Deciphering the monocyte-targeting mechanisms of PEGylated cationic liposomes by investigating the biomolecular corona.

Cationic liposomes specifically target monocytes in blood, rendering them promising drug-delivery tools for cancer immunotherapy, vaccines, and therapies for monocytic leukaemia. The mechanism behind this monocyte targeting ability is, however, not understood, but may involve plasma proteins adsorbed on the liposomal surfaces. To shed light on this, we investigated the biomolecular corona of three different types of PEGylated cationic liposomes, finding all of them to adsorb hyaluronan-associated proteins and proteoglycans upon incubation in human blood plasma. This prompted us to study the role of the TLR4 co-receptors CD44 and CD14, both involved in signalling and uptake pathways of proteoglycans and glycosaminoglycans. We found that separate inhibition of each of these receptors hampered the monocyte uptake of the liposomes in whole human blood. Based on clues from the biomolecular corona, we have thus identified two receptors involved in the targeting and uptake of cationic liposomes in monocytes, in turn suggesting that certain proteoglycans and glycosaminoglycans may serve as monocyte-targeting opsonins. This mechanistic knowledge may pave the way for rational design of future monocyte-targeting drug-delivery platforms.

Quantifying the transport of biologics across intestinal barrier models in real-time by fluorescent imaging.

Unsuccessful clinical translation of orally delivered biological drugs remains a challenge in pharmaceutical development and has been linked to insufficient mechanistic understanding of intestinal drug transport. Live cell imaging could provide such mechanistic insights by directly tracking drug transport across intestinal barriers at subcellular resolution, however traditional intestinal in vitro models are not compatible with the necessary live cell imaging modalities. Here, we employed a novel microfluidic platform to develop an in vitro intestinal epithelial barrier compatible with advanced widefield- and confocal microscopy. We established a quantitative, multiplexed and high-temporal resolution imaging assay for investigating the cellular uptake and cross-barrier transport of biologics while simultaneously monitoring barrier integrity. As a proof-of-principle, we use the generic model to monitor the transport of co-administrated cell penetrating peptide (TAT) and insulin. We show that while TAT displayed a concentration dependent difference in its transport mechanism and efficiency, insulin displayed cellular internalization, but was restricted from transport across the barrier. This illustrates how such a sophisticated imaging based barrier model can facilitate mechanistic studies of drug transport across intestinal barriers and aid in vivo and clinical translation in drug development.

Mechanisms of selective monocyte targeting by liposomes functionalized with a cationic, arginine-rich lipopeptide.

Stimulation of monocytes with immunomodulating agents can harness the immune system to treat a long range of diseases, including cancers, infections and autoimmune diseases. To this end we aimed to develop a monocyte-targeting delivery platform based on cationic liposomes, which can be utilized to deliver immunomodulators and thus induce monocyte-mediated immune responses while avoiding off-target side-effects. The cationic liposome design is based on functionalizing the liposomal membrane with a cholesterol-anchored tri-arginine peptide (TriArg). We demonstrate that TriArg liposomes can target monocytes with high specificity in both human and murine blood and that this targeting is dependent on the content of TriArg in the liposomal membrane. In addition, we show that the mechanism of selective monocyte targeting involves the CD14 co-receptor, and selectivity is compromised when the TriArg content is increased, resulting in complement-mediated off-target uptake in granulocytes. The presented mechanistic findings of uptake by peripheral blood leukocytes may guide the design of future drug delivery systems utilized for immunotherapy. STATEMENT OF SIGNIFICANCE: Monocytes are attractive targets for immunotherapies of cancers, infections and autoimmune diseases. Specific delivery of immunostimulatory drugs to monocytes is typically achieved using ligand-targeted drug delivery systems, but a simpler approach is to target monocytes using cationic liposomes. To achieve this, however, a deep understanding of the mechanisms governing the interactions of cationic liposomes with monocytes and other leukocytes is required. We here investigate these interactions using liposomes incorporating a cationic arginine-rich lipopeptide. We demonstrate that monocyte targeting can be achieved by fine-tuning the lipopeptide content in the liposomes. Additionally, we reveal that the CD14 receptor is involved in the targeting process, whereas the complement system is not. These mechanistic findings are critical for future design of monocyte-targeting liposomal therapies.

Unravelling Heterogeneities in Complement and Antibody Opsonization of Individual Liposomes as a Function of Surface Architecture.

Coating nanoparticles with poly(ethylene glycol) (PEG) is widely used to achieve long-circulating properties after infusion. While PEG reduces binding of opsonins to the particle surface, immunogenic anti-PEG side-effects show that PEGylated nanoparticles are not truly "stealth" to surface active proteins. A major obstacle for understanding the complex interplay between opsonins and nanoparticles is the averaging effects of the bulk assays that are typically applied to study protein adsorption to nanoparticles. Here, a microscopy-based method for directly quantifying opsonization at the single nanoparticle level is presented. Various surface coatings are investigated on liposomes, including PEG, and show that opsonization by both antibodies and complement C3b is highly dependent on the surface chemistry. It is further demonstrated that this opsonization is heterogeneous, with opsonized and non-opsonized liposomes co-existing in the same ensemble. Surface coatings modify the percentage of opsonized liposomes and/or opsonin surface density on the liposomes, with strikingly different patterns for antibodies and complement. Thus, this assay provides mechanistic details about opsonization at the single nanoparticle level previously inaccessible to established bulk assays.

Accelerated blood clearance and hypersensitivity by PEGylated liposomes containing TLR agonists.

Systemic administration of toll-like receptor (TLR) agonists have demonstrated impressive preclinical results as an anti-cancer therapy due to their potent innate immune-stimulatory properties. The clinical advancement has, however, been hindered by severe adverse effects due to systemic activation of the immune system. Liposomal drug delivery systems may modify biodistribution, cellular uptake, and extend blood circulation, and thus, potentially enable systemic administration of TLR agonists at therapeutic doses. In this study, we investigated potential barriers for the administration of TLR agonists formulated in polyethylene glycosylated (PEGylated) liposomes with regards to liposome formulation, TLR agonist, administration route, administration schedule, biodistribution, blood clearance, and anti-PEG antibodies. We found that administration of TLR agonists formulated in PEGylated liposomes led to high anti-PEG antibody titers, which upon multiple intravenous administrations, resulted in accelerated blood clearance and acute hypersensitivity reactions. The latter was found to be associated with anti-PEG IgG antibody and not anti-PEG IgM antibody opsonization. This study highlights the need to carefully design and evaluate nanoparticle delivery systems for immunotherapy as anti-nanoparticle immune responses may challenge the therapeutic application.

Imaging therapeutic peptide transport across intestinal barriers.

Oral delivery is a highly preferred method for drug administration due to high patient compliance. However, oral administration is intrinsically challenging for pharmacologically interesting drug classes, in particular pharmaceutical peptides, due to the biological barriers associated with the gastrointestinal tract. In this review, we start by summarizing the pharmacological performance of several clinically relevant orally administrated therapeutic peptides, highlighting their low bioavailabilities. Thus, there is a strong need to increase the transport of peptide drugs across the intestinal barrier to realize future treatment needs and further development in the field. Currently, progress is hampered by a lack of understanding of transport mechanisms that govern intestinal absorption and transport of peptide drugs, including the effects of the permeability enhancers commonly used to mediate uptake. We describe how, for the past decades, mechanistic insights have predominantly been gained using functional assays with end-point read-out capabilities, which only allow indirect study of peptide transport mechanisms. We then focus on fluorescence imaging that, on the other hand, provides opportunities to directly visualize and thus follow peptide transport at high spatiotemporal resolution. Consequently, it may provide new and detailed mechanistic understanding of the interplay between the physicochemical properties of peptides and cellular processes; an interplay that determines the efficiency of transport. We review current methodology and state of the art in the field of fluorescence imaging to study intestinal barrier transport of peptides, and provide a comprehensive overview of the imaging-compatible in vitro, ex vivo, and in vivo platforms that currently are being developed to accelerate this emerging field of research.

Protein Transduction Domain Mimics Facilitate Rapid Antigen Delivery into Monocytes.

Delivering peptides and proteins with intracellular function represents a promising avenue for therapeutics, but remains a challenge due to the selective permeability of the plasma membrane. The successful delivery of cytosolically active proteins would enable many opportunities, including improved vaccine development through major histocompatibility complex (MHC) class I antigen display. Extended research using cell-penetrating peptides (CPPs) has aimed to facilitate intracellular delivery of exogenous proteins with some success. A new class of polymer-based mimics termed protein transduction domain mimics (PTDMs), which maintain the positive charge and amphiphilic nature displayed by many CPPs, was developed using a poly-norbornene-based backbone. Herein, we use a previously characterized PTDM to investigate delivery of the model antigen SIINFEKL into leukocytes. Peptide delivery into over 90% of CD14+ monocytes was detected in less than 15 min with nominal inflammatory cytokine response and high cell viability. The co-delivery of a TLR9 agonist and antigen using the PTDM into antigen-presenting cells in vitro showed presentation of SIINFEKL in association with MHC class I molecules, in addition to upregulation of classical differentiation markers revealing the ability of the PTDM to successfully deliver cargo intracellularly and show application in the field of immunotherapy.

Lactate Dehydrogenase Assay for Assessment of Polycation Cytotoxicity.

Cellular toxicity and/or cell death entail complex mechanisms that require multifaceted characterization. A detailed mechanistic assessment of cytotoxicity is essential for design and construction of more effective polycations for nucleic acid delivery. A single toxicity assay cannot stand alone in determining the type and extent of damage or cell death mechanism. In this chapter we describe a lactate dehydrogenase (LDH) assay for high-throughput screening that can be used as a starting point for further detailed cytotoxicity determination. LDH release is considered an early event in necrosis but a late event in apoptosis. An accurate temporal assessment of the toxic responses is crucial as late apoptosis may convert into necrosis as well as in situations where cell death is initiated without any visible cell morphological changes or responses in assays measuring late events, resulting in early ongoing toxicity being overlooked.

Remote-loading of liposomes with manganese-52 and in vivo evaluation of the stabilities of 52Mn-DOTA and 64Cu-DOTA using radiolabelled liposomes and PET imaging.

Liposomes are nanoparticles used in drug delivery that distribute over several days in humans and larger animals. Radiolabeling with long-lived positron emission tomography (PET) radionuclides, such as manganese-52 (52Mn, T½=5.6days), allow the imaging of this biodistribution. We report optimized protocols for radiolabeling liposomes with 52Mn, through both remote-loading and surface labeling. For comparison, liposomes were also remote-loaded and surface labeled with copper-64 (64Cu, T½=12.7h) through conventional means. The chelator DOTA was used in all cases. The in vivo stability of radiometal chelates is widely debated but studies that mimic a realistic in vivo setting are lacking. Therefore, we employed these four radiolabeled liposome types as platforms to demonstrate a new concept for such in vivo evaluation, here of the chelates 52Mn-DOTA and 64Cu-DOTA. This was done by comparing "shielded" remote-loaded with "exposed" surface labeled variants in a CT26 tumor-bearing mouse model. Remote loading (90min at 55°C) and surface labeling (55°C for 2h) of 52Mn gave excellent radiolabeling efficiencies of 97-100% and 98-100% respectively, and the liposome biodistribution was imaged by PET for up to 8days. Liposomes with surface-conjugated 52Mn-DOTA exhibited a significantly shorter plasma half-life (T½=14.4h) when compared to the remote-loaded counterpart (T½=21.3h), whereas surface-conjugated 64Cu-DOTA cleared only slightly faster and non-significantly, when compared to remote-loaded (17.2±2.9h versus 20.3±1.2h). From our data, we conclude the successful remote-loading of liposomes with 52Mn, and furthermore that 52Mn-DOTA may be unstable in vivo whereas 64Cu-DOTA appears suitable for quantitative imaging.

Secretory phospholipase A2 responsive liposomes exhibit a potent anti-neoplastic effect in vitro, but induce unforeseen severe toxicity in vivo.

The clinical use of liposomal drug delivery vehicles is often hindered by insufficient drug release. Here we present the rational design of liposomes optimized for secretory phospholipase A2 (sPLA2) triggered drug release, and test their utility in vitro and in vivo. We hypothesized that by adjusting the level of cholesterol in anionic, unsaturated liposomes we could tune the enzyme specificity based on membrane fluidity, thus obtaining liposomes with an improved therapeutic outcome and reduced side effects. Cholesterol is generally important as a component in the membranes of liposome drug delivery systems due to its stabilizing effects in vivo. The incorporation of cholesterol in sPLA2 sensitive liposomes has not previously been possible due to reduced sPLA2 activity. However, in the present work we solved this challenge by optimizing membrane fluidity. In vitro release studies revealed enzyme specific drug release. Treatment of two different cancer cell lines with liposomal oxaliplatin revealed efficient growth inhibition compared to that of clinically used stealth liposomes. The in vivo therapeutic effect was evaluated in nude NMRI mice using the sPLA2 secreting mammary carcinoma cell line MT-3. Three days after first treatment all mice having received the novel sPLA2 sensitive liposome formulation were euthanized due to severe systemic toxicity. Thus the present study demonstrates that great caution should be implemented when utilizing sPLA2 sensitive liposomes and that the real utility can only be disclosed in vivo. The present studies have clinical implications, as sPLA2 sensitive formulations are currently undergoing clinical trials (LiPlaCis®).

AFM visualization of sub-50nm polyplex disposition to the nuclear pore complex without compromising the integrity of the nuclear envelope.

It has been questioned as to whether polyplexes in the cytoplasm can reach the nuclear compartment and if so in what form. By applying atomic force microscopy (AFM) to the nuclear envelope and the nuclear pore complexes, we demonstrate that disposition of polyethylenimine (PEI)/DNA polyplexes that were microinjected into the oocytes of Xenopus laevis, as an example of a non-dividing cell, is exclusive to the nuclear pore complex (NPC). AFM images show NPCs clogged only with sub-50nm polyplexes. This mode of disposition neither altered the morphology/integrity of the nuclear membrane nor the NPC. AFM images further show polyplexes on the nucleoplasmic side of the envelope, presumably indicating species in transit. Transmission electron microscopy studies of ruptured nuclei from transfected human cell lines demonstrate the presence of sub-50nm particles resembling polyplexes in morphology compared with control preparations.

Differential Modulation of Cellular Bioenergetics by Poly(L-lysine)s of Different Molecular Weights.

Poly(L-lysine)s (PLLs), and related derivatives, have received considerable attention as nonviral vectors. High molecular weight PLLs (H-PLLs) are superior transfectants compared with low Mw PLLs (L-PLLs), but suggested to be more cytotoxic. Through a pan-integrated metabolomic approach using Seahorse XF technology, we studied the impact of PLL size on cellular bioenergetic processes in two human cell lines. In contrast to L-PLLs (1-5 kDa), H-PLLs (15-30 kDa) were more detrimental to both mitochondrial oxidative phosphorylation (OXPHOS) and glycolytic activity resulting in considerable intracellular ATP depletion, thereby initiating necrotic-type cell death. The cellular differences to polycation sensitivity were further related to the mitochondrial state, where the impact was substantial on cells with hyperpolarized mitochondria. These medium-throughput approaches offer better opportunities for understanding inter-related intracellular and cell type-dependent processes instigating a bioenergetics crisis, thus, aiding selection (from available libraries) and improved design of safer biodegradable polycations for nucleic acid compaction and cell type-specific delivery.

Monocyte targeting and activation by cationic liposomes formulated with a TLR7 agonist.

OBJECTIVES: Monocytes are one of the major phagocytic cells that patrol for invading pathogens, and upon activation, differentiate into macrophages or antigen-presenting dendritic cells (DCs) capable of migrating to lymph nodes eliciting an adaptive immune response. The key role in regulating adaptive immune responses has drawn attention to modulate monocyte responses therapeutically within cancer, inflammation and infectious diseases. We present a technology for targeting of monocytes and delivery of a toll-like receptor (TLR) agonist in fresh blood using liposomes with a positively charged surface chemistry. METHODS: Liposomes were extruded at 100 nm, incubated with fresh blood, followed by leukocyte analyses by FACS. Liposomes with and without the TLR7 agonist TMX-202 were incubated with fresh blood, and monocyte activation measured by cytokine secretion by ELISA and CD14 and DC-SIGN expression. RESULTS: The liposomes target monocytes specifically over lymphocytes and granulocytes in human whole blood, and show association with 75 - 95% of the monocytes after 1 h incubation. Formulations of TMX-202 in cationic liposomes were potent in targeting and activation of monocytes, with strong induction of IL-6 and IL-12p40, and differentiation into CD14(+) and DC-SIGN+ DCs. CONCLUSION: Our present liposomes selectively target monocytes in fresh blood, enabling delivery of TLR7 agonists to the intracellular TLR7 receptor, with subsequent monocyte activation and boost in secretion of proinflammatory cytokines. We envision this technology as a promising tool in future cancer immunotherapy.

Low molecular weight poly (2-dimethylamino ethylmethacrylate) polymers with controlled positioned fluorescent labeling: Synthesis, characterization and in vitro interaction with human endothelial cells.

Poly (2-dimethylamino ethylmethacrylate) (PDMAEMA) is an attractive non-degradable polymer studied as nonviral vector for gene delivery but it can be also adopted for delivery of other biopharmaceutical drugs. As a parenteral carrier, the PDMAEMA free form (FF) might interact with tissues and cells. Few data are available on its selective internalization and efflux from cells, while the majority of studies published have followed the distribution of DNA complexed with PDMAEMA. In order to address polycation safety, the first aim was to synthesize by atom transfer radical polymerisation (ATRP) fluorescent labeled PDMAEMA of low molecular weight (Mw) (below 15 kDa), controlling the position and density of fluorescein. The second goal was to analyze the possible difference in uptake and subcellular distribution of this labeled FF polycation between human umbilical vein endothelial cells (HUVEC) and hCMEC/D3 cells. These two cell lines have been chosen in order to detect selectivity towards the blood-brain barrier (BBB). In both cases, polycation was detected along the plasma membrane followed by progressive migration to the peri-nuclear region, where it overlapped with lysosomal structures. The analysis by fluorescence-activated cell sorting (FACS) of the PDMAEMA uptake by hCMEC/D3 cells showed a significant (p<0.05) inhibition (40%) in presence of 2-dexoxy-D-glucose inhibitor, a result supporting an energy-dependence mechanism(s). Cytotoxicity study showed that low Mw PDMAEMA (10 kDa) lead to a minor cytotoxicity compared to the higher ones. As main conclusion this study highlights the similitude in cell trafficking of FF PDMAEMA and data previously reported for PDMAEMA/DNA complexes.

Polyethylenimine architecture-dependent metabolic imprints and perturbation of cellular redox homeostasis.

Polyethylenimines (PEIs) are among the most efficient polycationic non-viral transfectants. PEI architecture and size not only modulate transfection efficiency, but also cytotoxicity. However, the underlying mechanisms of PEI-induced multifaceted cell damage and death are largely unknown. Here, we demonstrate that the central mechanisms of PEI architecture- and size-dependent perturbations of integrated cellular metabolomics involve destabilization of plasma membrane and mitochondrial membranes with consequences on mitochondrial oxidative phosphorylation (OXPHOS), glycolytic flux and redox homeostasis that ultimately modulate cell death. In comparison to linear PEI, the branched architectures induced greater plasma membrane destabilization and were more detrimental to glycolytic activity and OXPHOS capacity as well as being a more potent inhibitor of the cytochrome c oxidase. Accordingly, the branched architectures caused a greater lactate dehydrogenase (LDH) and ATP depletion, activated AMP kinase (AMPK) and disturbed redox homeostasis through diminished availability of nicotinamide adenine dinucleotide phosphate (NADPH), reduced antioxidant capacity of glutathione (GSH) and increased burden of reactive oxygen species (ROS). The differences in metabolic and redox imprints were further reflected in the transfection performance of the polycations, but co-treatment with the GSH precursor N-acetyl-cysteine (NAC) counteracted redox dysregulation and increased the number of viable transfected cells. Integrated biomembrane integrity and metabolomic analysis provides a rapid approach for mechanistic understanding of multifactorial polycation-mediated cytotoxicity, and could form the basis for combinatorial throughput platforms for improved design and selection of safer polymeric vectors.

Polycation-mediated integrated cell death processes.

One of the major challenges in the field of nucleic acid delivery is the design of delivery vehicles with attributes that render them safe as well as efficient in transfection. To this end, polycationic vectors have been intensely investigated with native polyethylenimines (PEIs) being the gold standard. PEIs are highly efficient transfectants, but depending on their architecture and size they induce cytotoxicity through different modes of cell death pathways. Here, we briefly review dynamic and integrated cell death processes and pathways, and discuss considerations in cell death assay design and their interpretation in relation to PEIs and PEI-based engineered vectors, which are also translatable for the design and studying the safety of other transfectants.

Reciprocity in the developmental regulation of aquaporins 1, 3 and 5 during pregnancy and lactation in the rat.

Milk secretion involves significant flux of water, driven largely by synthesis of lactose within the Golgi apparatus. It has not been determined whether this flux is simply a passive consequence of the osmotic potential between cytosol and Golgi, or whether it involves regulated flow. Aquaporins (AQPs) are membrane water channels that regulate water flux. AQP1, AQP3 and AQP5 have previously been detected in mammary tissue, but evidence of developmental regulation (altered expression according to the developmental and physiological state of the mammary gland) is lacking and their cellular/subcellular location is not well understood. In this paper we present evidence of developmental regulation of all three of these AQPs. Further, there was evidence of reciprocity since expression of the rather abundant AQP3 and less abundant AQP1 increased significantly from pregnancy into lactation, whereas expression of the least abundant AQP5 decreased. It would be tempting to suggest that AQP3 and AQP1 are involved in the secretion of water into milk. Paradoxically, however, it was AQP5 that demonstrated most evidence of expression located at the apical (secretory) membrane. The possibility is discussed that AQP5 is synthesized during pregnancy as a stable protein that functions to regulate water secretion during lactation. AQP3 was identified primarily at the basal and lateral membranes of the secretory cells, suggesting a possible involvement in regulated uptake of water and glycerol. AQP1 was identified primarily at the capillary and secretory cell cytoplasmic level and may again be more concerned with uptake and hence milk synthesis, rather than secretion. The fact that expression was developmentally regulated supports, but does not prove, a regulatory involvement of AQPs in water flux through the milk secretory cell.

Live-cell fluorescent microscopy platforms for real-time monitoring of polyplex-cell interaction: basic guidelines.

A myriad of cationic polymeric delivery vehicles are currently being developed with the aim of transporting various forms of nucleic acids to mammalian cells. The complexes between polycations and nucleic acids are referred to as polyplexes. The screening for successful polyplex candidates requires interdisciplinary research platforms and techniques for a more profound understanding of biophysical properties of delivery vehicles and their biological performance, including stability, transfection efficacy and possible cytotoxicity. Fluorescent microscopy has proven to be a useful tool for real-time monitoring of performance and intracellular trafficking of polyplexes as well as for assessing cell functionality. This review highlights the application of some of the most promising fluorescent microscopy platforms in relation to polyplex-mediated transfection processes.

Poly(3-hydroxybutyrate-co-R-3-hydroxyhexanoate) nanoparticles with polyethylenimine coat as simple, safe, and versatile vehicles for cell targeting: population characteristics, cell uptake, and intracellular trafficking.

A simple and highly safe poly(3-hydroxybutyrate-co-R-3-hydroxyhexanoate) nanoparticulate delivery system that targets different cell types is developed. A sub-cytotoxic level of polyethylenimine coat mediates universal cell targeting. Internalized nanoparticles traffic along endolysosomal compartments, endoplasmic reticulum and the Golgi complex. Nanoparticles have no detrimental effects on cell morphology and respiration.

T cells expressing VHH-directed oligoclonal chimeric HER2 antigen receptors: towards tumor-directed oligoclonal T cell therapy.

BACKGROUND: Adoptive cell therapy with engineered T cells expressing chimeric antigen receptors (CARs) originated from antibodies is a promising strategy in cancer immunotherapy. Several unsuccessful trials, however, highlight the need for alternative conventional binding domains and the better combination of costimulatory endodomains for CAR construction to improve the effector functions of the engineered T cells. Camelid single-domain antibodies (VHHs), which are the smallest single domain antibodies, can endow great targeting ability to CAR-engineered T cells. METHODS: We have developed a method to generate genetically engineered Jurkat T cells armed with a CAR comprising the anti-HER2 VHH as targeting moiety. From an immune camel library, five VHH clones were selected as a set of oligoclonal anti-HER2 VHHs that exhibited diverse binding abilities and joined them to CD28-CD3ζ and CD28-OX40-CD3ζ signaling endodomains. Jurkat T cells expression of VHH-CARs and cell functions were evaluated. RESULTS: The oligoclonal engineered T cells showed higher proliferation, cytokine secretion and cytotoxicity than each individual VHH-CAR-engineered Jurkat T cells. CONCLUSIONS: The combination of superior targeting ability of oligoclonal VHHs with the third generation CAR can substantially improve the function of engineered T cells. GENERAL SIGNIFICANCE: Antigen-specific directed oligoclonal T cells are alternatively promising, but safer systems, to combat tumor cells.