Biology subjects

Chames, P.

Publications and source records attributed to Chames, P..

9 recordsLinked to original sources

bioRxiv2025

Cell-Based Immunization Combined with Single-Round Cell Panning Enables Discovery of PSMA-Targeting Nanobodies from Phage Display Libraries

There is a strong need for nanobodies targeting novel cancer-associated antigens to advance radioligand imaging and antibody-based therapeutics. In this study, we investigated whether non-targeted Llama immunization using tumor cells, combined with non-targeted phage display panning on human cell lines, could yield nanobodies specific to Prostate Specific Membrane Antigen (PSMA). Nanobody selection using both classical three round PSMA negative-positive panning and single round panning of cell lines positive or negative for PSMA showed clear enrichment for PSMA binders in both strategies. Using shRNA knockdown, flow cytometry, cell-ELISA, immunohistochemistry and structural modeling and docking, we confirmed the PSMA targeting of selected nanobodies. Two distinct epitopes were predicted to be bound by nanobodies PSMANb9 and A7 (JVZ-007), corroborated by epitope competition assays. These findings support the feasibility of non-targeted immunization and panning strategies for isolating clinically relevant cancer nanobodies.

cancer biology↗

bioRxiv2025

Bivalency of natalizumab promotes inhibition of dynamic VLA-4 adhesion beyond affinity gain

Natalizumab, a monoclonal IgG4 antibody used in the treatment of multiple sclerosis (MS), inhibits VLA-4 binding to VCAM-1, thereby reducing leukocyte recruitment to inflamed tissues. The intricate mechanisms underlying these effects remain unclear, particularly concerning the heavy-chain shuffling of IgG4. We conducted an in vitro study to quantify the impact of bivalent IgG and monovalent Fab forms of natalizumab on the capture and migration of human primary memory T lymphocytes under shear stress, using VCAM-1 and SDF-1-coated surfaces. IgG natalizumab at concentrations near its cell surface EC50 showed significant of capture and resistance of adherent cells to shear stress, whereas significantly higher doses of Fab natalizumab, up to 100-fold greater than its cell surface EC50, were needed to achieve similar effects. These findings highlight that receptor occupancy alone may not adequately predict the functional outcomes of inhibitor antibodies. For optimal therapeutic effect, inhibition of cell-surface adhesion may require specific kinetic and geometric binding properties of antibodies.

immunology↗

bioRxiv2025

Identification and optimization of trans-species reactive TfR1-binding VHH as tools for drug delivery across the blood brain barrier

The blood-brain barrier (BBB) is a major obstacle for delivering therapeutic agents to the central nervous system (CNS), posing significant challenges for treating neurological disorders. Among current strategies to improve brain drug exposure, hijacking physiological pathways involved in receptor-mediated transcytosis (RMT) has emerged as a promising strategy. While targeting transferrin receptor 1 (TfR1) is widely explored, many TfR1-antibodies lack cross-species reactivity, limiting translational development. In the present study, we identified and characterized camelid-derived single-domain antibodies (VHHs) with robust cross-reactivity to rodent, rhesus monkey, and human TfR1. Epitope mapping of the VHH revealed a novel binding site at the interface of the TfR1 dimer. When fused to a human IgG1 Fc domain, these VHHs, as monomers or homodimers, were efficiently internalized by engineered CHO cells and brain endothelial cells expressing TfR1 from different species. Systemic administration of VHH-Fc constructs in mice demonstrated significantly improved brain uptake compared to irrelevant controls. Functional delivery was confirmed using neurotensin (NT)-induced hypothermia, and we established correlations between in vivo effects and TfR1 VHH binding properties determined by surface plasmon resonance. Notably, efficient BBB transcytosis was associated with intermediate affinity and rapid dissociation rates. Engineered variants maintained favorable cross-species binding, including similar affinities to human and non-human primate TfR1, facilitating translational studies. The cross-reactive anti-TfR1 VHHs we developed offer a versatile and modular platform for CNS drug delivery and hold promise as molecular shuttles for transporting therapeutic agents across the BBB. Our work establishes a robust foundation for developing next-generation brain-targeted biotherapeutics, including peptides, enzyme replacement therapies, antibody-based treatments for neurodegenerative diseases, and oligonucleotide delivery for CNS disorders, enabling seamless translation from preclinical to clinical applications.

neuroscience↗

bioRxiv2024

Decoupling individual host response and immune cell engager cytotoxic potency

Immune cell engagers are molecular agents, usually antibody-based constructs, engineered to recruit immune cells against cancer cells and kill them. They represent a versatile and powerful tool for cancer immunotherapy. Despite the multiplication of new engagers tested and accepted in the clinics, how molecular and cellular parameters influence their action is poorly understood. In particular, disentangling the respective role of host immune cells and engager biophysical characteristics is needed to improve their design and efficiency. Focusing here on harnessing antibody dependent Natural Killer cell cytotoxicity, we measure the efficiency of 6 original bispecific antibodies (bsAb), associating an anti-HER2 nanobody and an anti-CD16 nanobody. In vitro cytotoxicity data using primary human NK cells on different target cell lines exposing different antigen densities were collected, exhibiting a wide range of bsAb dose response. In order to rationalize our observations, we introduce a simple multiscale model, postulating that the density of bsAb bridging the two cells is the main parameter triggering the cytotoxic response. We introduce two new microscopic parameters: the surface cooperativity describing bsAb affinity at the bridging step and the threshold of bridge density determining the donor-dependent response. Both parameters permit to rank Abs and donors and to predict bsAb potency as a function of antibodies bulk affinities and receptor surface densities on cells. Our approach thus provides a general way to decouple donor response from immune engagers characteristics, rationalizing the landscape of molecule design.

biophysics↗

bioRxiv2024

Celldetective: an AI-enhanced image analysis toolfor unraveling dynamic cell interactions

Analysis of multimodal and multidimensional data capturing dynamic interactions between diverse cell populations is a current challenge in bioimaging, especially in the context of immunology and immunotherapy research. Here, we introduce Celldetective, an open-source Python-based software tool designed for high-performance, end-to-end analysis of image-based in vitro immune and immunotherapy assays. Celldetective is purpose-built for multicondition, 2D multi-channel time-lapse microscopy of mixed cell populations. Although it is optimised for the needs of immunology assays, it is nevertheless broadly applicable to any biological system involving interacting cell populations. The software seamlessly integrates AI-based segmentation, tracking, and automated single-cell event detection, all within an intuitive graphical interface that supports interactive visualisation, annotation, and training options. We showcase its capabilities with original datasets of single immune effector cell interactions with an activating surface mediated by bispecific antibodies, and pairwise interactions in antibody-dependent cell cytotoxicity events.

bioinformatics↗

bioRxiv2024

Chronic potentiation of metabotropic glutamate receptor 2 with a nanobody accelerates amyloidogenesis in Alzheimer's disease.

Immunotherapy of Alzheimers disease (AD) is a promising approach to reduce the accumulation of amyloid-beta (A{beta}), a critical event in the onset of the disease. Targeting the group II metabotropic glutamate receptors, mGlu2 and mGlu3, could be important in controlling A{beta} production, although their respective contribution remains unclear due to the lack of selective tools. Here, we show that enhancing mGlu2 receptor activity increases A{beta}1-42 peptide production whereas activation of mGlu3 has no effect. We show that such a difference likely results from the direct interaction of APP with mGlu3, but not with mGlu2 receptors, that prevents APP amyloidogenic cleavage and A{beta}1-42 peptides production. We then show that chronic treatments of the AD model 5xFAD mice with a brain-penetrating mGlu2-potentiating nanobody accelerated amyloid aggregation and exacerbated memory deficits, but had no effect in control mice. Our results confirm that a selective mGluR2 activation exacerbates AD disease development, suggesting that therapeutic benefices could be obtained with blockers of this receptor. Our study also provides the proof-of-concept that chronic administration of nanobodies targeting neuroreceptors can be envisioned to treat brain diseases.

neuroscience↗

bioRxiv2024

Antigen density and applied force control enrichment of nanobody-expressing yeast cells in microfluidics

In vitro display technologies such as yeast display have been instrumental in developing the selection of new antibodies, antibody fragments or nanobodies that bind to a specific target, with affinity towards the target being the main factor that influences selection outcome. However, the roles of mechanical forces are being increasingly recognized as a crucial factor in the regulation and activation of effector cell function. It would thus be of interest to isolate binders behaving optimally under the influence of mechanical forces. We developed a microfluidic assay allowing the selection of yeast displaying nanobodies through antigen-specific immobilization on a surface under controlled hydrodynamic flow. This approach enabled enrichment of model yeast mixtures using tunable antigen density and applied force. This new force-based selection method opens the possibility of selecting binders by relying on both their affinity and force resistance, with implications for the design of more efficient immunotherapeutics.

bioengineering↗

bioRxiv2022

Combining DNA scaffolds and acoustic force spectroscopy to characterize individual protein bonds

Single-molecule data are of great significance in biology, chemistry, and medicine. However, experimental tools to characterize, in a multiplexed manner, protein bond rupture under force are needed. Acoustic force spectroscopy (AFS) is an emerging manipulation technique which generates acoustic waves to apply force in parallel on a large population of microbeads tethered to a surface. We have exploited this configuration on a recently developed modular Junctured-DNA (J-DNA) scaffold designed to study protein-protein interactions at the single-molecule level. By applying repetitive constant force steps on the FKBP12-rapamycin-FRB complex, we measured its unbinding kinetics under force at the single-bond level. Special effort was made in analyzing the data in order to identify potential pitfalls. We established a calibration method allowing in situ force determination during the course of the unbinding measurement. We compare our results with well established techniques, such as magnetic tweezers, to ensure their accuracy. We also apply our strategy for measuring the force dependent rupture of a single domain antibody with its antigen. We get a good agreement with standard measurement at zero force. Our technique offers single molecule precision for multiplexed measurements of interactions of biotechnological and medical interest.

biophysics↗

bioRxiv2018

Nanobody-antigen catch-bond reveals NK cell mechanosensitivity

Antibodies 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 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, while the Fc domain binds to the activating receptor CD16 (also known as FcgRIII) of an immune effector cell, in a quasi bi-dimensional environment (2D). Therefore, there is a strong need to investigating 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 behaviour where off-rate increases with force; the other exhibits a catch bond behaviour with off-rate decreasing with force. This is the first time, to our knowledge, that catch bond behaviour was reported for antigen-antibody bond. We further exploit this property to show how Natural Killer cells spread differentially on surfaces coated with these molecules, revealing NK cells mechanosensitivity. Our results may also have strong implications for the design of efficient bispecific antibodies for therapeutic applications.

biophysics↗