Nanobody-CD16 Catch Bond Reveals NK Cell Mechanosensitivity

International audienceAntibodies are key tools in biomedical research and medicine. Their binding properties are classically measured in solution and characterized by an affinity. However, in physiological conditions, antibodies can bridge an immune effector cell and an antigen-presenting cell, implying that mechanical forces may apply to the bonds. For example, in antibody-dependent cell cytotoxicity—a major mode of action of therapeutic monoclonal antibodies—the Fab domains bind the antigens on the target cell, whereas the Fc domain binds to the activating receptor CD16 (also known as FcgRIII) of an immune effector cell, in a quasi-bidimensional environment (2D). Therefore, there is a strong need to investigate antigen/antibody binding under force (2D) to better understand and predict antibody activity in vivo. We used two anti-CD16 nanobodies targeting two different epitopes and laminar flow chamber assay to measure the association and dissociation of single bonds formed between microsphere-bound CD16 antigens and surface-bound anti-CD16 nanobodies (or single-domain antibodies), simulating 2D encounters. The two nanobodies exhibit similar 2D association kinetics, characterized by a strong dependence on the molecular encounter duration. However, their 2D dissociation kinetics strongly differ as a function of applied force: one exhibits a slip bond behavior in which off rate increases with force, and the other exhibits a catch-bond behavior in which off rate decreases with force. This is the first time, to our knowledge, that catch-bond behavior was reported for antigen-antibody bond. Quantification of natural killer cells spreading on surfaces coated with the nanobodies provides a comparison between 2D and three-dimensional adhesion in a cellular context, supporting the hypothesis of natural killer cell mechanosensitivity. Our results may also have strong implications for the design of efficient bispecific antibodies for therapeutic applications

In vivo selection of engineered homing endonucleases using double-strand break induced homologous recombination

Homing endonucleases, endonucleases capable of recognizing long DNA sequences, have been shown to be a tool of choice for precise and efficient genome engineering. Consequently, the possibility to engineer novel endonucleases with tailored specificities is under strong investigation. In this report, we present a simple and efficient method to select meganucleases from libraries of variants, based on their cleavage properties. The method has the advantage of directly selecting for the ability to induce double-strand break induced homologous recombination in a eukaryotic environment. Model selections demonstrated high levels of enrichments. Moreover, this method compared favorably with phage display for enrichment of active mutants from a mutant library. This approach makes possible the exploration of large sequence spaces and thereby represents a valuable tool for genome engineering

A combinatorial approach to create artificial homing endonucleases cleaving chosen sequences

Meganucleases, or homing endonucleases (HEs) are sequence-specific endonucleases with large (>14 bp) cleavage sites that can be used to induce efficient homologous gene targeting in cultured cells and plants. These findings have opened novel perspectives for genome engineering in a wide range of fields, including gene therapy. However, the number of identified HEs does not match the diversity of genomic sequences, and the probability of finding a homing site in a chosen gene is extremely low. Therefore, the design of artificial endonucleases with chosen specificities is under intense investigation. In this report, we describe the first artificial HEs whose specificity has been entirely redesigned to cleave a naturally occurring sequence. First, hundreds of novel endonucleases with locally altered substrate specificity were derived from I-CreI, a Chlamydomonas reinhardti protein belonging to the LAGLIDADG family of HEs. Second, distinct DNA-binding subdomains were identified within the protein. Third, we used these findings to assemble four sets of mutations into heterodimeric endonucleases cleaving a model target or a sequence from the human RAG1 gene. These results demonstrate that the plasticity of LAGLIDADG endonucleases allows extensive engineering, and provide a general method to create novel endonucleases with tailored specificities

Adaptation of HIV-1 Envelope Glycoprotein gp120 to Humoral Immunity over the Course of the Epidemic

Since 2009, a large panel of broad and potent monoclonal neutralizing antibodies (MoNAbs) against HIV-1 have been isolated. These MoNAbs can protect from lllV-1 infection and suppress established infection in animal models. Because their efficacy should be evaluated in human clinical trials, it is of importance to define the sensitivity of the most contemporary transmitted variants to these MoNAbs. We, and others previously, reported that HIV-1 has become more resistant to neutralization over the course of the epidemic (Bunnik et al., Nature Med 2010, Bouvin-Pley et al., PloS Pathog 2013). Methods: Here we extended the analyses to the most potent MoNAbs described since then, either more recently isolated or improved by structure-based gene modifications. Results: We fully confirmed the first observations showing an increasing resistance of HIV-1 clade B over time to MoNAbs targeting the major gp l20 epitopes but not to MoNAbs targeting the gp41 MPER. Despite this evolution, some MoNAbs still were able to neutralize efficiently the most recently transmitted HIV-1 variants (2006-2010). The most potent MoNAbs were the bi-specific PG9- and PG16-iMab that alone were able to neutralize an variants at less than 0.4 mg/mL. The sensitivity to iMAb remained similar over time, suggesting that the trend of increasing resistance to PG9-/PG16-iMAb may be attributed only to die antigen binding domain of PG9/PG16. NIH45-46m2 (and -m7), 10-1074 and 10E8 were also highly potent and, if combined, reached the potency of PG9-/PG16-iMAb. We also observed that 3BNC 117 was almost as potent as the modified NIH45-46 antibodies, and that the lama-derived JM4IgG2b was the most potent Ab among those that do not target the major gp 120 neutralizing epitopes. Conclusions: These data clearly suggest a continuous drift of the env gene of HIV-1 elude B over the epidemic, and that not a single epitope is concerned but the entire gp120 as a whole. The consequences of this adaptation on the envelope functionality are being explored

Chronic Delivery of Antibody Fragments Using Immunoisolated Cell Implants as a Passive Vaccination Tool

BACKGROUND: Monoclonal antibodies and antibody fragments are powerful biotherapeutics for various debilitating diseases. However, high production costs, functional limitations such as inadequate pharmacokinetics and tissue accessibility are the current principal disadvantages for broadening their use in clinic. METHODOLOGY AND PRINCIPAL FINDINGS: We report a novel method for the long-term delivery of antibody fragments. We designed an allogenous immunoisolated implant consisting of polymer encapsulated myoblasts engineered to chronically release scFv antibodies targeted against the N-terminus of the Aβ peptide. Following a 6-month intracerebral therapy we observed a significant reduction of the production and aggregation of the Aβ peptide in the APP23 transgenic mouse model of Alzheimer's disease. In addition, functional assessment showed prevention of behavioral deficits related to anxiety and memory traits. CONCLUSIONS AND SIGNIFICANCE: The chronic local release of antibodies using immunoisolated polymer cell implants represents an alternative passive vaccination strategy in Alzheimer's disease. This novel technique could potentially benefit other diseases presently treated by local and systemic antibody administration

Fragments d’anticorps à domaine unique

En dépit des succès obtenus dans l’utilisation thérapeutique d’anticorps monoclonaux depuis une dizaine d’années, notamment en cancérologie, plusieurs obstacles restent difficiles à franchir : la difficile pénétration de ces molécules de grande taille dans les tissus qui compromet leur accès notamment aux cellules tumorales, et le coût très élevé de leur production. Il s’avère possible d’utiliser des fragments d’anticorps constitués d’un seul domaine, appelés domaines antibodies (dAb). Certains de ces fragments dérivent d’anticorps dénués de chaîne légère, naturellement présents chez les camélidés. Les approches d’ingénierie des anticorps ont depuis leur découverte permis de construire des dAb humains stables, solubles, produits facilement, et dont l’affinité pour leur cible est tout à fait comparable à celle des anticorps conventionnels. Des molécules multivalentes et multispécifiques peuvent aussi être conçues en assemblant ces domaines uniques. L’efficacité de ces dAb dans des modèles précliniques chez la souris, confirmée dans des essais cliniques chez l’homme, laissent entrevoir une alternative efficace aux anticorps conventionnels

[The future of antibody fragments, made of a single immunoglobulin domain.]

International audienceMonoclonal antibodies are now established as key therapeutics for a range of diseases including cancer and auto-immunity. However, despite important improvements, these molecules still face several serious limitations including production costs and tumor penetration. A new class of antibody fragments, made of a single immunoglobulin domain, is emerging as an exciting alternative. This review describes the outstanding properties and the first achievements of these domain antibodies. double dagger

Nanobody Engineering: Toward Next Generation Immunotherapies and Immunoimaging of Cancer

In the last decade, cancer immunotherapies have produced impressive therapeutic results. However, the potency of immunotherapy is tightly linked to immune cell infiltration within the tumor and varies from patient to patient. Thus, it is becoming increasingly important to monitor and modulate the tumor immune infiltrate for an efficient diagnosis and therapy. Various bispecific approaches are being developed to favor immune cell infiltration through specific tumor targeting. The discovery of antibodies devoid of light chains in camelids has spurred the development of single domain antibodies (also called VHH or nanobody), allowing for an increased diversity of multispecific and/or multivalent formats of relatively small sizes endowed with high tissue penetration. The small size of nanobodies is also an asset leading to high contrasts for non-invasive imaging. The approval of the first therapeutic nanobody directed against the von Willebrand factor for the treatment of acquired thrombotic thrombocypenic purpura (Caplacizumab, Ablynx), is expected to bolster the rise of these innovative molecules. In this review, we discuss the latest advances in the development of nanobodies and nanobody-derived molecules for use in cancer immunotherapy and immunoimaging

Bispecific antibodies for cancer therapy: The light at the end of the tunnel?

With 23 approvals in the US and other countries and four approvals outside US, antibodies are now widely recognized as therapeutic molecules. The therapeutic and commercial successes met by rituximab, trastuzumab, cetuximab and other mAbs have inspired antibody engineers to improve the efficacy of these molecules. Consequently, a new wave of antibodies with engineered Fc leading to much higher effector functions such as antibody-dependent cell-mediated cytotoxicity or complement-dependent cytotoxicity is being evaluated in the clinic, and several approvals are expected soon. In addition, research on a different class of antibody therapeutics, bispecific antibodies, has recently led to outstanding clinical results, and the first approval of the bispecific antibody catumaxomab, a T cell retargeting agent that was approved in the European Union in April 2009. This review describes the most recent advances and clinical study results in the field of bispecific antibodies, a new class of molecules that might outshine conventional mAbs as cancer immunotherapeutics in a near future

Special Issue: Nanobody

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