Camelid Antibodies Research Articles

Two VHH Antibodies Neutralize Botulinum Neurotoxin E1 by Blocking Its Membrane Translocation in Host Cells.

Botulinum neurotoxin serotype E (BoNT/E) is one of the major causes of human botulism, which is a life-threatening disease caused by flaccid paralysis of muscles. After receptor-mediated toxin internalization into motor neurons, the translocation domain (HN) of BoNT/E transforms into a protein channel upon vesicle acidification in endosomes and delivers its protease domain (LC) across membrane to enter the neuronal cytosol. It is believed that the rapid onset of BoNT/E intoxication compared to other BoNT serotypes is related to its swift internalization and translocation. We recently identified two neutralizing single-domain camelid antibodies (VHHs) against BoNT/E1 termed JLE-E5 and JLE-E9. Here, we report the crystal structures of these two VHHs bound to the LCHN domain of BoNT/E1. The structures reveal that these VHHs recognize two distinct epitopes that are partially overlapping with the putative transmembrane regions on HN, and therefore could physically block membrane association of BoNT/E1. This is confirmed by our in vitro studies, which show that these VHHs inhibit the structural change of BoNT/E1 at acidic pH and interfere with BoNT/E1 association with lipid vesicles. Therefore, these two VHHs neutralize BoNT/E1 by preventing the transmembrane delivery of LC. Furthermore, structure-based sequence analyses show that the 3-dimensional epitopes of these two VHHs are largely conserved across many BoNT/E subtypes, suggesting a broad-spectrum protection against the BoNT/E family. In summary, this work improves our understanding of the membrane translocation mechanism of BoNT/E and paves the way for developing VHHs as diagnostics or therapeutics for the treatment of BoNT/E intoxication.

Nanobodies: The Future of Antibody-Based Immune Therapeutics.

Targeted therapy is a fast evolving treatment strategy to reduce unwanted damage to healthy tissues, while increasing efficacy and specificity. Driven by state-of-the-art technology, this therapeutic approach is especially true of cancer. Antibodies with their remarkable specificity have revolutionized therapeutic strategies for autoimmune conditions and cancer, particularly blood-borne cancers, but have severe limitations in treating solid tumors. This is mainly due to their large molecular size, low stability, tumor-tissue penetration difficulties, and pharmacokinetic properties. The tumor microenvironment, rich in immune-suppressing molecules is also a major barrier in targeting solid tumors by antibody-based drugs. Nanobodies have recently emerged as an alternative therapeutic agent to overcome some of the drawbacks of traditional antibody treatment. Nanobodies are the VHH domains found on the heavy-chain only antibodies of camelids and are the smallest naturally available antibody fragments with excellent antigen-binding specificity and affinity, equivalent to conventional antibodies but with molecular weights as low as 15 kDa. The compact size, high stability, enhanced hydrophilicity, particularly in framework regions, excellent epitope interactions with protruding CDR3 regions, and improved tissue penetration make nanobodies the next-generation therapeutics (Nano-BioDrugs). In this review, the authors discuss the interesting properties of nanobodies and their advantages over their conventional counterparts and provide insight into how nanobodies are being utilized as agonists and antagonists, bispecific constructs, and drug and enzyme-conjugates to combat the tumor microenvironment and treat disease.

Cats and llamas could offer a path to coronavirus therapies

Computation and experimentation also yield possible therapeutic compounds for COVID-19 [ABSTRACT FROM AUTHOR] Copyright of Physics Today is the property of American Institute of Physics and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission However, users may print, download, or email articles for individual use This abstract may be abridged No warranty is given about the accuracy of the copy Users should refer to the original published version of the material for the full abstract (Copyright applies to all Abstracts )

Synthetic nanobodies as angiotensin receptor blockers

There is considerable interest in developing antibodies as functional modulators of G protein-coupled receptor (GPCR) signaling for both therapeutic and research applications. However, there are few antibody ligands targeting GPCRs outside of the chemokine receptor group. GPCRs are challenging targets for conventional antibody discovery methods, as many are highly conserved across species, are biochemically unstable upon purification, and possess deeply buried ligand-binding sites. Here, we describe a selection methodology to enrich for functionally modulatory antibodies using a yeast-displayed library of synthetic camelid antibody fragments called "nanobodies." Using this platform, we discovered multiple nanobodies that act as antagonists of the angiotensin II type 1 receptor (AT1R). Following angiotensin II infusion in mice, we found that an affinity matured nanobody antagonist has comparable antihypertensive activity to the angiotensin receptor blocker (ARB) losartan. The unique pharmacology and restricted biodistribution of nanobody antagonists may provide a path for treating hypertensive disorders when small-molecule drugs targeting the AT1R are contraindicated, for example, in pregnancy.

Engineering an anti-granulocyte colony stimulating factor receptor nanobody for improved affinity

AimsGranulocyte colony-stimulating factor (G-CSF) is a cytokine that induces proliferation and differentiation of hematopoietic precursor cells and activation of mature neutrophils. G-CSF is overexpressed in several malignant tumors and blocking its binding to the receptor can lead to significant decrease in tumor growth, vascularization and metastasis. Furthermore, targeting G-CSF receptor has shown therapeutic benefit in other diseases such as rheumatoid arthritis, progressive neurodegenerative disorder and uveitis. Camelid single-chain antibodies (nanobodies) have exceptional properties making them appropriate for tumor imaging and therapeutic application. In this study we aim to use the rational design approach to engineer a previously described G-CSF-R targeting nanobody (VHH1), to improve its affinity toward G-CSF-R. Main methodsWe redesigned the complementary determining region 3 (CDR3) domain of the VHH1 nanobody to mimic G-CSF interaction to its receptor and developed five new engineered nanobodies. Binding affinity of the engineered nanobodies was evaluated by ELISA (Enzyme-linked immunosorbent assay) on NFS60 cells. Key findingsEnzyme-linked immunosorbent assay (ELISA) confirmed the specificity of the engineered nanobodies and ELISA-based determination of affinity revealed that two of the engineered nanobodies (1c and 5a) bind to G-CSF-R on the surface of NFS60 cells in a dose-dependent manner and with a higher potency compared to the parental nanobody. SignificanceAdditional studies are required to better characterize these nanobodies and assess their interaction with G-CSF-R in vitro and in vivo. These newly developed nanobodies could be beneficial in tumor imaging and therapy and make a basis for development of additional engineered nanobodies.

Finding Camel-ot: A Holy Grail against pandemic SARS-CoV-2?

Engineered camelid antibody multimers can potently block SARS-CoV-2 viral entry.

Generation and characterization of dromedary Tenascin-C and Tenascin-W specific antibodies

Tenascin-C (TNC) and tenascin-W (TNW), large hexameric glycoproteins overexpressed in the tumor microenvironment, are useful tumor biomarkers for theranostic applications. For now, polyclonal and monoclonal antibodies, as well as aptamers targeting TNC and TNW have been developed. However, the immunostaining sensitivity of antibodies is very heterogenous. The main aim of this study was to generate antibodies in dromedary that detect TNC and TNW, respectively. We show that immune sera from immunized dromedaries are able to specifically bind native TNC and TNW by ELISA and also to detect TNC and TNW in matrix tracks of mammary tumors by immunostaining. Furthermore, we demonstrate that purified IgG subtypes are able to interact specifically with TNC or TNW by ELISA and immunostaining. These camelid antibodies are a good basis to develop tools for the detection of TNC and TNW in the tumor microenvironment and could potentially have a broader application for early diagnosis of solid cancers.

Phase I dose escalation study of immunoconjugate L-DOS47 in combination with pemetrexed/carboplatin in non-squamous non-small cell lung cancer (NSCLC) patients.

e21680 Background: L-DOS47, a cancer therapeutic designed to exploit the acidic tumour extracellular environment, consists of urease conjugated to an anti-CEACAM6 camelid monoclonal antibody. The antibody serves as a targeting agent to deliver urease to tumor sites, while the urease enzyme converts urea, an abundant natural metabolite, into ammonia and generates a local pH increase. The combined effect of ammonia toxicity and pH increase is cytotoxic to cancer cells. An additive cytotoxic effect of L-DOS47 and pemetrexed/carboplatin has been observed against the A549 human lung adenocarcinoma cell line. L-DOS47 has been shown in a previous phase I clinical trial to be safe and well tolerated at doses up to 13.55 µg/kg. The current phase I study was designed to define the maximum tolerated dose of L-DOS47 in combination with pemetrexed/carboplatin. Methods: Stage IV (TNM M1a and M1b) histologically confirmed non-squamous NSCLC patients (aged ≥18 yrs, ECOG PS ≤1) received up to four cycles of L-DOS47 in combination with standard of care doses of pemetrexed/carboplatin. L-DOS47 was administered weekly over 21 days in each treatment cycle. Seven planned dosing cohorts (0.59, 0.78, 1.5, 3.0, 6.0, 9.0, 12.0 μg/kg) employed a standard 3+3 design for the first two and the last two cohorts, and an accelerated 1 + 2 design for the middle three cohorts. Dose levels of L-DOS47 were escalated following a review of safety data by the Trial Steering Committee. Results: Fourteen (14) patients were enrolled across 6 dosing cohorts before recruitment was halted. Median age was 65.1 years, with 50.0% male and 78.6% Caucasian. 57.1% of patients had an ECOG score of 0, and 42.9% had an ECOG score of 1. The maximum tolerated dose (MTD) could not be determined as none of the patients experienced a dose limiting toxicity (DLT). 50.0% of patients experienced at least one treatment emergent adverse event (TEAE) assessed as study drug-related, with 14.3% of patients experiencing at least one grade 3/4 drug-related toxicity. Of 12 patients evaluable for efficacy, 5 patients (41.7%) had a partial response (PR), 4 patients (33.3%) experienced stable disease (SD) and 3 patients (25.0%) had progressive disease (PD). The objective response rate is 41.7%. The clinical benefit rate is 75.0%. Conclusions: L-DOS47, in combination with pemetrexed/carboplatin, appears to be well tolerated with promising anti-tumor activity against non-squamous NSCLC. Clinical trial information: NCT02309892.

Tumor-specific near-infrared nanobody probe rapidly labels tumors in an orthotopic mouse model of pancreatic cancer

BackgroundNanobodies, derived from camelid antibodies made of only heavy chains, are the smallest, biologic, antigen-binding fragments (~15kDa) with faster pharmacokinetics and better tumor penetration efficiency than standard antibodies. The present study evaluates the efficacy of a fluorescent, anti-carcinoembryonic antigen (CEA) nanobody for rapid tumor labeling in an orthotopic mouse model of pancreatic cancer. MethodsAnti-CEA or control nanobodies were conjugated with the near-infrared fluorophore IRDye 800CW. Fragments of BxPC-3 (high-CEA expressing) or MiaPACA-2 (low-CEA expressing) human pancreatic cancer cell lines were orthotopically implanted into the pancreatic tail of nude mice. After tumors reached 7 to 10 mm in size, 2 nmol anti-CEA or control nanobody-IRDye800CW were injected intravenously. Mice were imaged at various time points hours post-injection. ResultsAnti-CEA nanobodies clearly labeled BxPC3 orthotopic pancreatic tumors 3 hours after injection. The signal was present as early as 15 minutes after injection and was robust at 1 to 3 hours after injection with a tumor-to-background ratio of 2.66. In contrast, there was very low accumulation in the low CEA-expressing, MiaPACA2 pancreatic orthotopic tumors. The fluorophore-conjugated nanobody was specific for CEA-expressing tumors, while the control nanobody did not show any tumor-specific signal. Both nanobodies had strong kidney uptake as expected for small-molecule probes. The fluorescence signal was detectable using 2 clinical, Food and Drug Administration-approved, 800 nm imaging devices as well as small animal imaging systems. ConclusionThis anti-CEA, nanobody-based, fluorescent probe labeled pancreatic orthotopic tumors within 15 minutes of intravenous injection. Fluorescent anti-CEA nanobodies have labeling kinetics that approach the speed of nonspecific dyes such as indocyanine green but with the specificity of antibodies. The use of fluorescently-labeled, intact antibodies leads to a labeling delay of 48 to 96 hours between probe administration and the necessarily delayed time of operation, which can be avoided with nanobodies. The kinetics of a nanobody-based probe makes it a practical agent for same-day, patient administration and fluorescence-guided surgery.

Structural Basis for Potent Neutralization of Betacoronaviruses by Single-Domain Camelid Antibodies

Coronaviruses make use of a large envelope protein called spike (S) to engage host cell receptors and catalyze membrane fusion. Because of the vital role that these S proteins play, they represent a vulnerable target for the development of therapeutics. Here, we describe the isolation of single-domain antibodies (VHHs) from a llama immunized with prefusion-stabilized coronavirus spikes. These VHHs neutralize MERS-CoV or SARS-CoV-1 S pseudotyped viruses, respectively. Crystal structures of these VHHs bound to their respective viral targets reveal two distinct epitopes, but both VHHs interfere with receptor binding. We also show cross-reactivity between the SARS-CoV-1 S-directed VHH and SARS-CoV-2 S and demonstrate that this cross-reactive VHH neutralizes SARS-CoV-2 S pseudotyped viruses as a bivalent human IgG Fc-fusion. These data provide a molecular basis for the neutralization of pathogenic betacoronaviruses by VHHs and suggest that these molecules may serve as useful therapeutics during coronavirus outbreaks.

Serum albumin-binding VH Hs with variable pH sensitivities enable tailored half-life extension of biologics.

Prolonged serum half-life is required for the efficacy of most protein therapeutics. One strategy for half-life extension is to exploit the long circulating half-life of serum albumin by incorporating a binding moiety that recognizes albumin. Here, we describe camelid single-domain antibodies (VH Hs) that bind the serum albumins of multiple species with moderate to high affinity at both neutral and endosomal pH and significantly extend the serum half-lives of multiple proteins in rats from minutes to days. We serendipitously identified an additional VH H (M75) that is naturally pH-sensitive: at endosomal pH, binding affinity for human serum albumin (HSA) was dramatically weakened and binding to rat serum albumin (RSA) was undetectable. Domain mapping revealed that M75 bound to HSA domain 1 and 2. Moreover, alanine scanning of HSA His residues suggested a critical role for His247, located in HSA domain 2, in M75 binding and its pH dependence. Isothermal titration calorimetry experiments were suggestive of proton-linked binding of M75 to HSA, with differing binding enthalpies observed for full-length HSA and an HSA domain 1-domain 2 fusion protein in which surface-exposed His residues were substituted with Ala. M75 conferred moderate half-life extension in rats, from minutes to hours, likely due to rapid dissociation from RSA during FcRn-mediated endosomal recycling in tandem with albumin conformational changes induced by M75 binding that prevented interaction with FcRn. Humanized VH Hs maintained in vivo half-life extension capabilities. These VH Hs represent a new set of tools for extending protein therapeutic half-life and one (M75)demonstrates a unique pH-sensitive binding interaction that can be exploited to achieve modest in vivo half-life.

Lessons learned from immunoadsorption for hyperviscosity in IgM multiple myeloma—A case report

We report the case of a 63‐year‐old Caucasian woman with multiple relapsed IgM multiple myeloma (MM) and elevated free kappa light chains (fκLC). Due to hyperviscosity syndrome with visual impairment, regular plasma exchanges were performed. As part of her 11th line of therapy, an experimental protocol consisting of pembrolizumab, pomalidomide, and dexamethasone was initiated. To reduce fκLC and immunoglobulin (Ig) M, we performed immunoadsorption (IA) using columns containing recombinant single domain camelid antibody fragments as ligands. We measured pembrolizumab (humanized IgG4 kappa anti‐PD1 antibody) levels before and after each IA session and found a 98.1% reduction from baseline with five sessions of IA. Comparable elimination kinetics were observed for serum IgG, whereas fκLC and IgM were eliminated to a substantially lesser extent. These findings highlight that in hyperviscosity syndrome due to IgM MM, broad spectrum IA columns might be only moderately effective compared to total plasma exchange or double filtration plasmapheresis. Monoclonal antibodies are efficiently reduced by extracorporeal therapies and re‐dosing is necessary to provide sufficient efficacy. In the case of serious adverse events such as immune‐related adverse events, IA might be used to eliminate the monoclonal antibody. Measuring IgG levels might be a reasonable strategy for monitoring drug levels of monoclonal antibodies during IA.

Nanobody\u2011horseradish peroxidase and -EGFP fusions as reagents to detect porcine parvovirus in the immunoassays

BackgroundAntibodies are an important reagent to determine the specificity and accuracy of diagnostic immunoassays for various diseases. However, traditional antibodies have several shortcomings due to their limited abundance, difficulty in permanent storage, and required use of a secondary antibody. Nanobodies, which are derived from single-chain camelid antibodies, can circumvent many of these limitations and, thus, appear to be a promising substitute. In the presented study, a sandwich ELISA-like immunoassay and direct fluorescent assay with high sensitivity, good specificity, and easy operation were the first time to develop for detecting porcine parvovirus (PPV). After screening PPV viral particles 2 (VP2) specific nanobodies, horseradish peroxidase (HRP) and enhanced green fluorescent protein (EGFP) fusions were derived from the nanobodies by recombinant technology. Finally, using the nanobody-HRP and -EGFP fusions as probes, the developed immunoassays demonstrate specific, sensitive, and rapid detection of PPV.ResultsIn the study, five PPV-VP2 specific nanobodies screened from an immunised Bactrian camel were successfully expressed with the bacterial system and purified with a Ni–NTA column. Based on the reporter-nanobody platform, HRP and EGFP fusions were separately produced by transfection of HEK293T cells. A sandwich ELISA-like assay for detecting PPV in the samples was firstly developed using PPV-VP2-Nb19 as the capture antibody and PPV-VP2-Nb56-HRP fusions as the detection antibody. The assay showed 92.1% agreement with real-time PCR and can be universally used to surveil PPV infection in the pig flock. In addition, a direct fluorescent assay using PPV-VP2-Nb12-EGFP fusion as a probe was developed to detect PPV in ST cells. The assay showed 81.5% agreement with real-time PCR and can be used in laboratory tests.ConclusionsFor the first time, five PPV-VP2 specific nanobody-HRP and -EGFP fusions were produced as reagents for developing immunoassays. A sandwich ELISA-like immunoassay using PPV-VP2-Nb19 as the capture antibody and PPV-VP2-Nb56-HRP fusion as the detection antibody was the first time to develop for detecting PPV in different samples. Results showed that the immunoassay can be universally used to surveil PPV infection in pig flock. A direct fluorescent assay using PPV-VP2-Nb12-EGFP as a probe was also developed to detect PPV in ST cells. The two developed immunoassays eliminate the use of commercial secondary antibodies and shorten detection time. Meanwhile, both assays display great developmental prospect for further commercial production and application.

Mass spectrometric analysis of core fucosylation and sequence variation in a human-camelid monoclonal antibody.

Electrospray mass spectrometry (ESI-MS) was used to measure the masses of an intact dimeric monoclonal antibody (Mab) and assess the fucosylation level. The Mab under study was EG2-hFc, a chimeric human-camelid antibody of about 80 kDa (A. Bell et al., Cancer Lett., 2010, 289(1), 81-90). It was obtained from cell culture with and without a fucosylation inhibitor, and treated with EndoS which cleaves between the two core N-acetyl glucosamine (GlcNAc) residues. It is the first time that this combined approach with a unique mass spectrometer was used to measure 146 Da differences as part of a large intact dimeric antibody. Results showed that in the dimer, both heavy chains were fucosylated on the core GlcNAc of the Fc Asn site equivalent to Asn297. In the presence of the fucosylation inhibitor, fucosylation was lost on both subunits. Following reduction, monomers were analyzed and the masses obtained corroborated the dimer results. Dimeric EG2-hFc Mab treated with PNGase F, to deglycosylate the protein, was also measured by MS for mass comparison. In spite of the success of fucosylation level measurements, the experimental masses of deglycosylated dimers and GlcNAc-Fuc bearing dimers did not correspond to masses of our sequence of reference (A. Bell et al., Cancer Lett., 2010, 289(1), 81-90; ; ), which prompted experiments to determine the protein backbone sequence. Digest mixtures from trypsin, GluC, as well as trypsin + GluC proteolysis were analyzed by matrix-assisted laser desorption/ionization (MALDI) MS and MS/MS. A few variations were found relative to the reference sequence, which are discussed in detail herein. These measurements allowed us to build a new "experimental" sequence for the EG2-hFc samples investigated in this work, although there are still ambiguities to be resolved in this new sequence. MALDI-MS/MS also confirmed the fucosylation pattern in the Fc tryptic peptide EEQYNSTYR.

Nanobodies as versatile tools: A focus on targeted tumor therapy, tumor imaging and diagnostics.

Monoclonal antibodies and vaccines have widely been studied for the immunotherapy of cancer, though their large size appears to limit their functionality in solid tumors, in large part due to unique properties of tumor microenvironment. Smaller formats of antibodies have been developed to throw such restrictions. These small format antibodies include antigen binding fragments, single-chain variable fragments, single variable domain of camelid antibody (so-called nanobody (Nb) or VHH). Since their serendipitous discovery, nanobodies have been studies at length in the fields of research, diagnostics and therapy. These antigen binding fragments, originating from camelid heavy-chain antibodies, possess unusual hallmarks in terms of (small) size, stability, solubility and specificity, hence allowing cost-effective production and sometimes out performing monoclonal antibodies. In addition, these small camelid heavy-chain antibodies are highly adaptable tools for cancer research as they enable specific modulation of targets, enzymatic and non-enzymatic proteins alike. Molecular imaging studies benefit from the rapid, homogeneous tumor accumulation of nanobodies and their fast blood clearance, permitting previously unattainable fast tumor visualization. Moreover, they are endowed with considerable therapeutic potential as inhibitors of receptor-ligand pairs and deliverers of drugs or drug-loaded nanoparticles towards tumors. In this review, we shed light on the current status of nanobodies in diagnosis and imaging of tumor and exploiting nanobodies revert immunosuppressive events, modulation of immune checkpoints, and as deliverers of drugs for targeted tumor therapy.

Computer-guided library generation applied to the optimization of single-domain antibodies.

Computer-guided library generation is a plausible strategy to optimize antibodies. Herein, we report the improvement of the affinity of a single-domain camelid antibody for its antigen using such approach. We first conducted experimental and computational alanine scanning to describe the precise energetic profile of the antibody-antigen interaction surface. Based on this characterization, we hypothesized that in-silico mutagenesis could be employed to guide the development of a small library for phage display with the goal of improving the affinity of an antibody for its antigen. Optimized antibody mutants were identified after three rounds of selection, in which an alanine residue at the core of the antibody-antigen interface was substituted by residues with large side-chains, generating diverse kinetic responses, and resulting in greater affinity (>10-fold) for the antigen.

Development of a specific nanobody and its application in rapid and selective determination of Salmonella enteritidis in milk

The variable domain of heavy chain only antibody (VHH), also named as nanobody obtained from camelid antibody libraries, has shown great potential in immunochemistry, with many advantages over conventional antibodies. Here, nanobodies towards Salmonella enteritidis (S. enteritidis) were isolated for the first time after biopanning of an immune camelid nanobody library. Those nanobodies showed high thermostability and good specificity. A sandwich-ELISA was developed with nanobody Nb 13, which exhibited a detection limit of 1.4 × 105 CFU/mL. The proposed assay was also applied to detect S. enteritidis in milk samples, which could detect 6 CFU/mL of S. enteritidis in milk after 10 h of enrichment. This study demonstrated an alternative strategy for S. enteritidis determination, and further proved the utility and practicality of nanobody-based reagents in bioanalytical chemistry.

A Novel Nanobody Scaffold Optimized for Bacterial Expression and Suitable for the Construction of Ribosome Display Libraries.

Single-domain antigen-binding fragments of camelid antibodies, known as VHHs or nanobodies, are widely used affinity reagents. However, their production involving animal immunization is time- and resource-intensive. Starting from a sequence dataset of llama VHHs, we designed a novel scaffold, based on conserved framework sequences, suitable for bacterial nanobody expression and synthetic library construction. The consensus scaffold was validated by grafting the CDRs from two known nanobodies. While maintaining their binding properties, the two chimeric nanobodies showed higher levels of expression and solubility in E. coli when compared to the corresponding wild types. A proof-of-concept synthetic combinatorial library, suitable for ribosome display (RD) selection, was obtained by encoding three randomized complementarity determining regions within the consensus framework. The library, made of linear DNA fragments, has an estimated complexity of > 1012 that is three orders of magnitude higher than common phage display libraries. The bacterial expression of several library clones showed a high production of soluble recombinant proteins. The high complexity of the library, confirmed by sequencing of a subset of clones, as well as a preliminary RD selection of a maltose binding protein binder, indicated this approach as a starting point in the construction of synthetic combinatorial libraries to be used as animal-free tools for the low-cost selection of target-specific nanobodies.

Structural and thermodynamic basis for the recognition of the substrate-binding cleft on hen egg lysozyme by a single-domain antibody

Single-domain antibodies (VHHs or nanobodies), developed from heavy chain-only antibodies of camelids, are gaining attention as next-generation therapeutic agents. Despite their small size, the high affinity and specificity displayed by VHHs for antigen molecules rival those of IgGs. How such small antibodies achieve that level of performance? Structural studies have revealed that VHHs tend to recognize concave surfaces of their antigens with high shape-complementarity. However, the energetic contribution of individual residues located at the binding interface has not been addressed in detail, obscuring the actual mechanism by which VHHs target the concave surfaces of proteins. Herein, we show that a VHH specific for hen egg lysozyme, D3-L11, not only displayed the characteristic binding of VHHs to a concave region of the surface of the antigen, but also exhibited a distribution of energetic hot-spots like those of IgGs and conventional protein-protein complexes. The highly preorganized and energetically compact interface of D3-L11 recognizes the concave epitope with high shape complementarity by the classical lock-and-key mechanism. Our results shed light on the fundamental basis by which a particular VHH accommodate to the concave surface of an antigens with high affinity in a specific manner, enriching the mechanistic landscape of VHHs.

Identification of New DR5 Agonistic Nanobodies and Generation of Multivalent Nanobody Constructs for Cancer Treatment.

Current cancer therapeutics suffer from a lack of specificity in targeting tumor cells and cause severe side effects. Therefore, the design of highly specialized drugs comprising antibody derivatives inducing apoptosis in targeted cancer cells is considered to be a promising strategy. Drugs acting on death receptor 5 (DR5) such as DR5 agonist antibodies replacing “TNF-related apoptosis-inducing ligand” (TRAIL) offer feasible opportunities in this direction. Although such agonists provided good antitumor activity in preclinical studies, they were less effective in clinical studies, possibly due to a disturbed Fc interaction with Fc-γ receptors. Thus, multimerized antigen binding fragments without Fc have been proposed to increase their efficacy. We generated nanobodies (Nbs), recombinant variable domains of heavy chain-only antibodies of camelids, against the DR5 ectodomain. Nb24 and Nb28 had an affinity in the nM and sub-nM range, but only Nb28 competes with TRAIL for binding to DR5. Bivalent, trivalent, and tetravalent constructs were generated, as well as an innovative pentameric Nb complex, to provoke avidity effects. In our cellular assays, these trimeric, tetrameric, and pentameric Nbs have a higher apoptotic capacity than monomeric Nbs and seem to mimic the activity of the natural TRAIL ligand on various cancer cells.