Nanobody Research Articles

Isolation of camel single domain antibodies against Yersinia pestis V270 antigen based on a semi-synthetic single domain antibody library and development of a VHH-based lateral flow assay.

Antibodies have been proven effective as diagnostic agents for detecting zoonotic diseases. The variable domain of camel heavy chain antibody (VHH), as an antibody derivative, may be used as an alternative for traditional antibodies in existing immunodiagnostic reagents for detecting rapidly spreading infectious diseases. To expedite the isolation of specific antibodies for diagnostic purposes, we constructed a semi-synthetic camel single domain antibody library based on the phage display technique platform (PDT) and verified the validity of this study. The semi-synthetic single domain antibody sequences consist of two parts: one is the FR1-FR3 region amplified by RT-PCR from healthy camel peripheral blood lymphocytes (PBLs), and the other part is the CDR3-FR4 region synthesised as an oligonucleotide containing CDR3 randomised region. The two parts were fused by overlapping PCR, resulting in the rearranged variabledomain of heavy-chain antibodies (VHHs). Y. pestis low-calcium response V protein (LcrV) is an optional biomarker to detect the Y. pestis infection. The semi-synthetic library herein was screened using recombinant (LcrV) as a target antigen. After four cycles of panning the library, four VHH binders targeting 1-270 aa residues of LcrV were isolated. The four VHH genes with unique sequences were recloned into an expression vector and expressed as VHH-hFc chimeric antibodies. The purified antibodies were identified and used to develop a lateral flow immunoassay (LFA) test strip using latex microspheres (LM) for the rapid and visual detection of Y. pestis infection. These data demonstrate the great potential of the semi-synthetic library for use in isolation of antigen-specific nanobodies and the isolated specific VHHs can be used in antigen-capture immunoassays.

Identification and biophysical characterization of a novel domain-swapped camelid antibody specific for fentanyl

Opioid use disorders (OUD) and overdoses are ever-evolving public health threats that continue to grow in incidence and prevalence in the United States and abroad. Current treatments consist of opioid receptor agonists and antagonists, which are safe and effective but still suffer from some limitations. Murine and humanized monoclonal antibodies (mAb) have emerged as an alternative and complementary strategy to reverse and prevent opioid-induced respiratory depression. To explore antibody applications beyond traditional heavy-light chain mAbs, we identified and biophysically characterized a novel single-domain antibody specific for fentanyl from a camelid variable-heavy-heavy (VHH) domain phage display library. Structural data suggested that VHH binding to fentanyl was facilitated by a unique domain-swapped dimerization mechanism, which accompanied a rearrangement of complementarity-determining region loops leading to the formation of a fentanyl-binding pocket. Structure-guided mutagenesis further identified an amino acid substitution that improved the affinity and relaxed the requirement for dimerization of the VHH in fentanyl binding. Our studies demonstrate VHH engagement of an opioid and inform on how to further engineer a VHH for enhanced stability and efficacy, laying the groundwork for exploring the invivo applications of VHH-based biologics against OUD and overdose.

A Potent Sybody Selectively Inhibits α-Synuclein Amyloid Formation by Binding to the P1 Region.

Increasing research efforts focus on exploiting antibodies to inhibit the amyloid formation of neurodegenerative proteins. Nevertheless, it is challenging to discover antibodies that inhibit this process in a specific manner. Using ribosome display, we screened for synthetic single-domain antibodies, i.e., sybodies, of the P1 region of α-synuclein (residues 36-42), a protein that forms amyloid in Parkinson's disease and multiple-system atrophy. Hits were assessed for direct binding to a P1 peptide and the inhibition of amyloid formation. We discovered a sybody, named αSP1, that inhibits amyloid formation of α-synuclein at substoichiometric concentrations in a specific manner, even within highly crowded heterogeneous mixtures. Fluorescence resonance energy transfer-based binding assays and seeding experiments with and without αSP1 further demonstrate the importance of the P1 region for both primary and secondary nucleation mechanisms of amyloid assembly.

Investigating the Diagnostic and Therapeutic Potential of a T Cell Receptor (TCR)-like single Domain Antibody (sDAb)-Human IgG1 Antibody against Heat Shock Protein (HSP) 16KDa/HLA-A2 for Latent Tuberculosis.

Heat shock protein 16-kDa (HSP 16-kDa) is essential for the survival of Mycobacterium tuberculosis (M. tuberculosis) during the latent period; hence, a peptide-MHC presentation of HSP 16-kDa could be a potential diagnostic and therapeutic target for latent tuberculosis (LTB). This study aimed to generate a TCR-like single-domain antibody (sDAb)-human IgG1 antibody and subsequently investigate its diagnostic and therapeutic potential in LTB, utilizing a model cell presenting the target peptide. A previously generated TCR-like sDAB that can bind to HSP 16-kDa was first fused to a human IgG1 Fc-receptor via a linker. The fusion product, sDAb-IgG1, was expressed with HEK293-F and was subsequently purified. Its diagnostic potential was investigated via cell-based ELISA utilizing MCF-7 cells peptide-pulsed with HSP 16-kDa peptides. Investigation into the antibody-dependent cell-mediated cytotoxicity (ADCC) of MCF-7 cells was also conducted to investigate its therapeutic potential. Finally, TCR-like sDAb-IgG1 was successfully produced transiently with HEK-293F and was purified using protein A chromatography. The generated antibody was tested using cell-based ELISA, which demonstrated the effective binding of the TCR-like sDAb-IgG1 to the 16-kDa peptide-MHC on the cell surface. The ADCC assay also showed that the antibody effectively mediated the ADCC of MCF-7 cells with the help of 16-kDa peptide-MHC. This allows us to hypothesize the possible utility of the said antibody for both diagnostics and therapeutics of latent tuberculosis after more investigations with clinical samples.

Fit-for-purpose heterodivalent single-domain antibody for gastrointestinal targeting of toxin B from Clostridium difficile.

Single-domain antibodies (sdAbs), such as VHHs, are increasingly being developed for gastrointestinal (GI) applications against pathogens to strengthen gut health. However, what constitutes a suitable developability profile for applying these proteins in a gastrointestinal setting remains poorly explored. Here, we describe an in vitro methodology for the identification of sdAb derivatives, more specifically divalent VHH constructs, that displayextraordinary developability properties for oral delivery and functionality in the GI environment. We showcase this by developing a heterodivalent VHH construct that cross-inhibits the toxic activity of the glycosyltransferase domains (GTDs) from three different toxinotypes of cytotoxin B (TcdB) from lineages of Clostridium difficile. We show that the VHH construct possesseshigh stability and binding activity under gastric conditions, in the presence of bile salts, and at high temperatures. We suggest that the incorporation of early developability assessment could significantly aid in the efficient discovery of VHHs and related constructs fit for oral delivery and GI applications.

Reversal of carbapenem resistance in Pseudomonas aeruginosa by camelid single domain antibody fragment (VHH) against the C4-dicarboxylate transporter.

Antimicrobial resistance is emerging as the new healthcare crisis necessitating the development of newer classes of drugs using non-traditional approaches. Pseudomonas aeruginosa, one of the most common pathogens involved in nosocomial infections, is extremely difficult to treat even with the last resort frontline drug, the carbapenems. As the pathogen has the ability to acquire resistance to new small-molecule antibiotics, being deployed, a novel biological approach has been tried using antibody fragments in combination with carbapenems and β-lactams as adjunct therapy for an enduring solution to the problem. In this study, we developed a camelid antibody fragment (VHH) library against P. aeruginosa and isolated a highly potent hit, PsC23. Mass spectrometry identified the target to be a component of the C4-dicarboxylate transporter that feeds metabolites to the glyoxylate shunt particularly under conditions of oxidative stress. PsC23 is bacteriostatic at a concentration of 1.66 µM (25 µg ml-1) and shows a synergistic effect with both the classes of drugs at an effective concentration of 100-200 nM (1.5-3.0 µg ml-1) when co administered with them. In combination with meropenem the VHH completely cleared the infection from a neutropenic mouse with a carbapenem-resistant P. aeruginosa systemic infection. Blocking the glyoxylate shunt by PsC23 resulted in disruption of energy transduction due to a respiratory shift to the oxygen-depleted TCA cycle causing inhibition of efflux and increased free radical generation from carbapenems and β-lactams exerting a strong bactericidal effect that reversed the resistance to multiple unrelated drugs.

Selection and evaluation of single domain antibody against p19 subunit of IL-23 by phage display for potential use as an autoinflammatory therapeutic

IL-23 is a double-subunit cytokine that plays an important role in shaping the immune response. IL-23 was found to be associated with several autoinflammatory diseases by generating sustained inflammatory loops that lead to tissue damage. Antibody neutralization of IL-23 was proven to be effective in ameliorating associated diseases. However, antibodies as large proteins have limited tissue penetration and tend to elicit anti-drug antibodies. Additionally, anti-IL-23 antibodies target only one subunit of IL-23 leaving the other one unneutralized. Here, we attempted to isolate a recycling single domain antibody by phage display. One of IL-23 subunits, p19, was expressed in E. coli fused to Gamillus protein to stabilize the α-helix-only p19. To remove Gamillus binders, two biopanning methods were investigated, first, preselection with Gamillus and second, challenge with IL-23 then on the subsequent round challenge with p19-Gam. The isolation of calcium-dependent and pH-dependent recycling binders was performed with EDTA and citrate buffers respectively. Both methods of panning failed to isolate high-affinity and specific p19 recycling binders, while from the second panning method, a high affinity and specific p19 standard binder, namely H11, was successfully isolated. H11 significantly inhibited the gene expression of IL-17 and IL-22 in IL-23-challenged PBMCs indicating H11 specificity and neutralizing ability for IL-23. The new binder due to its small size can overcome antibodies limitations, also, it can be further engineered in the future for antigen clearance such as fusing it to cell penetrating peptides, granting H11 the ability to clear excess IL-23 and enhancing its potential therapeutic effect.

High efficacy of huCD20-targeted AcTaferon in humanized patient derived xenograft models of aggressive B cell lymphoma

Type I interferon (IFN) is a potent antitumoral drug, with an important history in the treatment of hematologic malignancies. However, its pleiotropic nature leads to severe dose-limiting toxicities that blunt its therapeutic potential. To achieve selective targeting of specific immune or tumor cells, AcTakines (Activity-on-Target Cytokines), i.e., immunocytokines utilizing attenuated cytokines, and clinically optimized A-Kines™ were developed. In syngeneic murine models, the CD20-targeted murine IFNα2-based AcTaferons (AFNs) have demonstrated clear antitumoral effects, with excellent tolerability. The current study explores the antitumoral potential of the humanized huCD20-Fc-AFN in 5 different humanized patient derived xenograft (PDX) models of huCD20+ aggressive B non-Hodgkin lymphomas (B-NHLs). The huCD20-Fc-AFN consists of a huCD20-specific single-domain antibody (VHH) linked through a heterodimeric ‘knob-in-hole’ human IgG1 Fc molecule to an attenuated huIFNα2 sequence. An in vitro targeting efficacy of up to 1.000-fold could be obtained, without detectable in vivo toxicities, except for selective (on-target) and reversible B cell depletion. Treatment with huCD20-Fc-AFN significantly increased the median overall survival (mOS) in both non-humanized (mOS 31 to 45 days; HR = 0.26; p = 0.001), and humanized NSG/NOG mice (mOS 34 to 80 days; HR = 0.37; p < 0.0001). In humanized mice, there was a trend for increased survival when compared to equimolar rituximab (mOS 49 to 80 days; HR = 0.73; p = 0.09). The antitumoral effects of huCD20-Fc-AFN were partly due to direct effects of type I IFN on the tumor cells, but additional effects via the human immune system are essential to obtain long-term remissions. To conclude, huCD20-Fc-AFN could provide a novel therapeutic strategy for huCD20-expressing aggressive B-NHLs.Graphical

Design of targeted antiviral polypeptides specific to SARS-CoV-2. Challenges and prospects

The COVID-19 epidemic demanded the rapid development of high-affinity molecules of different types aimed at a single target, the S-protein of SARS-CoV-2. The simultaneous development and testing of such molecules provide a unique opportunity to compare the features of biotechnological platforms for creating therapeutic proteins. This review considers classical antibodies, variable lymphocyte receptors, single-domain antibodies, and artificial scaffolds (DARPins, affibodies, VH), that are compared in terms of affinity, neutralizing activity, size and compatibility with different delivery methods. It can be concluded that all platforms used have produced high-affinity proteins that specifically bind to the coronavirus S-protein. The highest affinity of the targeting molecules with the virus protein was achieved by developing classical antibodies, nanobodies and by combining several binding modules into multivalent constructs with high avidity. Based on the results of &lt;i&gt;in vivo&lt;/i&gt; experiments, it can be concluded that a high affinity of the therapeutic protein for the surface antigens of SARS-CoV-2 is a necessary but not sufficient condition for suppression of COVID-19 due to the peculiarities of the biology of this virus. The experience gained in the development of therapeutic agents against coronavirus will be useful for design of effective targeted drugs for the treatment of known and new viral infections.&lt;br&gt;The bibliography includes 126 references.

A phase I/II open label, multicenter study to assess the safety, tolerability, pharmacokinetics, and efficacy of HB0028 in patients with advanced solid tumors.

2528 Background: HB0028 is a bifunctional fusion protein consisting of an anti-PD-L1 IgG1 single domain antibody and TGF-β RII receptor extracellular domain (TGFβRII-ECD). This is the first dose escalation and expansion phase I/II study to evaluate the safety and preliminary anti-tumor activity of HB0028 in advanced solid tumors. Methods: In the phase I portion of the study, the patients (pts) with advanced solid tumor were enrolled, and an accelerated titration followed by a standard 3+3 design was applied to dose escalation. The objectives were toxicity evaluation, maximum tolerable dose (MTD), and recommended phase 2 dose (RP2D). Eligible pts with advanced solid cancers of any histologic subtype, and ECOG performance status ≤ 1 were treated with HB0028 (0.3mg, 1mg, 3mg, 10mg, 20mg, Q3W), until unacceptable toxicity or disease progression. Radiologic tumor assessments (by RECIST v1.1) were performed every 6 weeks while on treatment. Results: 16 pts [12 females and 4 males; ECOG 0/1, 9/7; median age 52 years (range 36 – 60)] were enrolled in phase I (0.3 mg/kg [n = 1], 1 mg/kg [n = 3], 3 mg/kg [n = 3], 10 mg/kg [n = 3], 20 mg/kg [n = 6]). Median number of prior lines of systemic therapy was 2 (range 1-4). 8(50%) pts received and progressed upon prior anti-PD-1/L1 therapy. All pts had measurable metastatic disease. No DLT and Death occurred. MTD was not reached. As of DEC 25,2023, among 16 patients' safety data are evaluable for toxicity. Any grade treatment‐related adverse events (TRAEs) occurred in 15 subjects (93.8%), with 2 subjects (12.5%) reported with ≥ Grade 3 (G3 Anemia and G3 γ-glutamyltransferase). The most common TRAEs (all grades) were AST increased (n = 4, 25%), anemia (n = 7, 43.8%) and infusion-related response (n = 5, 31.3%). Two SAEs (G2 AST increased and G3 Neoplastic fevers) were reported, only AST increased was considered as possibly related to study drug, but recovered shortly. The efficacy data cutoff date of DEC 25,2023, 16 patients are evaluable for radiologic response. 1 subject with cervical cancer in HB0028 20 mg/kg group had a partial response (PR), 3 subjects (liver cancer, lung cancer, left submandibular gland cancer) had stable disease (SD) and 12 subjects had progressive disease (PD). The ORR per RECIST 1.1 by investigator was 6.25% (1/16; 95% CI, 0%-30.2%), and the DCR was 25% (4/16; 95% CI, 7.3%-52.4%). Durable clinical benefit (SD for &gt; 32 weeks) was seen in 1 subject with malignant tumor in the left submandibular gland. To date, 2 responders are still on treatment. Conclusions: HB0028 was generally well tolerated, and therapy provided modest antitumor activity in patients with heavily pretreated advanced solid tumors. Based on these data, a phase II prospective clinical trial is being planned in specific cancer. Clinical trial information: NCT06223308 .

A β-catenin chromobody-based probe highlights endothelial maturation during vascular morphogenesis in vivo.

Visualization of protein dynamics is a crucial step in understanding cellular processes. Chromobodies, fluorescently labelled single-domain antibodies, have emerged as versatile probes for live cell imaging of endogenous proteins. However, how these chromobodies behave in vivo and how accurately they monitor tissue changes remain poorly explored. Here, we generated an endothelial-specific β-catenin chromobody-derived probe and analyzed its expression pattern during cardiovascular development in zebrafish. Using high-resolution confocal imaging, we show that the chromobody signal correlates with the localization of β-catenin in the nucleus and at cell-cell junctions, and thereby can be used to assess endothelial maturation. Loss of Cadherin 5 strongly affects the localization of the chromobody at the cell membrane, confirming the cadherin-based adherens junction role of β-catenin. Furthermore, using a genetic model to block blood flow, we observed that cell junctions are compromised in most endothelial cells but not in the endocardium, highlighting the heterogeneous response of the endothelium to the lack of blood flow. Overall, our data further expand the use of chromobodies for in vivo applications and illustrate their potential to monitor tissue morphogenesis at high resolution.

Shark-derived single-domain antibody as a new and effective recognition element for norfloxacin detection

Low specificity and sensitivity often restrict the fluoroquinolones (FQs) detection when traditional IgG antibodies are used as recognition elements in immunoassay. Herein, shark-derived single-domain antibodies (ssdAbs) were used as new recognition elements to enhance the sensitivity and specificity for norfloxacin detection in immunoassay. Firstly, NOR-specific clones were isolated by phage display technology. Finally, five clones were successfully isolated from a NOR-immunized library. Secondly, a soluble expression system was constructed to produce ssdAbs. The 1N9 ssdAb exhibits the best affinity to the NOR complete antigen, with a limit of detection of 0.392 ng/mL and the cross-reaction rates of less than 8.240 %, indicating its high sensitivity and specificity as a new recognition element in NOR detection. More importantly, the ELISA result proved that in NOR-negative fish matrix, no serious interference was observed, the 1N9 ssdAb could still recognize complete antigen NOR-OVA effectively. While in icELISA, 1N9 ssdAb could recognize NOR in different NOR-spiked concentrations between 10 and 1000 ng/mL, with the recovery rates between 93.66 % and 116.49 % with high reproducibility. Our study provides a new and effective recognition element with great potential for NOR detection.

Single-domain antibody-based protein degrader for synucleinopathies

Synucleinopathies are a group of neurodegenerative diseases characterized by the accumulation of α-synuclein (α-syn) in the brain, leading to motor and neuropsychiatric symptoms. Currently, there are no known cures for synucleinopathies, and treatments mainly focus on symptom management. In this study, we developed a single-domain antibody (sdAb)-based protein degrader with features designed to enhance proteasomal degradation of α-syn. This sdAb derivative targets both α-syn and Cereblon (CRBN), a substrate-receptor for the E3-ubiquitin ligase CRL4CRBN, and thereby induces α-syn ubiquitination and proteasomal degradation. Our results indicate that this therapeutic candidate enhances proteasomal degradation of α-syn, in addition to the endogenous lysosomal degradation machinery. By promoting proteasomal degradation of α-syn, we improved clearance of α-syn in primary culture and mouse models of synucleinopathy. These findings indicate that our sdAb-based protein degrader is a promising therapeutic candidate for synucleinopathies. Considering that only a small percentage of antibodies enter the brain, more potent sdAbs with greater brain entry than whole antibodies could enhance clinical benefits of antibody-based therapies.

Structural basis for selectivity and antagonism in extracellular GPCR-nanobodies

G protein-coupled receptors (GPCRs) are pivotal therapeutic targets, but their complex structure poses challenges for effective drug design. Nanobodies, or single-domain antibodies, have emerged as a promising therapeutic strategy to target GPCRs, offering advantages over traditional small molecules and antibodies. However, an incomplete understanding of the structural features enabling GPCR-nanobody interactions has limited their development. In this study, we investigate VUN701, a nanobody antagonist targeting the atypical chemokine receptor 3 (ACKR3). We determine that an extended CDR3 loop is required for ACKR3 binding. Uncommon in most nanobodies, an extended CDR3 is prevalent in GPCR-targeting nanobodies. Combining experimental and computational approaches, we map an inhibitory ACKR3-VUN701 interface and define a distinct conformational mechanism for GPCR inactivation. Our results provide insights into class A GPCR-nanobody selectivity and suggest a strategy for the development of these new therapeutic tools.

Welding PROxAb Shuttles: A Modular Approach for Generating Bispecific Antibodies via Site-Specific Protein-Protein Conjugation.

Targeted protein degradation is an innovative therapeutic strategy to selectively eliminate disease-causing proteins. Exemplified by proteolysis-targeting chimeras (PROTACs), they have shown promise in overcoming drug resistance and targeting previously undruggable proteins. However, PROTACs face challenges, such as low oral bioavailability and limited selectivity. The recently published PROxAb Shuttle technology offers a solution enabling the targeted delivery of PROTACs using antibodies fused with PROTAC-binding domains derived from camelid single-domain antibodies (VHHs). Here, a modular approach to quickly generate PROxAb Shuttles by enzymatically coupling PROTAC-binding VHHs to off-the-shelf antibodies was developed. The resulting conjugates retained their target binding and internalization properties, and incubation with BRD4-targeting PROTACs resulted in formation of defined PROxAb-PROTAC complexes. These complexes selectively induced degradation of the BRD4 protein, resulting in cytotoxicity specifically to cells expressing the antibody's target. The chemoenzymatic approach described herein provides a versatile and efficient solution for generating antibody-VHH conjugates for targeted protein degradation applications, but it could also be used to combine antibodies and VHH binders to generate bispecific antibodies for further applications.

Development of a single-domain antibody to target a G-quadruplex located on the hepatitis B virus covalently closed circular DNA genome.

To achieve a virological cure for hepatitis B virus (HBV), innovative strategies are required to target the covalently closed circular DNA (cccDNA) genome. Guanine-quadruplexes (G4s) are a secondary structure that can be adopted by DNAand play a significant role in regulating viral replication, transcription, and translation. Antibody-based probes and small molecules have been developed to study the role of G4s in the context of the human genome, but none have been specifically made to target G4s in viral infection. Herein,we describe the development of a humanized single-domain antibody (S10) that can target a G4 located in the PreCore (PreC) promoter of the HBV cccDNA genome. MicroScale Thermophoresis demonstrated that S10 has a strong nanomolar affinity to the PreC G4 in its quadruplex form and a structural electron density envelope of the complex was determined using Small-Angle X-ray Scattering. Lentiviral transduction of S10 into HepG2-NTCP cells shows nuclear localization, and chromatin immunoprecipitation coupled with next-generation sequencing demonstrated that S10 can bind to the HBV PreC G4 present on the cccDNA. This research validates the existence of a G4 in HBV cccDNA and demonstrates that this DNA secondary structure can be targeted with high structural and sequence specificity using S10.

Camelid-derived Tcell engagers harnessing human γδ Tcells as promising antitumor immunotherapeutic agents.

In the last decade, there has been a surge in developing immunotherapies to enhance the immune system's ability to eliminate tumor cells. Bispecific antibodies known as Tcell engagers (TCEs) present an attractive strategy in this pursuit. TCEs aim to guide cytotoxic Tcells toward tumor cells, thereby inducing a strong activation and subsequent tumor cell lysis. In this study, we investigated the activity of different TCEs on both conventional alpha-beta (αβ) Tcells and unconventional gamma delta (γδ) Tcells. TCEs were built using camelid single-domain antibodies (VHHs) targeting the tumor-associated antigen CEACAM5 (CEA), together with Tcell receptor chains or a CD3 domain. We show that Vγ9Vδ2 Tcells display stronger in vitro antitumor activity than αβ Tcells when stimulated with a CD3xCEA TCE. Furthermore, restricting the activation of fresh human peripheral Tcells to Vγ9Vδ2 Tcells limited the production of protumor factors and proinflammatory cytokines, commonly associated with toxicity in patients. Taken together, our findings provide further insights that γδ Tcell-specific TCEs hold promise as specific, effective, and potentially safe molecules to improve antitumor immunotherapies.

Nanobody-liposomes as novel cancer vaccine platform to efficiently stimulate T cell immunity

Cancer vaccines can be utilized in combination with checkpoint inhibitors to optimally stimulate the anti-tumor immune response. Uptake of vaccine antigen by antigen presenting cells (APCs) is a prerequisite for T cell priming, but often relies on non-specific mechanisms. Here, we have developed a novel vaccination strategy consisting of cancer antigen-containing liposomes conjugated with CD169- or DC-SIGN-specific nanobodies (single domain antibodies) to achieve specific uptake by APCs. Our studies demonstrate efficient nanobody liposome uptake by human and murine CD169+ and DC-SIGN+ APCs in vitro and in vivo when compared to control liposomes or liposomes with natural ligands for CD169 and DC-SIGN. Uptake of CD169 nanobody liposomes resulted in increased T cell activation by human APCs and stimulated naive T cell priming in mouse models. In conclusion, while nanobody liposomes have previously been utilized to direct drugs to tumors, here we show that nanobody liposomes can be applied as vaccination strategy that can be extended to other receptors on APCs in order to elicit a potent immune response against tumor antigens.

Single domain antibodies from camelids in the treatment of microbial infections.

Infectious diseases continue to pose significant global health challenges. In addition to the enduring burdens of ailments like malaria and HIV, the emergence of nosocomial outbreaks driven by antibiotic-resistant pathogens underscores the ongoing threats. Furthermore, recent infectious disease crises, exemplified by the Ebola and SARS-CoV-2 outbreaks, have intensified the pursuit of more effective and efficient diagnostic and therapeutic solutions. Among the promising options, antibodies have garnered significant attention due to their favorable structural characteristics and versatile applications. Notably, nanobodies (Nbs), the smallest functional single-domain antibodies of heavy-chain only antibodies produced by camelids, exhibit remarkable capabilities in stable antigen binding. They offer unique advantages such as ease of expression and modification and enhanced stability, as well as improved hydrophilicity compared to conventional antibody fragments (antigen-binding fragments (Fab) or single-chain variable fragments (scFv)) that can aggregate due to their low solubility. Nanobodies directly target antigen epitopes or can be engineered into multivalent Nbs and Nb-fusion proteins, expanding their therapeutic potential. This review is dedicated to charting the progress in Nb research, particularly those derived from camelids, and highlighting their diverse applications in treating infectious diseases, spanning both human and animal contexts.

Structural basis of the Meinwald rearrangement catalysed by styrene oxide isomerase

Membrane-bound styrene oxide isomerase (SOI) catalyses the Meinwald rearrangement—a Lewis-acid-catalysed isomerization of an epoxide to a carbonyl compound—and has been used in single and cascade reactions. However, the structural information that explains its reaction mechanism has remained elusive. Here we determine cryo-electron microscopy (cryo-EM) structures of SOI bound to a single-domain antibody with and without the competitive inhibitor benzylamine, and elucidate the catalytic mechanism using electron paramagnetic resonance spectroscopy, functional assays, biophysical methods and docking experiments. We find ferric haem b bound at the subunit interface of the trimeric enzyme through H58, where Fe(III) acts as the Lewis acid by binding to the epoxide oxygen. Y103 and N64 and a hydrophobic pocket binding the oxygen of the epoxide and the aryl group, respectively, position substrates in a manner that explains the high regio-selectivity and stereo-specificity of SOI. Our findings can support extending the range of epoxide substrates and be used to potentially repurpose SOI for the catalysis of new-to-nature Fe-based chemical reactions.