Due to its restricted expression in normal tissues, its frequent overexpression in various aggressive malignancies, and its involvement in several pro-tumorigenic signaling pathways, mesothelin, a glycosylphosphatidylinositol-anchored cell surface protein, has been identified as a promising tumor-associated antigen. This review provides an up-to-date comprehensive overview regarding the role of mesothelin in cancer. It encompasses the protein structural characteristics, expression profiles, glycosylation patterns, shedding mechanisms, prognostic significance, and functional roles. Subsequently, therapeutic strategies targeting mesothelin that have advanced to clinical trial stage are discussed, including vaccine-based approaches, antibody-mediated immunotherapies, and cell therapies. The remaining challenges of mesothelin targeted treatments are highlighted, along with the ongoing options aimed at overcoming these limitations.

Mesothelin is a tumor-associated antigen highly expressed in various cancers, with limited presence in normal tissues, making it an ideal target for cancer diagnosis and therapy. Nanobodies, small single-domain antibody fragments, possess favorable properties such as excellent tumor penetration, low immunogenicity, and versatility in design. This study engineered six anti-mesothelin nanobody-based constructs differing in format, valency, molecular weight, and half-life extension strategies, using either albumin-binding domains or Fc-fusion. All constructs were labeled with a near-infrared dye for functional evaluation. Binding affinity was measured, and tumor penetration was assessed in vitro using three-dimensional tumor models combining cancer cells and fibroblasts, as well as in vivo in xenografted mouse models. Key findings demonstrate that i) the enhancement of functional affinity is influenced by both the molecule format and the tumor cell type, ii) below a certain molecular size, bivalency can compensate size-related limitations, iii) the flexible "pearl necklace" architecture is the most suitable option for achieving optimal short-term imaging, and iv) the incorporation of half-life extension modules extends imaging and therapeutic windows, with albumin-binding-based strategies outperforming Fc-fusion in tumor biodistribution, which must be carefully balanced with specificity to maximize efficacy and minimize adverse effects. In conclusion, this study contributes to the development of more effective next-generation anti-mesothelin theranostic approaches and highlights that nanobody properties and optimal design strategies may be target specific, emphasizing the need for context-dependent development.

Natalizumab, a monoclonal IgG4 antibody used in the treatment of multiple sclerosis (MS), inhibits α4β1 (very late antigen-4, VLA-4)-binding to vascular cell adhesion molecule-1 (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 CXCL12-coated surfaces. IgG natalizumab at concentrations near its cell surface EC50 (half-maximal effective concentration) 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. Insight Box This study shows how natalizumab functionally inhibits α4β1 (very late antigen-4, VLA-4)-mediated T-cell adhesion by integrating quantitative affinity measurements with dynamic assays performed under shear stress. We show that the antibody's bivalency provides a major functional advantage, enabling strong inhibition of cell capture and adhesion at concentrations near its EC50 (half-maximal effective concentration), whereas monovalent Fab fragments require 10- to 100-fold higher doses. These findings offer biological insight into why receptor occupancy alone cannot predict adhesion inhibition. By combining cell-based affinity quantification with real-time adhesion and migration assays, our work reveals how kinetic and geometric properties of antibody-integrin interactions shape immune cell dynamics, informing the rational design of next-generation integrin-targeting therapeutics.

Small extracellular vesicles (sEVs) are promising vehicles for targeted therapeutic delivery, but strategies for their surface functionalization remain limited. Here, we present a reliable and simple genetic approach that enables customized modification of sEV surfaces and supports enhanced sEV uptake by recipient cells. This strategy is based on the fusion of targeting moieties to the C-terminal fragment of syndecan-1 (SDC1-CTF), a peptide naturally enriched in sEVs. Combining various analytical approaches including single-vesicle analysis, we establish that this strategy enables decoration of up to 20% of secreted sEVs with Nanobodies (Nbs). In quantitative bioluminescence assays, using concentrated conditioned media, we demonstrate that sEV-coating with anti-EGFR Nb supports enhanced sEV uptake by EGFR-expressing cells. This new strategy thus offers a robust and modular solution for endowing sEV surfaces with defined targeting properties to support further sEV-based therapeutic applications.

There is a strong need for nanobodies that target 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 of 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), and this was corroborated by epitope competition assays. These findings support the feasibility of non-targeted immunization and panning strategies for isolating antigen-targeting cancer nanobodies.

Despite vaccine availability, the morbilliviruses measles virus and canine distemper virus (CDV) are still causing major health impairments in human and animal populations. Here, we identified two potent, neutralizing single domain antibodies directed against the tetrameric receptor binding (H) protein of CDV. Structural analyses spotlighted two vulnerable sites within the H protein. While the first overlaps with the receptor binding site, the second encompasses amino acid residues of two protomers located at the distal dimeric head interface, which supports distinct mechanisms of neutralization. Upon application of an engineered tetravalent and biparatopic antibody, ferrets were protected at a remarkably low antibody dose (1 mg/kg) administered intra-peritoneally on days 3 and 7 post-exposure of a lethal CDV challenge. Collectively, this study spotlights the power of integrating multiple mechanisms of neutralization in a single format and provides a roadmap to design next-generation therapeutics against morbilliviral infections as well as other infectious pathogens. Measles virus and canine distemper virus (CDV) are still causing health impairments in humans and animals. Here, the authors develop a synthetic antibody protecting ferrets from lethal CDV infections, providing a roadmap for antiviral drug design.

Abstract 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.

Immunotherapy is a promising avenue for reducing amyloid-β (Aβ) accumulation, a hallmark of Alzheimer's disease (AD) pathology. Camelid single domain antibodies, called nanobodies, offer several advantages over conventional monoclonal antibodies, including improved brain penetration and fine-tuning of the targeted neuroreceptors, and may represent an effective strategy to modulate Aβ production. Among potential therapeutic targets, group II metabotropic glutamate receptors (mGluR2 and mGluR3) have been implicated in Aβ regulation, though their individual contributions remain unclear. Here, we showed that activation of mGluR2 significantly increases Aβ peptides and sAPPβ production in a cellular model, by enhancing the internalization of amyloid precursor protein (APP) and its subsequent amyloidogenic processing. In contrast, mGluR3 directly interacts with APP, protecting it from amyloidogenic cleavage and favoring its non-amyloidogenic processing. We used a brain-penetrant nanobody acting as a selective positive allosteric modulator of mGluR2 to validate its role in Aβ dynamics in vivo. Chronic administration of this nanobody in 5xFAD mice accelerated amyloid plaque deposition and worsened cognitive deficits. These findings establish mGluR2 as a target in AD and demonstrate that its selective modulation by nanobodies influences Aβ pathology. This also highlights the potential of nanobodies as next-generation therapeutic agents for modulating neuroreceptors activity in AD.

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.

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 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-induced hypothermia, and we established correlations between in vivo effects and 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.

There is an urgent need for efficient and innovative therapies to treat brain disorders such as psychiatric and neurodegenerative diseases. Immunotherapies have proved to be efficient in many medical areas, but have not been considered to treat brain diseases due to the poor brain penetration of immunoglobulins1,2. Here we developed a bivalent biparatopic antibody, made of two camelid heavy-chain antibodies (called nanobodies)3, one binding to, and the other potentiating the activity of, homodimeric metabotropic glutamate receptor 2. We show that this bivalent nanobody, given peripherally, reaches the brain and corrects cognitive deficits in two preclinical mouse models with endophenotypes resulting from NMDA receptor hypofunction. Notably, these in vivo effects last for at least 7 days after a single intraperitoneal injection and are maintained after subchronic treatment. Our results establish a proof of concept that nanobodies can target brain receptors, and pave the way for nanobody-based therapeutic strategies for the treatment of brain disorders.

Background:Rap1 is critical for platelet activation, functioning as a key node of the platelet activation pathways. Objectives:This study aimed to develop VHH-Fc (minibodies) against Rap1 for the purpose to quantify active Rap1 levels in platelets. Methods:We have produced the first generation of VHHs against active Rap1 through a series of negative and positive screenings of a synthetic phage display library, utilizing both inactive and active Rap1B. We performed random mutagenesis, followed by yeast 2-hybrid screening to optimize variants. Results:Among 122 VHH clones, 2 with the highest redundancy were subcloned as VHH-Fc. Both selectively detected active Rap1B G12V in HeLa cells but failed to recognize the inactive Rap1B S17N isoform. They successfully captured Rap1 from platelet lysates incubated with GTPγS and from thrombin receptor activator peptide 6-stimulated platelets. Further optimization yielded 2 superbinder VHH clones: VHH-Fc B89 and VHH-Fc B14. VHH-Fc B89 exhibited a K D of 4.4 nM and showed enhanced capacity to capture active GTP-bound Rap1 compared with the original VHH-Fc. Additionally, it was able to detect overexpressed active Rap1B G12V in HeLa cells. An ELISA setup combining VHH-Fc B14 and a commercial monoclonal antibody targeting total Rap1 was highly effective in detecting both GTPγS-bound Rap1 and endogenous active Rap1 in platelets. Conclusion:This study identifies, that accurately capture active Rap1 in platelets, thereby establishing them as promising tools for future research. We also developed a reliable ELISA test that can facilitate clinical studies to monitor platelet Rap1 activation in various medical contexts.

The heparan sulphate proteoglycan, Glypican-4 (GPC-4), is an integral component of cell surfaces that fulfils key functions as a modulator of cell communication. Over time, human GPC-4 (hGPC4) has gained recognition as a valuable target for enhancing the therapeutic potential of human pluripotent stem cells (hPSCs). hGPC-4 is also a promising diagnostic and therapeutic target for a range of developmental and neurological disorders, as well as cancer. Its involvement in multiple biological processes and its impact on cellular signaling pathways make it a compelling candidate for future research and clinical applications. Here, we report RB1 and RB3 as the first hGPC-4-specific nanobodies. Both RB3 and RB1, bind recombinant hGPC4 with affinities in the tens of nanomolar range, whereas only RB1 recognizes native, cell-expressed hGPC4, highlighting its potential for functional studies. Notably, the bivalent nanobody Fc-fusion form of RB1, termed RB1-Fc, demonstrates a significant ∼14-fold increase in apparent binding affinity on cells when compared to the monovalent RB1. Furthermore, binding of RB1-Fc to hGPC-4 is dependent on the native conformation of hGPC-4, demonstrating that RB1-Fc is a conformational nanobody. Notably, RB1-Fc neutralizes the activity of GPC-4, as shown by our functional studies in hPSCs. These studies demonstrate the potent efficacy of the lead hGPC4 nanobodies, RB1-Fc and RB3. They also provide a solid rationale for using these nanobodies in the detection and characterization of physiologically and clinically relevant hGPC-4. Additionally, their potential as agents for therapeutic targeting of hGPC-4 opens new avenues for treating disorders associated with dysregulated hGPC-4 activity.

Although the demand for novel immunotherapies to treat companion dogs with spontaneously developing cancer is increasing in high-income countries, most options remain inaccessible. Dogs host a complete and functional immune system reacting to the presence of their tumor. As for humans, many canine neoplasms were shown to overexpress programmed death-ligand 1 (PD-L1), an immune checkpoint inhibitor (ICI) known to downregulate cytotoxic T cell activity upon interaction with its ligand PD-1. In this study, we used alpaca-derived single domain antibodies (sdAbs), also known as nanobodies (Nbs), to develop new ICI targeting canine PD-L1. We selected several clones binding to both recombinant soluble and cell membrane-anchored cPD-L1 forms. Next, their cPD-L1-binding affinities, cPD-1/cPD-L1-blocking abilities and epitope relationships were determined. Most effective Nbs binding to non-overlapping epitopes were combined as biparatopic Fc fusions to provide additional functionalities and improve their efficacy. Remarkably, all engineered Nb constructs efficiently interfered with the cPD-1/cPD-L1-induced signaling pathway, with some multivalent molecules displaying inhibitory concentrations reaching low picomolar range. Moreover, Fc-competent Nb constructs were also shown to induce tumor cell death by antibody-dependent cell-mediated cytotoxicity using human or canine models. Finally, using donor canine peripheral blood mononuclear cells (PBMCs), best candidates were favorably compared with atezolizumab in a Staphylococcal enterotoxin B (SEB)-based interferon-γ (IFNγ) secretion assay.

Cell-surface and cell-cell interaction assays are fundamental for studying receptor-ligand interactions and characterizing cellular responses and functions. They play a critical role in diagnostics and in modulating immune system activity for therapeutic applications, notably in cancer immunotherapy. By providing time-lapsed and cell-level direct observation of the sample, optical microscopy offers strong advantages compared to current go-to techniques, which are typically either ensemble methods (e.g., measuring cell populations) or indirect readouts (e.g., impedance for adherent cells). This protocol describes two complementary microscopy-based assays: (1) a cell-surface ligand binding assay to quantify dynamic interactions between human primary Natural Killer (NK) cells and a cancer-mimicking surface, and (2) a cell-cell interaction assay to evaluate antibody-dependent cell cytotoxicity (ADCC) mediated by NK cells targeting tumor cells. Additionally, the protocol uses Celldetective, a new open graphical user interface for quantitative analysis of cell interaction dynamics from 2D time-lapse microscopy datasets. Although applied here to primary immune cells, these methods are adaptable to various cell types, including other immune cells, fibroblasts, and cancer cells. This approach enables direct observation and quantification of cellular morphology, motility, cell-cell interactions, and dynamic behaviors at single-cell resolution over time, facilitating detailed analysis of mechanisms such as cell death, migration, and immune synapse formation. Key features • End-to-end protocol for antibody evaluation by optical microscopy on living cells using simple reagents, followed by full open-source software image analysis and data rendering • Quantitative analysis of cell-surface interactions using label-free imaging to study the dynamic spreading of NK cells on antibody-coated surfaces under different antibody concentrations. • High-resolution evaluation of antibody-dependent cell cytotoxicity in NK-cancer cells co-culture using fluorescence imaging, deep learning-based death detection, and synchronized single-cell measurements.

Immune cell engagers are molecular agents, usually antibody-based constructs, engineered to recruit immune cells against cancer cells and kill them. They are versatile and powerful tools for cancer immunotherapy. Despite the multiplication of engagers tested and accepted in the clinic, how molecular and cellular parameters influence their actions is poorly understood. In particular, disentangling the respective roles 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 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 ranking Abs and donors and predicting 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 engager characteristics, rationalizing the landscape of molecule design.

The treatment of brain diseases is hindered by the blood-brain barrier (BBB), a major obstacle for efficient brain exposure of therapeutic agents, in particular biotherapeutics. Different strategies are currently evaluated to enhance drug delivery across the BBB, among which the development of vector molecules that target specific receptors expressed by BBB endothelial cells and involved in receptor-mediated transcytosis (RMT). In this process, therapeutic cargos conjugated to optimized molecular vectors can undergo trans-endothelial transport and delivery in the brain parenchyma. The transferrin receptor 1 (TfR1) is enriched in brain endothelial cells and is one of the most studied receptors for drug delivery to the central nervous system (CNS). Several antibodies and molecules derived thereof that target TfR1 have been developed, but few display trans-species reactivity, hindering the transition from preclinical to clinical development. In the present study, we selected and characterized cross-species reactive Variable domain of Heavy chain only antibody from camelids (VHH), and in particular C5 and B8, that bind rodent, rhesus monkey and human TfR1. When fused to a human Immunoglobulin 1 (IgG1) Fc region, C5 or B8 monomers or homodimers were taken up by engineered CHO cells expressing the rodent, human and rhesus monkey TfR1, and by rodent or human brain endothelial cells. Key human TfR1 amino-acid residues of the C5 and B8 epitopes were identified based on the generation of human TfR1 mutants. Assessment of their functional binding revealed an original binding region at the interface of the TfR1 dimer. Following systemic injection in mice of VHH-Fc fusions displaying different properties in terms of affinity and avidity toward TfR1, we demonstrated improved brain uptake compared to control molecules encompassing an irrelevant VHH. Brain delivery efficiency of the parental and optimized VHHTfR1 was further demonstrated using Neurotensin (NT)-induced hypothermia (HT) as a read-out in wild-type mice and in B-hTfR transgenic mice expressing the human TfR1 ectodomain. We established in vitro-in vivo correlations between the human TfR1 binding properties determined by surface plasmon resonance (SPR) of VHH-NT affinity variants, and their potential to induce HT in B-hTfR mice. We identified key human TfR1 binding parameters leading to efficient BBB transcytosis i.e.: a rapid dissociation rate and intermediate affinity. Some of the engineered C5 and B8 variants presented promising cross-species reactivity towards the murine, but also rhesus monkey TfR1. We also generated variants with very similar human-rhesus monkey TfR1 binding properties and optimal affinities for TfR1-dependant transcytosis. These VHHs could be further developed as molecular shuttles for the transport of imaging or therapeutic agents, including biomolecules, across the rodent, non-human primate and human BBB, allowing straightforward preclinical to clinical translation. ### Competing Interest Statement Competing interests MK was director of the Institute of Neurophysiopathology, UMR7051 academic neuroscience laboratory supported by the CNRS and Aix-Marseille University, co-founder, shareholder and scientific counsel of the VECT-HORUS biotechnology company. MD, RC, DB, AF, KB, GG, MM, YM, SDG, IJ, CB, CF, KV, MT, GJ, PL, BS, AR, GDP, MG, JT are currently or were previously paid employees of VECT-HORUS. VS is currently a paid employee of Deeptope and RS is CEO of Deeptope. Financial support was provided by the French National Agency for Research (ANR NANOVECTOR Project coordinated by MK), by the CNRS, Aix Marseille University and Vect-Horus., NANOVECTOR ANR-15-CE18-0010-03

Due to their small size, high specificity, and modularity, nanobodies, or single-domain antibodies, have emerged as versatile tools in cancer immunotherapy. This review provides a comprehensive and up-to-date overview of nanobody-based strategies designed to enhance T cell-mediated anti-tumor responses. It first outlines the structural and functional characteristics of nanobodies, highlighting their tumor penetration and modular design potential. Then, three major nanobody-driven therapeutic approaches are discussed in detail: immune checkpoint inhibition, T cell engagement, and chimeric antigen receptor (CAR)-T cell engineering. For each strategy, current preclinical and clinical developments are reviewed. Challenges are explored, along with emerging strategies to overcome them and expand the therapeutic applicability of nanobody-based immunotherapy, including format choice, valency tuning, pharmacokinetic optimization, and safety enhancements.

The world has witnessed a revolution in therapeutics with the development of biological medicines such as antibodies and antibody fragments, notably nanobodies. These nanobodies possess unique characteristics including high specificity and modulatory activity, making them promising candidates for therapeutic applications. Identifying their binding mode is essential for their development. Experimental structural techniques are effective to get such information, but they are expensive and time-consuming. Here, we propose a computational approach, aiming to identify the epitope of a nanobody that acts as an agonist and a positive allosteric modulator at the rat metabotropic glutamate receptor 5. We employed multiple structure modeling tools, including various artificial intelligence algorithms for epitope mapping. The computationally identified epitope was experimentally validated, confirming the success of our approach. Additional dynamics studies provided further insights on the modulatory activity of the nanobody. The employed methodologies and approaches initiate a discussion on the efficacy of diverse techniques for epitope mapping and later nanobody engineering.