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BACKGROUND: The pro-inflammatory ATP-gated P2X7 receptor is widely expressed by immune and non-immune cells. Nanobodies targeting P2X7, with potentiating or antagonistic effects, have been developed. Adeno-associated virus (AAV)-mediated gene transfer represents an efficient approach to achieve long-term in vivo expression of selected nanobody-based biologics. This approach (AAVnano) was used to validate the relevance of P2X7 as a target in dextran sodium sulfate (DSS)-induced colitis in mice. RESULTS: Mice received an intramuscular injection of AAV vectors coding for potentiating (14D5-dimHLE) or antagonistic (13A7-Fc) nanobody-based biologics targeting P2X7. Long-term modulation of P2X7 activity was evaluated ex vivo from blood samples. Colitis was induced with DSS in mice injected with AAV vectors coding for nanobody-based biologics. Severity of colitis, colon histopathology and expression of chemokines and cytokines were determined to evaluate the impact of P2X7 modulation. A single injection of an AAV vector coding for 13A7-Fc or 14D5-dimHLE efficiently modulated P2X7 function in vivo from day 15 up to day 120 post-injection in a dose-dependent manner. An AAV vector coding for 13A7-Fc significantly ameliorated DSS-induced colitis and significantly reduced immune cell infiltration and expression of chemokines and proinflammatory cytokines in colonic tissue. CONCLUSIONS: We have demonstrated the validity of AAVnano methodology to modulate P2X7 functions in vivo. Applying this methodological approach to a DSS-induced colitis model, we have shown that P2X7 blockade reduces inflammation and disease severity. Hence, this study confirms the importance of P2X7 as a pharmacological target and suggests the use of nanobody-based biologics as potential therapeutics in inflammatory bowel disease.
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Productos Biológicos , Colitis , Ratones , Animales , Colon/metabolismo , Colitis/inducido químicamente , Colitis/tratamiento farmacológico , Citocinas/metabolismo , Quimiocinas/metabolismo , Ratones Endogámicos C57BL , Modelos Animales de EnfermedadRESUMEN
P2X7, an ion channel gated by extracellular ATP, is widely expressed on the plasma membrane of immune cells and plays important roles in inflammation and apoptosis. Several single nucleotide polymorphisms have been identified in the human P2RX7 gene. In contrast to other members of the P2X family, non-synonymous polymorphisms in P2X7 are common. Three of these occur at overall frequencies of more than 25% and affect residues in the extracellular "head"-domain of P2X7 (155 Y/H), its "lower body" (270 R/H), and its "tail" in the second transmembrane domain (348 T/A). Comparison of the P2X7 orthologues of human and other great apes indicates that the ancestral allele is Y-R-T (at 155-270-348). Interestingly, each single amino acid variant displays lower ATP-sensitivity than the ancestral allele. The originally published reference sequence of human P2X7, often referred to as "wildtype," differs from the ancestral allele at all three positions, i.e. H-H-A. The 1,000 Genome Project determined the sequences of both alleles of 2,500 human individuals, including roughly 500 persons from each of the five major continental regions. This rich resource shows that the ancestral alleles Y155, R270, and T348 occur in all analyzed human populations, albeit at strikingly different frequencies in various subpopulations (e.g., 25%-59% for Y155, 59%-77% for R270, and 13%-47% for T348). BLAST analyses of ancient human genome sequences uncovered several homozygous carriers of variant P2X7 alleles, possibly reflecting a high degree of inbreeding, e.g., H-R-T for a 50.000 year old Neanderthal, H-R-A for a 24.000 year old Siberian, and Y-R-A for a 7,000 year old mesolithic European. In contrast, most present-day individuals co-express two copies of P2X7 that differ in one or more amino acids at positions 155, 270, and 348. Our results improve the understanding of how P2X7 structure affects its function and suggest the importance of considering P2X7 variants of participants when designing clinical trials targeting P2X7.
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The P2X7 ion channel is a key sensor for extracellular ATP and a key trigger of sterile inflammation. Intravenous injection of nanobodies that block P2X7 has shown to be beneficial in mouse models of systemic inflammation. P2X7 has also emerged as an attractive therapeutic target for inflammatory brain diseases. However, little is known about the ability of nanobodies to cross the BBB. Here we evaluated the ability of P2X7-specific nanobodies to reach and to block P2X7 on microglia following intravenous or intracerebral administration. For this study, we reformatted and sequence-optimized P2X7 nanobodies for higher stability and elevated isoelectric point. Following injection of nanobodies or nanobody-encoding adeno-associated viral vectors (AAV), we monitored the occupancy and blockade of microglial P2X7 in vivo using ex vivo flow cytometry. Our results show that P2X7 on microglia was within minutes completely occupied and blocked by intracerebroventricularly injected nanobodies, even at low doses. In contrast, very high doses were required to achieve similar effects when injected intravenously. The endogenous production of P2X7-antagonistic nanobodies following intracerebral or intramuscular injection of nanobody-encoding AAVs resulted in a long-term occupancy and blockade of P2X7 on microglia. Our results provide new insights into the conditions for the delivery of nanobodies to microglial P2X7 and point to AAV-mediated delivery of P2X7 nanobodies as a promising strategy for the treatment of sterile brain inflammation.
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BACKGROUND: Previous studies have demonstrated that purinergic receptors could be therapeutic targets to modulate the inflammatory response in multiple models of brain diseases. However, tools for the selective and efficient targeting of these receptors are lacking. The development of new P2X7-specific nanobodies (nbs) has enabled us to effectively block the P2X7 channel. METHODS: Temporary middle cerebral artery occlusion (tMCAO) in wild-type (wt) and P2X7 transgenic (tg) mice was used to model ischemic stroke. Adenosine triphosphate (ATP) release was assessed in transgenic ATP sensor mice. Stroke size was measured after P2X7-specific nbs were injected intravenously (iv) and intracerebroventricularly (icv) directly before tMCAO surgery. In vitro cultured microglia were used to investigate calcium influx, pore formation via 4,6-diamidino-2-phenylindole (DAPI) uptake, caspase 1 activation and interleukin (IL)-1ß release after incubation with the P2X7-specific nbs. RESULTS: Transgenic ATP sensor mice showed an increase in ATP release in the ischemic hemisphere compared to the contralateral hemisphere or the sham-treated mice up to 24 h after stroke. P2X7-overexpressing mice had a significantly greater stroke size 24 h after tMCAO surgery. In vitro experiments with primary microglial cells demonstrated that P2X7-specific nbs could inhibit ATP-triggered calcium influx and the formation of membrane pores, as measured by Fluo4 fluorescence or DAPI uptake. In microglia, we found lower caspase 1 activity and subsequently lower IL-1ß release after P2X7-specific nb treatment. The intravenous injection of P2X7-specific nbs compared to isotype controls before tMCAO surgery did not result in a smaller stroke size. As demonstrated by fluorescence-activated cell sorting (FACS), after stroke, iv injected nbs bound to brain-infiltrated macrophages but not to brain resident microglia, indicating insufficient crossing of the blood-brain barrier of the nbs. Therefore, we directly icv injected the P2X7-specific nbs or the isotype nbs. After icv injection of 30 µg of P2X7 specific nbs, P2X7 specific nbs bound sufficiently to microglia and reduced stroke size. CONCLUSION: Mechanistically, we can show that there is a substantial increase of ATP locally after stroke and that blockage of the ATP receptor P2X7 by icv injected P2X7-specific nbs can reduce ischemic tissue damage.
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Receptores Purinérgicos P2 , Anticuerpos de Dominio Único , Accidente Cerebrovascular , Adenosina Trifosfato/farmacología , Animales , Calcio/metabolismo , Caspasa 1/metabolismo , Infarto de la Arteria Cerebral Media/patología , Interleucina-1beta/metabolismo , Ratones , Microglía/metabolismo , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Anticuerpos de Dominio Único/metabolismo , Accidente Cerebrovascular/metabolismoRESUMEN
Extracellular ATP activates the P2X7 receptor, leading to inflammasome activation and release of pro-inflammatory cytokines in monocytes. However, a detailed analysis of P2X7 receptor expression and function in the human T cell compartment has not been reported. Here, we used a P2X7-specific nanobody to assess cell membrane expression and function of P2X7 on peripheral T lymphocyte subsets. The results show that innate-like T cells, which effectively react to innate stimuli by secreting high amounts of pro-inflammatory cytokines, have the highest expression of P2X7 in the human T cell compartment. Using Tγδ cells as example for an innate-like lymphocyte population, we demonstrate that these cells are more sensitive to P2X7 receptor activation than conventional T cells, affecting fundamental cellular mechanisms like calcium signaling and ATP-induced cell death. The increased susceptibility of innate-like T cells to P2X7-mediated cell death provides a mechanism to control their homeostasis under inflammatory conditions. Understanding the expression and function of P2X7 on human immune cells is essential to assume the benefits and consequences of newly developed P2X7-based therapeutic approaches.
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Adenosina Trifosfato , Receptores Purinérgicos P2X7 , Humanos , Receptores Purinérgicos P2X7/genética , Receptores Purinérgicos P2X7/metabolismo , Adenosina Trifosfato/metabolismo , Muerte Celular , Monocitos/metabolismo , Citocinas/metabolismoRESUMEN
On murine T cells, mono-ADP ribosyltransferase ARTC2.2 catalyzes ADP-ribosylation of various surface proteins when nicotinamide adenine dinucleotide (NAD+) is released into the extracellular compartment. Covalent ADP-ribosylation of the P2X7 receptor by ARTC2.2 thereby represents an additional mechanism of activation, complementary to its triggering by extracellular ATP. P2X7 is a multifaceted receptor that may represents a potential target in inflammatory, and neurodegenerative diseases, as well as in cancer. We present herein an experimental approach using intramuscular injection of recombinant AAV vectors (rAAV) encoding nanobody-based biologics targeting ARTC2.2 or P2X7. We demonstrate the ability of these in vivo generated biologics to potently and durably block P2X7 or ARTC2.2 activities in vivo, or in contrast, to potentiate NAD+- or ATP-induced activation of P2X7. We additionally demonstrate the ability of rAAV-encoded functional heavy chain antibodies to elicit long-term depletion of T cells expressing high levels of ARTC2.2 or P2X7. Our approach of using rAAV to generate functional nanobody-based biologics in vivo appears promising to evaluate the role of ARTC2.2 and P2X7 in murine acute as well as chronic disease models.
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ADP Ribosa Transferasas , Productos Biológicos/inmunología , Dependovirus , Vectores Genéticos , Depleción Linfocítica , Receptores Purinérgicos P2X7/inmunología , Anticuerpos de Dominio Único , ADP Ribosa Transferasas/antagonistas & inhibidores , ADP Ribosa Transferasas/inmunología , Animales , Ratones , Anticuerpos de Dominio Único/genética , Anticuerpos de Dominio Único/inmunologíaRESUMEN
Adenosine triphosphate (ATP) represents a danger signal that accumulates in injured tissues, in inflammatory sites, and in the tumor microenvironment. Extracellular ATP is known to signal through plasma membrane receptors of the P2Y and P2X families. Among the P2X receptors, P2X7 has attracted increasing interest in the field of inflammation as well as in cancer. P2X7 is expressed by immune cells and by most malignant tumor cells where it plays a crucial yet complex role that remains to be clarified. P2X7 activity has been associated with production and release of pro-inflammatory cytokines, modulation of the activity and survival of immune cells, and the stimulation of proliferation and migratory properties of tumor cells. Hence, P2X7 plays an intricate role in the tumor microenvironment combining beneficial and detrimental effects that need to be further investigated. For this, we developed a novel methodology termed AAVnano based on the use of Adeno-associated viral vectors (AAV) encoding nanobodies targeting P2X7. We discuss here the advantages of this tool to study the different functions of P2X7 in cancer and other pathophysiological contexts.
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Antibody-based biologics are the corner stone of modern immunomodulatory therapy. Though highly effective in dampening systemic inflammatory processes, their large size and Fc-fragment mediated effects hamper crossing of the blood brain barrier (BBB). Nanobodies (Nbs) are single domain antibodies derived from llama or shark heavy-chain antibodies and represent a new generation of biologics. Due to their small size, they display excellent tissue penetration capacities and can be easily modified to adjust their vivo half-life for short-term diagnostic or long-term therapeutic purposes or to facilitate crossing of the BBB. Furthermore, owing to their characteristic binding mode, they are capable of antagonizing receptors involved in immune signaling and of neutralizing proinflammatory mediators, such as cytokines. These qualities combined make Nbs well-suited for down-modulating neuroinflammatory processes that occur in the context of brain ischemia. In this review, we summarize recent findings on Nbs in preclinical stroke models and how they can be used as diagnostic and therapeutic reagents. We further provide a perspective on the design of innovative Nb-based treatment protocols to complement and improve stroke therapy.
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Targeting the P2X7 ion channel, a danger sensor for extracellular nucleotides, improves outcomes in models of inflammation, cancer, and brain-diseases. Antibodies and nanobodies have been developed that antagonize or potentiate gating of P2X7. Their potential advantages over small-molecule drugs include high specificity, lower off-target effects, and tunable in vivo half-life. Genetic fusion of P2X7-specific biologics to binding modules may enable targeting of specific cell subsets. Besides directly modulating P2X7 function, antibodies can also initiate specific depletion of P2X7-expressing cells. Adeno-associated viral vectors (AAV) can be used to express P2X7-specific antibodies in vivo to achieve long-lasting biological effects. Furthermore, if successfully targeted to P2X7-expressing cells, AAVs may enable modulation of the function of P2X7-expressing immune cells via encoded transgenic RNA or proteins.
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Productos Biológicos/farmacología , Receptores Purinérgicos P2X7/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Anticuerpos/uso terapéutico , Productos Biológicos/uso terapéutico , Dependovirus , Humanos , Inflamación/tratamiento farmacológico , NAD/metabolismo , Neoplasias/tratamiento farmacológico , Enfermedades del Sistema Nervioso/tratamiento farmacológico , Dolor/tratamiento farmacológico , Anticuerpos de Dominio Único/uso terapéuticoRESUMEN
Immune cells express various voltage-gated and ligand-gated ion channels that mediate the influx and efflux of charged ions across the plasma membrane, thereby controlling the membrane potential and mediating intracellular signal transduction pathways. These channels thus present potential targets for experimental modulation of immune responses and for therapeutic interventions in immune disease. Small molecule drugs and natural toxins acting on ion channels have illustrated the potential therapeutic benefit of targeting ion channels on immune cells. Unwanted side effects and immunogenicity have however hampered the application of these molecules. Owing to their high specificity, low immunogenicity and beneficial pharmacodynamics, antibodies targeting membrane and secretory proteins have emerged as potent therapeutics in oncology and inflammation. Nanobodies-single domain fragments derived from heavy chain antibodies naturally occurring in camelids-offer additional benefits versus antibodies, including protrusion into cryptic epitopes and easy formatting of multi-specific reagents. Here we review recent progress in the development and application of antibodies and Nanobodies targeting ion channels on immune cells.