDMDNT and pyroptosis. The nanobody-stabilized GSDMDNT monomers partition into the plasma membrane, suggesting that membrane insertion precedes oligomerization. Inhibition of GSDMD pore formation switches cell death from pyroptosis to apoptosis, likely driven by the enhanced caspase-1 activity required to activate caspase-3. Recombinant antagonistic nanobodies added to the extracellular space prevent pyroptosis and exhibit unexpected therapeutic potential. They may thus be suitable to treat the ever-growing list of diseases caused by activation of (non-) canonical inflammasomes. Inflammasome assembly promotes the cleavage and oligomerisation of gasdermin D (GSDMD) and subsequent pore formation. Here the authors raise nanobodies to human gasdermin and characterize the pore formation process mediated by GSDMD and how antagonistic nanobodies prevent pyroptosis.