Biology subjects

Limozin, L.

Publications and source records attributed to Limozin, L..

12 recordsLinked to original sources

bioRxiv2026

Tuning T-cell immunological synapse by modular DNA-Nanobody engagers for precision immunotherapy

Bispecific T-cell engagers (TCEs) are a promising class of cancer immunotherapies, but their clinical use is limited by toxicity and insufficient specificity. Tuning the T cell- tumor interface through engager architecture may address these drawbacks. To this end, we engineered hybrid constructs composed of two nanobodies targeting CD3 and the model tumor antigen HER2, respectively, connected by rigid DNA linkers of variable length. Using cytotoxicity assays and hybrid biophysical platforms, we demonstrate a linker-length dependence of cell spreading on antigen, target killing and cytokine release, revealing a functional decoupling between killing and cytokine secretion, and implicating the glycocalyx as a key player. Through the addition of EGFR targeting, we also generate trispecific constructs implementing an "OR-gate" logic to address tumor heterogeneity and reduce resistance due to antigen loss. Overall, these versatile constructs show great therapeutic promise, and at the same time serve as platforms to test hypotheses on biophysical mechanisms.

synthetic biology↗

bioRxiv2026

Antibody maturation in germinal centers selects mutants based on BCR-antigen bond mechanical resistance

Antibody maturation in germinal centers (GCs) is traditionally viewed as a process that enhances antigen-binding affinity of B cell receptors (BCR). However, recent studies challenge this paradigm, revealing no systematic antibody affinity improvement during GC selection. Here, we investigate whether mechanical resistance of antibody-antigen bonds, rather than affinity, is the selective parameter driving GC maturation. Using biolayer interferometry measurements for assessing affinity and laminar flow chamber for mechanical resistance, we analyzed three lineages of mouse antibodies generated after immunization with ovalbumin. While affinity changes were heterogeneous, ranging from gains to losses across lineages, mechanical resistance consistently increased after maturation. Bond lifetimes under physiological forces (10-70 pN) converged to similar values across lineages, suggesting a selective pressure for mechanical stability. Functionally, NK cell activation in a surrogate in vitro ADCC assay correlated with bond lifetimes under force, but not with affinity. This indicates that lymphocytes are sensitive to the mechanical stability of antibody-antigen interactions, which may underlie GC selection. Our findings reconcile antibody maturation with the idea of enhanced binding, while aligning with the role of mechanical forces in B cell signaling and antigen uptake. These insights provide a framework for understanding GC biology and may inform the design of therapeutic antibodies.

immunology↗

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

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

Probing mechanical interaction of immune receptors and cytoskeleton by membrane nanotube extraction

The role of force application in immune cell recognition is now well established, the force being transmitted between the actin cytoskeleton to the anchoring ligands through receptors such as integrins. In this chain, the mechanics of the cytoskeleton to receptor link, though clearly crucial, remains poorly understood. To probe this link, we combine mechanical extraction of membrane tubes from T cells using optical tweezers, and fitting of the resulting force curves with a viscoelastic model taking into account the cell and relevant molecules. We solicit this link using four different antibodies against various membrane bound receptors: antiCD3 to target the T Cell Receptor (TCR) complex, antiCD45 for the long sugar CD45, and two clones of antiCD11 targeting open or closed conformation of LFA1 integrins. Upon disruption of the cytoskeleton, the stiffness of the link changes for two of the receptors, exposing the existence of a receptor to cytoskeleton link - namely TCR-complex and open LFA1, and does not change for the other two where no such a link was expected. Our integrated approach allows us to probe, for the first time, the mechanics of the intracellular receptor-cytoskeleton link in immune cells.

biophysics↗

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↗

bioRxiv2022

Mechanical forces impair antigen discrimination by reducing differences in T cell receptor off-rates

T cells use their T cell receptors (TCRs) to discriminate between lower-affinity self and higher-affinity foreign peptide major-histocompatibility-complexes (pMHCs) based on the TCR/pMHC off-rate. It is now appreciated that T cells generate mechanical forces during this process but how force impacts the TCR/pMHC off-rate remains unclear. Here, we measured the effect of mechanical force on the off-rate of multiple TCR/pMHC interactions. Unexpectedly, we found that lower-affinity pMHCs with faster solution off-rates were more resistant to mechanical force (weak slip or catch bonds) than higher-affinity interactions (strong slip bonds), and this was confirmed by molecular dynamic simulations. Consistent with these findings, we show that the best characterized catch-bond, involving the OT-I TCR, has a low affinity and an exceptionally fast solution off-rate. Our findings imply that reducing forces on the TCR/pMHC interaction improves antigen discrimination and we suggest this new force-shielding role for the adhesion receptors CD2 and LFA-1. One sentence summaryMechanical forces disproportionately accelerate the off-rates of higher-affinity antigens reducing T cell antigen discrimination

immunology↗

bioRxiv2020

Controlling T cells shape, mechanics and activation by micropatterning.

We designed a strategy, based on a careful examination of the activation capabilities of proteins and antibodies used as substrates for adhering T cells, coupled to protein microstamping. This allowed us to control at the same time the position, shape, mechanics and activation state of T cells. Once adhered on shaped patterns we examined the capacities of T cells to be activated with soluble aCD3, in comparison to T cells adhered to a continuously decorated substrate with the same density of ligands. We show that, in our hand, adhering onto an anti CD45 (aCD45) antibody decorated surface is not affecting T cell calcium fluxes, even adhered on variable size micro-patterns. We further demonstrate this by expressing MEGF10 as a non immune adhesion receptor in T cells to obtain the very same spreading area on PLL substrates and Young modulus than immobilized cells on aCD45, while retaining similar activation capabilities using soluble aCD3 or through model APC contacts. We propose that our system is a way to test activation or anergy of T cells with defined adhesion and mechanical characteristics, and may allow to dissect fine details of these mechanisms since it allows to observe homogenised populations in standardized T cell activation assays.

biophysics↗

bioRxiv2020

CD8 co-receptor enhances T cell activation without any effect on initial attachment

The scanning of surrounding tissues by T lymphocytes to detect cognate antigen is a highly demanding process that requires high rapidity, sensitivity and specificity. Co-receptors such as CD8 are known to increase detection performance, but the exact mechanism of this role remains incompletely understood. Here, we used interference reflection microscopy to image the initial spreading of 1G4 receptor transfected CD8+ and CD8- Jurkat cells dropped on surfaces exposing five cognate antigens of varying activating power, and we used a laminar flow chamber to measure the influence of CD8 on the kinetics of bond formation and rupture between cell-born T cell receptors (TCRs) and peptide-exposing major histocompatibility complex antigens (pMHCs) at the single molecule level. It is concluded that CD8 did not influence TCR-pMHC interaction during the first seconds following cell surface encounter, but it promoted the spreading responses during the first minutes, thus suggesting that CD8 was involved in early activation rather than binding. In addition, presented results were quantitatively compared with a recent report on the cell-free interaction between the same ligand-receptor couples : it is concluded that bond formation was strongly impaired by cell molecular environment, while bond rupture was comparable in both systems. Results from this and previous reports were used to propose a quantitative scheme of the strategy used by T lymphocytes to scan foreign surfaces. It is suggested that the understanding of the strategy used by cells to perform their basic functions may be a prerequisite to understand the function of molecular networks revealed by high throughput methods.

immunology↗

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↗

bioRxiv2017

Biphasic mechanosensitivity of TCR mediated adhesion of T lymphocytes

Force sensitivity of the T cell receptor (TCR) is now believed to be essential for immune recognition, but cellular mechanosensitivity of T cells is still poorly understood. Here we show that T cells adhering via the TCR-complex respond to environmental stiffness in an unusual biphasic fashion. As the stiffness increases, adhesion and spreading first increase, then decrease, attaining their maximal values on an optimally stiff surface, with stiffness comparable to certain antigen presenting cells. Remarkably, in presence of additional ligands for the integrin LFA-1, spreading increases monotonously with stiffness up to a saturation value. Using a mesoscopic semi-analytical model linking spreading to molecular characteristics of bonds, we identify force sensitivity of the off-rate and the effective bond stiffness as the crucial parameters that determine monotonic or biphasic mechanosensitive behavior.

biophysics↗