Goods and Bads of the Endocannabinoid System as a Therapeutic Target: Lessons Learned after 30 Years.

The cannabis derivative marijuana is the most widely used recreational drug in the Western world and is consumed by an estimated 83 million individuals (∼3% of the world population). In recent years, there has been a marked transformation in society regarding the risk perception of cannabis, driven by its legalization and medical use in many states in the United States and worldwide. Compelling research evidence and the Food and Drug Administration cannabis-derived cannabidiol approval for severe childhood epilepsy have confirmed the large therapeutic potential of cannabidiol itself, Δ9-tetrahydrocannabinol and other plant-derived cannabinoids (phytocannabinoids). Of note, our body has a complex endocannabinoid system (ECS)-made of receptors, metabolic enzymes, and transporters-that is also regulated by phytocannabinoids. The first endocannabinoid to be discovered 30 years ago was anandamide (N-arachidonoyl-ethanolamine); since then, distinct elements of the ECS have been the target of drug design programs aimed at curing (or at least slowing down) a number of human diseases, both in the central nervous system and at the periphery. Here a critical review of our knowledge of the goods and bads of the ECS as a therapeutic target is presented to define the benefits of ECS-active phytocannabinoids and ECS-oriented synthetic drugs for human health. SIGNIFICANCE STATEMENT: The endocannabinoid system plays important roles virtually everywhere in our body and is either involved in mediating key processes of central and peripheral diseases or represents a therapeutic target for treatment. Therefore, understanding the structure, function, and pharmacology of the components of this complex system, and in particular of key receptors (like cannabinoid receptors 1 and 2) and metabolic enzymes (like fatty acid amide hydrolase and monoacylglycerol lipase), will advance our understanding of endocannabinoid signaling and activity at molecular, cellular, and system levels, providing new opportunities to treat patients.

2-AG-Mediated Control of GABAergic Signaling Is Impaired in a Model of Epilepsy.

Repeated seizures result in a persistent maladaptation of endocannabinoid (eCB) signaling, mediated part by anandamide signaling deficiency in the basolateral amygdala (BLA) that manifests as aberrant synaptic function and altered emotional behavior. Here, we determined the effect of repeated seizures (kindling) on 2-arachidonoylglycerol (2-AG) signaling on GABA transmission by directly measuring tonic and phasic eCB-mediated retrograde signaling in an in vitro BLA slice preparation from male rats. We report that both activity-dependent and muscarinic acetylcholine receptor (mAChR)-mediated depression of GABA synaptic transmission was reduced following repeated seizure activity. These effects were recapitulated in sham rats by preincubating slices with the 2-AG synthesizing enzyme inhibitor DO34. Conversely, preincubating slices with the 2-AG degrading enzyme inhibitor KML29 rescued activity-dependent 2-AG signaling, but not mAChR-mediated synaptic depression, over GABA transmission in kindled rats. These effects were not attributable to a change in cannabinoid type 1 (CB1) receptor sensitivity or altered 2-AG tonic signaling since the application of the highly selective CB1 receptor agonist CP55,940 provoked a similar reduction in GABA synaptic activity in both sham and kindled rats, while no effect of either DO34 or of the CB1 inverse agonist AM251 was observed on frequency and amplitude of spontaneous IPSCs in either sham or kindled rats. Collectively, these data provide evidence that repeated amygdala seizures persistently alter phasic 2-AG-mediated retrograde signaling at BLA GABAergic synapses, probably by impairing stimulus-dependent 2-AG synthesis/release, which contributes to the enduring aberrant synaptic plasticity associated with seizure activity.SIGNIFICANCE STATEMENT The plastic reorganization of endocannabinoid (eCB) signaling after seizures and during epileptogenesis may contribute to the negative neurobiological consequences associated with seizure activity. Therefore, a deeper understanding of the molecular basis underlying the pathologic long-term eCB signaling remodeling following seizure activity will be crucial to the development of novel therapies for epilepsy that not only target seizure activity, but, most importantly, the epileptogenesis and the comorbid conditions associated with epilepsy.

Targeting Endocannabinoid Signaling: FAAH and MAG Lipase Inhibitors.

Inspired by the medicinal properties of the plant Cannabis sativa and its principal component (-)-trans-Δ9-tetrahydrocannabinol (THC), researchers have developed a variety of compounds to modulate the endocannabinoid system in the human brain. Inhibitors of fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), which are the enzymes responsible for the inactivation of the endogenous cannabinoids anandamide and 2-arachidonoylglycerol, respectively, may exert therapeutic effects without inducing the adverse side effects associated with direct cannabinoid CB1 receptor stimulation by THC. Here we review the FAAH and MAGL inhibitors that have reached clinical trials, discuss potential caveats, and provide an outlook on where the field is headed.

Discovery of a Photoaffinity Probe that Captures the Active Conformation of the Cannabinoid CB2 Receptor.

The cannabinoid receptor type 2 (CB2R) is a G protein-coupled receptor with therapeutic potential for the treatment of inflammatory disorders. Fluorescent probes are desirable to study its receptor localization, expression and occupancy. Previously, we have reported a photoaffinity probe LEI-121 that stabilized the inactive conformation of the CB2R. Here, we report the structure-based design of a novel bifunctional probe that captures the active conformation of the CB2R upon irradiation with light. An alkyne handle was incorporated to visualize the receptor using click-chemistry with fluorophore-azides. These probes may hold promise to study different receptor conformations in relation to their cellular localization and function.

Enhancing Drug Discovery and Development through the Integration of Medicinal Chemistry, Chemical Biology, and Academia-Industry Partnerships: Insights from Roche's Endocannabinoid System Projects.

The endocannabinoid system (ECS) is a critical regulatory network composed of endogenous cannabinoids (eCBs), their synthesizing and degrading enzymes, and associated receptors. It is integral to maintaining homeostasis and orchestrating key functions within the central nervous and immune systems. Given its therapeutic significance, we have launched a series of drug discovery endeavors aimed at ECS targets, including peroxisome proliferator-activated receptors (PPARs), cannabinoid receptors types 1 (CB1R) and 2 (CB2R), and monoacylglycerol lipase (MAGL), addressing a wide array of medical needs. The pursuit of new therapeutic agents has been enhanced by the creation of specialized labeled chemical probes, which aid in target localization, mechanistic studies, assay development, and the establishment of biomarkers for target engagement. By fusing medicinal chemistry with chemical biology in a comprehensive, translational end-to-end drug discovery strategy, we have expedited the development of novel therapeutics. Additionally, this strategy promises to foster highly productive partnerships between industry and academia, as will be illustrated through various examples.

Chemical Proteomics Reveals Antibiotic Targets of Oxadiazolones in MRSA.

Phenotypic screening is a powerful approach to identify novel antibiotics, but elucidation of the targets responsible for the antimicrobial activity is often challenging in the case of compounds with a polypharmacological mode of action. Here, we show that activity-based protein profiling maps the target interaction landscape of a series of 1,3,4-oxadiazole-3-ones identified in a phenotypic screen to have high antibacterial potency against multidrug-resistant Staphylococcus aureus. In situ competitive and comparative chemical proteomics with a tailor-made activity-based probe, in combination with transposon and resistance studies, revealed several cysteine and serine hydrolases as relevant targets. Our data showcase oxadiazolones as a novel antibacterial chemotype with a polypharmacological mode of action, in which FabH, FphC, and AdhE play a central role.

EFMC: Trends in Medicinal Chemistry and Chemical Biology.

Ground-breaking research in disease biology and continuous efforts in method development have uncovered a range of potential new drug targets. Increasingly, the drug discovery process is informed by technologies involving chemical probes as tools. Applications for chemical probes comprise target identification and assessment, as well as the qualification of small molecules as chemical starting points and drug candidates. Progress in probe chemistry has opened the way to novel assay formats and pharmaceutical compound classes. The European Federation of Medicinal Chemistry and Chemical Biology (EFMC) has launched the Chemical Biology Initiative to advance science in the field of medicinal chemistry and chemical biology, while representing all members of this extended scientific community. This review provides an overview of the many important developments in the field of chemical biology that have happened at the lively interface of academic and industrial research.

Chemical Probes to Control and Visualize Lipid Metabolism in the Brain.

Signaling lipids, such as the endocannabinoids, play an important role in the brain. They regulate synaptic transmission and control various neurophysiological processes, including pain sensation, appetite, memory formation, stress, and anxiety. Unlike classical neurotransmitters, lipid messengers are produced on demand and degraded by metabolic enzymes to control their lifespan and signaling actions. Chemical biology approaches have become one of the main driving forces to study and unravel the physiological role of lipid messengers in the brain. Here, we review how the development and use of chemical probes has allowed one to study endocannabinoid signaling by (i) inhibiting the biosynthetic and metabolic enzymes; (ii) visualizing the activity of these enzymes; and (iii) controlling the release and transport of the endocannabinoids. Activity-based probes were instrumental to guide the discovery of highly selective and in vivo active inhibitors of the biosynthetic (DAGL, NAPE-PLD) and metabolic (MAGL, FAAH) enzymes of endocannabinoids. These inhibitors allowed one to study the role of these enzymes in animal models of disease. For instance, the DAGL-MAGL axis was shown to control neuroinflammation and the NAPE-PLD-FAAH axis to regulate emotional behavior. Activity-based protein profiling and chemical proteomics were essential to guide the drug discovery and development of compounds targeting MAGL and FAAH, such as ABX-1431 (Lu AG06466) and PF-04457845, respectively. These experimental drugs are now in clinical trials for multiple indications, including multiple sclerosis and post-traumatic stress disorders. Activity-based probes have also been used to visualize the activity of these lipid metabolizing enzymes with high spatial resolution in brain slices, thereby showing the cell type-specific activity of these lipid metabolizing enzymes. The transport, release, and uptake of signaling lipids themselves cannot, however, be captured by activity-based probes in a spatiotemporal controlled manner. Therefore, bio-orthogonal lipids equipped with photoreactive, photoswitchable groups or photocages have been developed. These chemical probes were employed to investigate the protein interaction partners of the endocannabinoids, such as putative membrane transporters, as well as to study the functional cellular responses within milliseconds upon irradiation. Finally, genetically encoded sensors have recently been developed to monitor the real-time release of endocannabinoids with high spatiotemporal resolution in cultured neurons, acute brain slices, and in vivo mouse models. It is anticipated that the combination of chemical probes, highly selective inhibitors, and sensors with advanced (super resolution) imaging modalities, such as PharmacoSTORM and correlative light-electron microscopy, will uncover the fundamental basis of lipid signaling at nanoscale resolution in the brain. Furthermore, chemical biology approaches enable the translation of these fundamental discoveries into clinical solutions for brain diseases with aberrant lipid signaling.

Stress-induced modulation of endocannabinoid signaling leads to delayed strengthening of synaptic connectivity in the amygdala.

Even a brief exposure to severe stress strengthens synaptic connectivity days later in the amygdala, a brain area implicated in the affective symptoms of stress-related psychiatric disorders. However, little is known about the synaptic signaling mechanisms during stress that eventually culminate in its delayed impact on the amygdala. Hence, we investigated early stress-induced changes in amygdalar synaptic signaling in order to prevent its delayed effects. Whole-cell recordings in basolateral amygdala (BLA) slices from rats revealed higher frequency of miniature excitatory postsynaptic currents (mEPSCs) immediately after 2-h immobilization stress. This was replicated by inhibition of cannabinoid receptors (CB1R), suggesting a role for endocannabinoid (eCB) signaling. Stress also reduced N-arachidonoylethanolamine (AEA), an endogenous ligand of CB1R. Since stress-induced activation of fatty acid amide hydrolase (FAAH) reduces AEA, we confirmed that oral administration of an FAAH inhibitor during stress prevents the increase in synaptic excitation in the BLA soon after stress. Although stress also caused an immediate reduction in synaptic inhibition, this was not prevented by FAAH inhibition. Strikingly, FAAH inhibition during the traumatic stressor was also effective 10 d later on the delayed manifestation of synaptic strengthening in BLA neurons, preventing both enhanced mEPSC frequency and increased dendritic spine-density. Thus, oral administration of an FAAH inhibitor during a brief stress prevents the early synaptic changes that eventually build up to hyperexcitability in the amygdala. This framework is of therapeutic relevance because of growing interest in targeting eCB signaling to prevent the gradual development of emotional symptoms and underlying amygdalar dysfunction triggered by traumatic stress.

Comparative Photoaffinity Profiling of Omega-3 Signaling Lipid Probes Reveals Prostaglandin Reductase 1 as a Metabolic Hub in Human Macrophages.

The fish oil constituent docosahexaenoic acid (DHA, 22:6 n-3) is a signaling lipid with anti-inflammatory properties. The molecular mechanisms underlying the biological effect of DHA are poorly understood. Here, we report the design, synthesis, and application of a complementary pair of bio-orthogonal, photoreactive probes based on the polyunsaturated scaffold DHA and its oxidative metabolite 17-hydroxydocosahexaenoic acid (17-HDHA). In these probes, an alkyne serves as a handle to introduce a fluorescent reporter group or a biotin-affinity tag via copper(I)-catalyzed azide-alkyne cycloaddition. This pair of chemical probes was used to map specific targets of the omega-3 signaling lipids in primary human macrophages. Prostaglandin reductase 1 (PTGR1) was identified as an interaction partner that metabolizes 17-oxo-DHA, an oxidative metabolite of 17-HDHA. 17-oxo-DHA reduced the formation of pro-inflammatory lipids 5-HETE and LTB4 in human macrophages and neutrophils. Our results demonstrate the potential of comparative photoaffinity protein profiling for the discovery of metabolic enzymes of bioactive lipids and highlight the power of chemical proteomics to uncover new biological insights.

Discovery of a NAPE-PLD inhibitor that modulates emotional behavior in mice.

N-acylethanolamines (NAEs), which include the endocannabinoid anandamide, represent an important family of signaling lipids in the brain. The lack of chemical probes that modulate NAE biosynthesis in living systems hamper the understanding of the biological role of these lipids. Using a high-throughput screen, chemical proteomics and targeted lipidomics, we report here the discovery and characterization of LEI-401 as a CNS-active N-acylphosphatidylethanolamine phospholipase D (NAPE-PLD) inhibitor. LEI-401 reduced NAE levels in neuroblastoma cells and in the brain of freely moving mice, but not in NAPE-PLD KO cells and mice, respectively. LEI-401 activated the hypothalamus-pituitary-adrenal axis and impaired fear extinction, thereby emulating the effect of a cannabinoid CB1 receptor antagonist, which could be reversed by a fatty acid amide hydrolase inhibitor. Our findings highlight the distinctive role of NAPE-PLD in NAE biosynthesis in the brain and suggest the presence of an endogenous NAE tone controlling emotional behavior.

Live-Cell Imaging of Sterculic Acid-a Naturally Occurring 1,2-Cyclopropene Fatty Acid-by Bioorthogonal Reaction with Turn-On Tetrazine-Fluorophore Conjugates.

In the field of lipid research, bioorthogonal chemistry has made the study of lipid uptake and processing in living systems possible, whilst minimising biological properties arising from detectable pendant groups. To allow the study of unsaturated free fatty acids in live cells, we here report the use of sterculic acid, a 1,2-cyclopropene-containing oleic acid analogue, as a bioorthogonal probe. We show that this lipid can be readily taken up by dendritic cells without toxic side effects, and that it can subsequently be visualised using an inverse electron-demand Diels-Alder reaction with quenched tetrazine-fluorophore conjugates. In addition, the lipid can be used to identify changes in protein oleoylation after immune cell activation. Finally, this reaction can be integrated into a multiplexed bioorthogonal reaction workflow by combining it with two sequential copper-catalysed Huisgen ligation reactions. This allows for the study of multiple biomolecules in the cell simultaneously by multimodal confocal imaging.

Protein Dynamics Influence the Enzymatic Activity of Phospholipase A/Acyltransferases 3 and 4.

Phospholipase A/acyltransferase 3 (PLAAT3) and PLAAT4 are enzymes involved in the synthesis of bioactive lipids. Despite sequential and structural similarities, the two enzymes differ in activity and specificity. The relation between the activity and dynamics of the N-terminal domains of PLAAT3 and PLAAT4 was studied. PLAAT3 has a much higher melting temperature and exhibits less nanosecond and millisecond dynamics in the active site, in particular in loop L2(B6), as shown by NMR spectroscopy and molecular dynamics calculations. Swapping the L2(B6) loops between the two PLAAT enzymes results in strongly increased phospholipase activity in PLAAT3 but no reduction in PLAAT4 activity, indicating that this loop contributes to the low activity of PLAAT3. The results show that, despite structural similarity, protein dynamics differ substantially between the PLAAT variants, which can help to explain the activity and specificity differences.

Cannabinoid type 1 receptor inverse agonism attenuates dyslipidemia and atherosclerosis in APOE∗3-Leiden.CETP mice.

Pharmacological blockade of the cannabinoid type 1 receptor, a G protein-coupled receptor expressed in the central nervous system and various peripheral tissues, reverses diet-induced obesity and dyslipidemia through the reduction of food intake and altered nutrient partitioning. This strategy is being explored for a number of therapeutic applications; however, its potency for the treatment of atherosclerotic cardiovascular disease via improvements in lipid metabolism remains unclear. Therefore, here, we aimed to investigate whether inhibition of the endocannabinoid system can attenuate atherosclerosis development through improvement of dyslipidemia. Lean, dyslipidemic female APOE∗3-Leiden.CETP transgenic mice were fed a Western-type diet supplemented with or without the cannabinoid type 1 receptor inverse agonist rimonabant (20 mg·kg body weight-1 day-1) for up to 20 weeks. Plasma lipids and bile acids were determined, and atherosclerotic lesions were scored in the aortic valve region. Rimonabant lowered plasma levels of triglyceride (TG) (-56%) and non-HDL-C (-19%) and increased HDL-C (+57%). These effects were explained by decreased VLDL-TG production (-52%) and accelerated VLDL-TG turnover accompanied by pronounced browning of white adipose tissue. In addition, rimonabant attenuated reverse cholesterol transport (-30%), increased plasma bile acid levels (+160%), and increased hepatic cholesterol accumulation (+88%). Importantly, rimonabant markedly lowered atherosclerotic lesion size (-64%), which coincided with decreased lesion severity (28% vs. 56% severe lesions) and which strongly correlated with non-HDL-C exposure (R2 = 0.60). Taken together, inhibition of the endocannabinoid system potently reverses dyslipidemia and prevents atherogenesis, even in the absence of obesity.

The Chemical Biology-Medicinal Chemistry Continuum: EFMC's Vision.

The European Federation for Medicinal chemistry and Chemical biology (EFMC) is a federation of learned societies. It groups organizations of European scientists working in a dynamic field spanning chemical biology and medicinal chemistry. New ideas, tools, and technologies emerging from a wide array of scientific disciplines continuously energize this rapidly evolving area. Medicinal chemistry is the design, synthesis, and optimization of biologically active molecules aimed at discovering new drug candidates - a mission that in many ways overlaps with the scope of chemical biology. Chemical biology is by now a mature field of science for which a more precise definition of what it encompasses, in the frame of EFMC, is timely. This article discusses chemical biology as currently understood by EFMC, including all activities dealing with the design and synthesis of biologically active chemical tools and their use to probe, characterize, or influence biological systems.

Manno-epi-cyclophellitols Enable Activity-Based Protein Profiling of Human α-Mannosidases and Discovery of New Golgi Mannosidase II Inhibitors.

Golgi mannosidase II (GMII) catalyzes the sequential hydrolysis of two mannosyl residues from GlcNAcMan5GlcNAc2 to produce GlcNAcMan3GlcNAc2, the precursor for all complex N-glycans, including the branched N-glycans associated with cancer. Inhibitors of GMII are potential cancer therapeutics, but their usefulness is limited by off-target effects, which produce α-mannosidosis-like symptoms. Despite many structural and mechanistic studies of GMII, we still lack a potent and selective inhibitor of this enzyme. Here, we synthesized manno-epi-cyclophellitol epoxide and aziridines and demonstrate their covalent modification and time-dependent inhibition of GMII. Application of fluorescent manno-epi-cyclophellitol aziridine derivatives enabled activity-based protein profiling of α-mannosidases from both human cell lysate and mouse tissue extracts. Synthesized probes also facilitated a fluorescence polarization-based screen for dGMII inhibitors. We identified seven previously unknown inhibitors of GMII from a library of over 350 iminosugars and investigated their binding modalities through X-ray crystallography. Our results reveal previously unobserved inhibitor binding modes and promising scaffolds for the generation of selective GMII inhibitors.

Development of High-Specificity Fluorescent Probes to Enable Cannabinoid Type 2 Receptor Studies in Living Cells.

Pharmacological modulation of cannabinoid type 2 receptor (CB2R) holds promise for the treatment of numerous conditions, including inflammatory diseases, autoimmune disorders, pain, and cancer. Despite the significance of this receptor, researchers lack reliable tools to address questions concerning the expression and complex mechanism of CB2R signaling, especially in cell-type and tissue-dependent contexts. Herein, we report for the first time a versatile ligand platform for the modular design of a collection of highly specific CB2R fluorescent probes, used successfully across applications, species, and cell types. These include flow cytometry of endogenously expressing cells, real-time confocal microscopy of mouse splenocytes and human macrophages, as well as FRET-based kinetic and equilibrium binding assays. High CB2R specificity was demonstrated by competition experiments in living cells expressing CB2R at native levels. The probes were effectively applied to FACS analysis of microglial cells derived from a mouse model relevant to Alzheimer's disease.

STA-55, an Easily Accessible, Broad-Spectrum, Activity-Based Aldehyde Dehydrogenase Probe.

Aldehyde dehydrogenases (ALDHs) convert aldehydes into carboxylic acids and are often upregulated in cancer. They have been linked to therapy resistance and are therefore potential therapeutic targets. However, only a few selective and potent inhibitors are currently available for this group of enzymes. Competitive activity-based protein profiling (ABPP) would aid the development and validation of new selective inhibitors. Herein, a broad-spectrum activity-based probe that reports on several ALDHs is presented. This probe was used in a competitive ABPP protocol against three ALDH inhibitors in lung cancer cells to determine their selectivity profiles and establish their target engagement.

Olaparib-Based Photoaffinity Probes for PARP-1 Detection in Living Cells.

The poly-ADP-ribose polymerase (PARP) is a protein from the family of ADP-ribosyltransferases that catalyzes polyadenosine diphosphate ribose (ADPR) formation in order to attract the DNA repair machinery to sites of DNA damage. The inhibition of PARP activity by olaparib can cause cell death, which is of clinical relevance in some tumor types. This demonstrates that quantification of PARP activity in the context of living cells is of great importance. In this work, we present the design, synthesis and biological evaluation of photo-activatable affinity probes inspired by the olaparib molecule that are equipped with a diazirine for covalent attachment upon activation by UV light and a ligation handle for the addition of a reporter group of choice. SDS-PAGE, western blotting and label-free LC-MS/MS quantification analysis show that the probes target the PARP-1 protein and are selectively outcompeted by olaparib; this suggests that they bind in the same enzymatic pocket. Proteomics data are available via ProteomeXchange with identifier PXD018661.

Improving CLL Vγ9Vδ2-T-cell fitness for cellular therapy by ex vivo activation and ibrutinib.

The efficacy of autologous (αß) T-cell-based treatment strategies in chronic lymphocytic leukemia (CLL) has been modest. The Vγ9Vδ2-T cell subset consists of cytotoxic T lymphocytes with potent antilymphoma activity via a major histocompatibility complex-independent mechanism. We studied whether Vγ9Vδ2-T cells can be exploited as autologous effector lymphocytes in CLL. Healthy control Vγ9Vδ2-T cells were activated by and had potent cytolytic activity against CLL cells. However, CLL-derived Vγ9Vδ2-T cells proved dysfunctional with respect to effector cytokine production and degranulation, despite an increased frequency of the effector-type subset. Consequently, cytotoxicity against malignant B cells was hampered. A comparable dysfunctional phenotype was observed in healthy Vγ9Vδ2-T cells after coculture with CLL cells, indicating a leukemia-induced mechanism. Gene-expression profiling implicated alterations in synapse formation as a conceivable contributor to compromised Vγ9Vδ2-T-cell function in CLL patients. Dysfunction of Vγ9Vδ2-T cells was fully reversible upon activation with autologous monocyte-derived dendritic cells (moDCs). moDC activation resulted in efficient expansion and predominantly yielded Vγ9Vδ2-T cells with a memory phenotype. Furthermore, ibrutinib treatment promoted an antitumor T helper 1 (TH1) phenotype in Vγ9Vδ2-T cells, and we demonstrated binding of ibrutinib to IL-2-inducible kinase (ITK) in Vγ9Vδ2-T cells. Taken together, CLL-mediated dysfunction of autologous Vγ9Vδ2-T cells is fully reversible, resulting in potent cytotoxicity toward CLL cells. Our data support the potential use of Vγ9Vδ2-T cells as effector T cells in CLL immunotherapy and favor further exploration of combining Vγ9Vδ2-T-cell-based therapy with ibrutinib.