RESUMEN
Clearance of apoptotic cells by macrophages prevents excessive inflammation and supports immune tolerance. Here, we examined the effect of blocking apoptotic cell clearance on anti-tumor immune response. We generated an antibody that selectively inhibited efferocytosis by phagocytic receptor MerTK. Blockade of MerTK resulted in accumulation of apoptotic cells within tumors and triggered a type I interferon response. Treatment of tumor-bearing mice with anti-MerTK antibody stimulated T cell activation and synergized with anti-PD-1 or anti-PD-L1 therapy. The anti-tumor effect induced by anti-MerTK treatment was lost in Stinggt/gt mice, but not in Cgas-/- mice. Abolishing cGAMP production in Cgas-/- tumor cells, depletion of extracellular ATP, or inactivation of the ATP-gated P2X7R channel also compromised the effects of MerTK blockade. Mechanistically, extracellular ATP acted via P2X7R to enhance the transport of extracellular cGAMP into macrophages and subsequent STING activation. Thus, MerTK blockade increases tumor immunogenicity and potentiates anti-tumor immunity, which has implications for cancer immunotherapy.
Asunto(s)
Macrófagos/inmunología , Proteínas de la Membrana/metabolismo , Neoplasias/inmunología , Nucleótidos Cíclicos/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Tirosina Quinasa c-Mer/inmunología , Adenosina Trifosfato/metabolismo , Animales , Apoptosis , Antígeno B7-H1/inmunología , Células Cultivadas , Femenino , Inmunidad Innata , Inmunoterapia , Interferón Tipo I/metabolismo , Macrófagos/metabolismo , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Neoplasias/metabolismo , Neoplasias/patología , Neoplasias/terapia , Nucleotidiltransferasas/deficiencia , Nucleotidiltransferasas/metabolismo , Fagocitosis , Receptor de Muerte Celular Programada 1/inmunología , Receptores Purinérgicos P2X7/deficiencia , Transducción de Señal/inmunología , Ensayos Antitumor por Modelo de Xenoinjerto , Tirosina Quinasa c-Mer/genéticaRESUMEN
Efferocytosis is a phagocytic process by which apoptotic cells are cleared by professional and nonprofessional phagocytic cells. In tumors, efferocytosis of apoptotic cancer cells by tumor-associated macrophages prevents Ag presentation and suppresses the host immune response against the tumor. Therefore, reactivating the immune response by blockade of tumor-associated macrophage-mediated efferocytosis is an attractive strategy for cancer immunotherapy. Even though several methods have been developed to monitor efferocytosis, an automated and high-throughput quantitative assay should offer highly desirable advantages for drug discovery. In this study, we describe a real-time efferocytosis assay with an imaging system for live-cell analysis. Using this assay, we successfully discovered potent anti-MerTK Abs that block tumor-associated macrophage-mediated efferocytosis in mice. Furthermore, we used primary human and cynomolgus monkey macrophages to identify and characterize anti-MerTK Abs for potential clinical development. By studying the phagocytic activities of different types of macrophages, we demonstrated that our efferocytosis assay is robust for screening and characterization of drug candidates that inhibit unwanted efferocytosis. Moreover, our assay is also applicable to investigating the kinetics and molecular mechanisms of efferocytosis/phagocytosis.
Asunto(s)
Apoptosis , Neoplasias , Ratones , Humanos , Animales , Tirosina Quinasa c-Mer , Macaca fascicularis , Fagocitosis , Macrófagos , Neoplasias/patologíaRESUMEN
Here, we explore whether PEGylation of antibodies can modulate their biodistribution to the eye, an organ once thought to be immune privileged but has recently been shown to be accessible to IV-administered large molecules, such as antibodies. We chose to PEGylate an anti-MerTK antibody, a target with known potential for ocular toxicity, to minimize biodistribution to retinal pigment epithelial cells (RPEs) in the eye by increasing the hydrodynamic volume of the antibody. We used site-specific conjugation to an engineered cysteine on anti-MerTK antibody to chemically attach 40-kDa branched or linear PEG polymers. Despite reduced binding to MerTK on cells, site-specifically PEGylated anti-MerTK retained similar potency in inhibiting MerTK-mediated macrophage efferocytosis of apoptotic cells. Importantly, we found that PEGylation of anti-MerTK significantly reduced MerTK receptor occupancy in RPE cells in both naïve mice and MC-38 tumor-bearing mice, with the branched PEG exhibiting a greater effect than linear PEG. Furthermore, similar to unconjugated anti-MerTK, PEGylated anti-MerTK antibody triggered type I IFN response and exhibited antitumor effect in syngeneic mouse tumor studies. Our results demonstrate the potential of PEGylation to control ocular biodistribution of antibodies.
Asunto(s)
Cisteína , Neoplasias , Ratones , Animales , Tirosina Quinasa c-Mer/metabolismo , Distribución Tisular , Cisteína/metabolismo , Fagocitosis/fisiología , Anticuerpos/metabolismo , Neoplasias/metabolismo , Polietilenglicoles/química , Polímeros/metabolismo , Pigmentos Retinianos/metabolismoRESUMEN
Chronic graft-versus-host disease (cGVHD) is a major complication of allogeneic hematopoietic cell transplantation (allo-HCT) and remains an area of unmet clinical need with few treatment options available. Notch blockade prevents acute GVHD in multiple mouse models, but the impact of Notch signaling on cGVHD remains unknown. Using genetic and antibody-mediated strategies of Notch inhibition, we investigated the role of Notch signaling in complementary mouse cGVHD models that mimic several aspects of human cGVHD in search of candidate therapeutics. In the B10.D2âBALB/c model of sclerodermatous cGVHD, Delta-like ligand 4 (Dll4)-driven Notch signaling was essential for disease development. Antibody-mediated Dll4 inhibition conferred maximum benefits when pursued early in a preventative fashion, with anti-Dll1 enhancing early protection. Notch-deficient alloantigen-specific T cells showed no early defects in proliferation or helper polarization in vivo but subsequently exhibited markedly decreased cytokine secretion and enhanced accumulation of FoxP3+ regulatory T cells. In the B6âB10.BR major histocompatibility complex-mismatched model with multi-organ system cGVHD and prominent bronchiolitis obliterans (BO), but not skin manifestations, absence of Notch signaling in T cells provided long-lasting disease protection that was replicated by systemic targeting of Dll1, Dll4, or both Notch ligands, even during established disease. Notch inhibition decreased target organ damage and germinal center formation. Moreover, decreased BO-cGVHD was observed upon inactivation of Notch1 and/or Notch2 in T cells. Systemic targeting of Notch2 alone was safe and conferred therapeutic benefits. Altogether, Notch ligands and receptors regulate key pathogenic steps in cGVHD and emerge as novel druggable targets to prevent or treat different forms of cGVHD.
Asunto(s)
Enfermedad Injerto contra Huésped/patología , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Péptidos y Proteínas de Señalización Intercelular/inmunología , Péptidos y Proteínas de Señalización Intracelular/inmunología , Proteínas de la Membrana/inmunología , Receptores Notch/inmunología , Proteínas Adaptadoras Transductoras de Señales , Animales , Bronquiolitis Obliterante/etiología , Bronquiolitis Obliterante/inmunología , Bronquiolitis Obliterante/patología , Proteínas de Unión al Calcio , Enfermedad Crónica , Enfermedad Injerto contra Huésped/etiología , Enfermedad Injerto contra Huésped/inmunología , Isoantígenos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Linfocitos T/inmunología , Linfocitos T/patología , Trasplante Homólogo/efectos adversosRESUMEN
BACKGROUND: Nutrients are transported through endothelial cells before being metabolized in muscle cells. However, little is known about the regulation of endothelial transport processes. Notch signaling is a critical regulator of metabolism and angiogenesis during development. Here, we studied how genetic and pharmacological manipulation of endothelial Notch signaling in adult mice affects endothelial fatty acid transport, cardiac angiogenesis, and heart function. METHODS: Endothelial-specific Notch inhibition was achieved by conditional genetic inactivation of Rbp-jκ in adult mice to analyze fatty acid metabolism and heart function. Wild-type mice were treated with neutralizing antibodies against the Notch ligand Delta-like 4. Fatty acid transport was studied in cultured endothelial cells and transgenic mice. RESULTS: Treatment of wild-type mice with Delta-like 4 neutralizing antibodies for 8 weeks impaired fractional shortening and ejection fraction in the majority of mice. Inhibition of Notch signaling specifically in the endothelium of adult mice by genetic ablation of Rbp-jκ caused heart hypertrophy and failure. Impaired heart function was preceded by alterations in fatty acid metabolism and an increase in cardiac blood vessel density. Endothelial Notch signaling controlled the expression of endothelial lipase, Angptl4, CD36, and Fabp4, which are all needed for fatty acid transport across the vessel wall. In endothelial-specific Rbp-jκ-mutant mice, lipase activity and transendothelial transport of long-chain fatty acids to muscle cells were impaired. In turn, lipids accumulated in the plasma and liver. The attenuated supply of cardiomyocytes with long-chain fatty acids was accompanied by higher glucose uptake, increased concentration of glycolysis intermediates, and mTOR-S6K signaling. Treatment with the mTOR inhibitor rapamycin or displacing glucose as cardiac substrate by feeding a ketogenic diet prolonged the survival of endothelial-specific Rbp-jκ-deficient mice. CONCLUSIONS: This study identifies Notch signaling as a novel regulator of fatty acid transport across the endothelium and as an essential repressor of angiogenesis in the adult heart. The data imply that the endothelium controls cardiomyocyte metabolism and function.
Asunto(s)
Endotelio Vascular/metabolismo , Ácidos Grasos/metabolismo , Miocardio/metabolismo , Receptores Notch/metabolismo , Transducción de Señal , Remodelación Vascular , Proteínas Adaptadoras Transductoras de Señales , Angiopoyetinas/genética , Angiopoyetinas/metabolismo , Animales , Antígenos CD36/genética , Antígenos CD36/metabolismo , Proteínas de Unión al Calcio , Endotelio Vascular/citología , Proteínas de Unión a Ácidos Grasos/genética , Proteínas de Unión a Ácidos Grasos/metabolismo , Ácidos Grasos/genética , Glucosa/genética , Glucosa/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Miocitos Cardíacos/metabolismo , Neovascularización Fisiológica , Receptores Notch/genética , Proteínas Quinasas S6 Ribosómicas/genética , Proteínas Quinasas S6 Ribosómicas/metabolismo , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Rejection remains a major clinical challenge limiting allograft survival after solid organ transplantation. Both cellular and humoral immunity contribute to this complication, with increased recognition of Ab-mediated damage during acute and chronic rejection. Using a mouse model of MHC-mismatched heart transplantation, we report markedly protective effects of Notch inhibition, dampening both T cell and Ab-driven rejection. T cell-specific pan-Notch blockade prolonged heart allograft survival and decreased IFN-γ and IL-4 production by alloreactive T cells, especially when combined with depletion of recipient CD8(+) T cells. These effects were associated with decreased infiltration by conventional T cells and an increased proportion of regulatory T cells in the graft. Transient administration of neutralizing Abs specific for delta-like (Dll)1/4 Notch ligands in the peritransplant period led to prolonged acceptance of allogeneic hearts, with superior outcome over Notch inhibition only in T cells. Systemic Dll1/4 inhibition decreased T cell cytokines and graft infiltration, germinal center B cell and plasmablast numbers, as well as production of donor-specific alloantibodies and complement deposition in the transplanted hearts. Dll1 or Dll4 inhibition alone provided partial protection. Thus, pathogenic signals delivered by Dll1/4 Notch ligands early after transplantation promote organ rejection through several complementary mechanisms. Transient interruption of these signals represents an attractive new therapeutic strategy to enhance long-term allograft survival.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Rechazo de Injerto/inmunología , Trasplante de Corazón/métodos , Inmunidad/inmunología , Péptidos y Proteínas de Señalización Intercelular/inmunología , Péptidos y Proteínas de Señalización Intracelular/inmunología , Proteínas de la Membrana/inmunología , Proteínas Adaptadoras Transductoras de Señales , Animales , Anticuerpos Neutralizantes/farmacología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Proteínas de Unión al Calcio , Citometría de Flujo , Rechazo de Injerto/prevención & control , Supervivencia de Injerto/inmunología , Inmunidad Celular/inmunología , Inmunidad Humoral/inmunología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Interferón gamma/inmunología , Interferón gamma/metabolismo , Interleucina-4/inmunología , Interleucina-4/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores Notch/antagonistas & inhibidores , Receptores Notch/inmunología , Receptores Notch/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/metabolismo , Factores de Tiempo , Trasplante HomólogoRESUMEN
Notch is long recognized as a signaling molecule important for stem cell self-renewal and fate determination. Here, we reveal a novel adhesive role of Notch-ligand engagement in hematopoietic stem and progenitor cells (HSPCs). Using mice with conditional loss of O-fucosylglycans on Notch EGF-like repeats important for the binding of Notch ligands, we report that HSPCs with faulty ligand binding ability display enhanced cycling accompanied by increased egress from the marrow, a phenotype mainly attributed to their reduced adhesion to Notch ligand-expressing stromal cells and osteoblastic cells and their altered occupation in osteoblastic niches. Adhesion to Notch ligand-bearing osteoblastic or stromal cells inhibits wild type but not O-fucosylglycan-deficient HSPC cycling, independent of RBP-JK -mediated canonical Notch signaling. Furthermore, Notch-ligand neutralizing antibodies induce RBP-JK -independent HSPC egress and enhanced HSPC mobilization. We, therefore, conclude that Notch receptor-ligand engagement controls HSPC quiescence and retention in the marrow niche that is dependent on O-fucosylglycans on Notch.
Asunto(s)
Células Madre Hematopoyéticas/metabolismo , Receptores Notch/metabolismo , Nicho de Células Madre/genética , Células del Estroma/metabolismo , Animales , Humanos , Ratones , Transducción de SeñalRESUMEN
Delta-like 4 (DLL4)-mediated Notch signalling has emerged as an attractive target for cancer therapy. However, the potential side effects of blocking this pathway remain uncertain. Here we show that chronic DLL4 blockade causes pathological activation of endothelial cells, disrupts normal organ homeostasis and induces vascular tumours, raising important safety concerns.
Asunto(s)
Antineoplásicos/efectos adversos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Proteínas de la Membrana/antagonistas & inhibidores , Neoplasias Vasculares/inducido químicamente , Proteínas Adaptadoras Transductoras de Señales , Animales , Antineoplásicos/farmacología , Proteínas de Unión al Calcio , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Enfermedad Hepática Inducida por Sustancias y Drogas/fisiopatología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/patología , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Macaca fascicularis , Proteínas de la Membrana/metabolismo , Ratones , Ratas , Receptores Notch/metabolismo , Transducción de SeñalRESUMEN
The four receptors of the Notch family are widely expressed transmembrane proteins that function as key conduits through which mammalian cells communicate to regulate cell fate and growth. Ligand binding triggers a conformational change in the receptor negative regulatory region (NRR) that enables ADAM protease cleavage at a juxtamembrane site that otherwise lies buried within the quiescent NRR. Subsequent intramembrane proteolysis catalysed by the gamma-secretase complex liberates the intracellular domain (ICD) to initiate the downstream Notch transcriptional program. Aberrant signalling through each receptor has been linked to numerous diseases, particularly cancer, making the Notch pathway a compelling target for new drugs. Although gamma-secretase inhibitors (GSIs) have progressed into the clinic, GSIs fail to distinguish individual Notch receptors, inhibit other signalling pathways and cause intestinal toxicity, attributed to dual inhibition of Notch1 and 2 (ref. 11). To elucidate the discrete functions of Notch1 and Notch2 and develop clinically relevant inhibitors that reduce intestinal toxicity, we used phage display technology to generate highly specialized antibodies that specifically antagonize each receptor paralogue and yet cross-react with the human and mouse sequences, enabling the discrimination of Notch1 versus Notch2 function in human patients and rodent models. Our co-crystal structure shows that the inhibitory mechanism relies on stabilizing NRR quiescence. Selective blocking of Notch1 inhibits tumour growth in pre-clinical models through two mechanisms: inhibition of cancer cell growth and deregulation of angiogenesis. Whereas inhibition of Notch1 plus Notch2 causes severe intestinal toxicity, inhibition of either receptor alone reduces or avoids this effect, demonstrating a clear advantage over pan-Notch inhibitors. Our studies emphasize the value of paralogue-specific antagonists in dissecting the contributions of distinct Notch receptors to differentiation and disease and reveal the therapeutic promise in targeting Notch1 and Notch2 independently.
Asunto(s)
Anticuerpos/farmacología , Anticuerpos/uso terapéutico , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Receptores Notch/antagonistas & inhibidores , Inhibidores de la Angiogénesis/inmunología , Inhibidores de la Angiogénesis/farmacología , Inhibidores de la Angiogénesis/uso terapéutico , Animales , Anticuerpos/efectos adversos , Anticuerpos/inmunología , Especificidad de Anticuerpos/inmunología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Células Caliciformes/efectos de los fármacos , Células Caliciformes/patología , Humanos , Ratones , Ratones Endogámicos BALB C , Células 3T3 NIH , Neoplasias/irrigación sanguínea , Neoplasias/patología , Neovascularización Patológica/tratamiento farmacológico , Biblioteca de Péptidos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Receptor Notch1/antagonistas & inhibidores , Receptor Notch1/inmunología , Receptor Notch2/antagonistas & inhibidores , Receptor Notch2/inmunología , Receptores Notch/genética , Receptores Notch/inmunología , Receptores Notch/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Many important signaling pathways rely on multiple ligands. It is unclear if this is a mechanism of safeguard via redundancy or if it serves other functional purposes. In this study, we report unique insight into this question by studying the activin receptor-like kinase 1 (ALK1) pathway. Despite its functional importance in vascular development, the physiological ligand or ligands for ALK1 remain to be determined. Using conventional knockout and specific antibodies against bone morphogenetic protein 9 (BMP9) or BMP10, we showed that BMP9 and BMP10 are the physiological, functionally equivalent ligands of ALK1 in vascular development. Timing of expression dictates the in vivo requisite role of each ligand, and concurrent expression results in redundancy. We generated mice (Bmp10(9/9)) in which the coding sequence of Bmp9 replaces that of Bmp10. Surprisingly, analysis of Bmp10(9/9) mice demonstrated that BMP10 has an exclusive function in cardiac development, which cannot be substituted by BMP9. Our study reveals context-dependent significance in having multiple ligands in a signaling pathway.
Asunto(s)
Receptores de Activinas Tipo I/metabolismo , Proteínas Morfogenéticas Óseas/metabolismo , Sistema Cardiovascular/embriología , Sistema Cardiovascular/crecimiento & desarrollo , Factor 2 de Diferenciación de Crecimiento/metabolismo , Transducción de Señal/fisiología , Receptores de Activinas Tipo II , Animales , Anticuerpos Neutralizantes , Proteínas Morfogenéticas Óseas/genética , Sistema Cardiovascular/metabolismo , Técnicas de Sustitución del Gen , Factor 2 de Diferenciación de Crecimiento/genética , Células Endoteliales de la Vena Umbilical Humana , Humanos , Hibridación in Situ , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Microscopía Fluorescente , Vasos Retinianos/crecimiento & desarrollo , Vasos Retinianos/metabolismoRESUMEN
Hematopoietic stem cells (HSCs) are the most primitive cells in the hematopoietic system and are under tight regulation for self-renewal and differentiation. Notch signals are essential for the emergence of definitive hematopoiesis in mouse embryos and are critical regulators of lymphoid lineage fate determination. However, it remains unclear how Notch regulates the balance between HSC self-renewal and differentiation in the adult bone marrow (BM). Here we report a novel mechanism that prevents HSCs from undergoing premature lymphoid differentiation in BM. Using a series of in vivo mouse models and functional HSC assays, we show that leukemia/lymphoma related factor (LRF) is necessary for HSC maintenance by functioning as an erythroid-specific repressor of Delta-like 4 (Dll4) expression. Lrf deletion in erythroblasts promoted up-regulation of Dll4 in erythroblasts, sensitizing HSCs to T-cell instructive signals in the BM. Our study reveals novel cross-talk between HSCs and erythroblasts, and sheds a new light on the regulatory mechanisms regulating the balance between HSC self-renewal and differentiation.
Asunto(s)
Proteínas de Unión al ADN/genética , Eritroblastos/metabolismo , Células Madre Hematopoyéticas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/genética , Factores de Transcripción/genética , Proteínas Adaptadoras Transductoras de Señales , Animales , Células de la Médula Ósea/metabolismo , Trasplante de Médula Ósea , Proteínas de Unión al Calcio , Diferenciación Celular/genética , Proliferación Celular , Microambiente Celular/genética , Proteínas de Unión al ADN/metabolismo , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , Receptor Notch1/genética , Receptor Notch1/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genética , Linfocitos T/metabolismo , Factores de Tiempo , Factores de Transcripción/metabolismo , Transcriptoma/genéticaRESUMEN
The Notch signaling pathway plays a fundamental role during blood vessel development. Notch signaling regulates blood vessel morphogenesis by promoting arterial endothelial differentiation and providing spatial and temporal control over "tip cell" phenotype during angiogenic sprouting. Components of the Notch signaling pathway have emerged as potential regulators of lymphatic development, joining the increasing examples of blood vessel regulators that are also involved in lymphatic development. However, in mammals a role for the Notch signaling pathway during lymphatic development remains to be demonstrated. In this report, we show that blockade of Notch1 and Dll4, with specific function-blocking antibodies, results in defective postnatal lymphatic development in mice. Mechanistically, Notch1-Dll4 blockade is associated with down-regulation of EphrinB2 expression, been shown to be critically involved in VEGFR3/VEGFC signaling, resulting in reduced lymphangiogenic sprouting. In addition, Notch1-Dll4 blockade leads to compromised expression of distinct lymphatic markers and to dilation of collecting lymphatic vessels with reduced and disorganized mural cell coverage. Finally, Dll4-blockade impairs wound closure and severely affects lymphangiogenesis during the wound healing in adult mouse skin. Thus, our study demonstrates for the first time in a mammalian system that Notch1-Dll4 signaling pathway regulates postnatal lymphatic development and pathologic lymphangiogenesis.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Linfangiogénesis , Vasos Linfáticos/metabolismo , Proteínas de la Membrana/metabolismo , Receptor Notch1/metabolismo , Transducción de Señal , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas de Unión al Calcio , Línea Celular , Efrina-B2/genética , Regulación del Desarrollo de la Expresión Génica , Humanos , Vasos Linfáticos/citología , Vasos Linfáticos/ultraestructura , RatonesRESUMEN
In vertebrates, endothelial cells form 2 hierarchical tubular networks, the blood vessels and the lymphatic vessels. Despite the difference in their structure and function and genetic programs that dictate their morphogenesis, common signaling pathways have been recognized that regulate both vascular systems. ALK1 is a member of the transforming growth factor-beta type I family of receptors, and compelling genetic evidence suggests its essential role in regulating blood vascular development. Here we report that ALK1 signaling is intimately involved in lymphatic development. Lymphatic endothelial cells express key components of the ALK1 pathway and respond robustly to ALK1 ligand stimulation in vitro. Blockade of ALK1 signaling results in defective lymphatic development in multiple organs of neonatal mice. We find that ALK1 signaling regulates the differentiation of lymphatic endothelial cells to influence the lymphatic vascular development and remodeling. Furthermore, simultaneous inhibition of ALK1 pathway increases apoptosis in lymphatic vessels caused by blockade of VEGFR3 signaling. Thus, our study reveals a novel aspect of ALK1 signaling in regulating lymphatic development and suggests that targeting ALK1 pathway might provide additional control of lymphangiogenesis in human diseases.
Asunto(s)
Receptores de Activinas Tipo II/metabolismo , Receptores de Activinas Tipo I/metabolismo , Diferenciación Celular/fisiología , Células Endoteliales/metabolismo , Linfangiogénesis/fisiología , Vasos Linfáticos/metabolismo , Animales , Animales Recién Nacidos , Apoptosis/fisiología , Células Cultivadas , Humanos , Ligandos , Ratones , Transducción de Señal , Receptor 3 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Haploinsufficiency of Dll4, a vascular-specific Notch ligand, has shown that it is essential for embryonic vascular development and arteriogenesis. Mechanistically, it is unclear how the Dll4-mediated Notch pathway contributes to complex vascular processes that demand meticulous coordination of multiple signalling pathways. Here we show that Dll4-mediated Notch signalling has a unique role in regulating endothelial cell proliferation and differentiation. Neutralizing Dll4 with a Dll4-selective antibody rendered endothelial cells hyperproliferative, and caused defective cell fate specification or differentiation both in vitro and in vivo. In addition, blocking Dll4 inhibited tumour growth in several tumour models. Remarkably, antibodies against Dll4 and antibodies against vascular endothelial growth factor (VEGF) had paradoxically distinct effects on tumour vasculature. Our data also indicate that Dll4-mediated Notch signalling is crucial during active vascularization, but less important for normal vessel maintenance. Furthermore, unlike blocking Notch signalling globally, neutralizing Dll4 had no discernable impact on intestinal goblet cell differentiation, supporting the idea that Dll4-mediated Notch signalling is largely restricted to the vascular compartment. Therefore, targeting Dll4 might represent a broadly efficacious and well-tolerated approach for the treatment of solid tumours.
Asunto(s)
Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/metabolismo , Neoplasias/irrigación sanguínea , Neoplasias/patología , Neovascularización Patológica , Transducción de Señal , Animales , Diferenciación Celular , Línea Celular , Proliferación Celular , Endotelio Vascular/citología , Homeostasis , Humanos , Intestino Delgado/citología , Intestino Delgado/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Ratones , Receptores Notch/metabolismo , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
While antibody-dependent cellular phagocytosis mediated by activating Fcγ receptor is a key mechanism underlying many antibody drugs, their full therapeutic activities can be restricted by the inhibitory Fcγ receptor IIB (FcγRIIB). Here, we describe a bispecific antibody approach that harnesses phagocytic receptor CLEC5A (C-type Lectin Domain Containing 5A) to drive Fcγ receptor-independent phagocytosis, potentially circumventing the negative impact of FcγRIIB. First, we established the effectiveness of such an approach by constructing bispecific antibodies that simultaneously target CLEC5A and live B cells. Furthermore, we demonstrated its in vivo application for regulatory T cell depletion and subsequent tumor regression.
Asunto(s)
Anticuerpos Biespecíficos , Anticuerpos Biespecíficos/farmacología , Linfocitos B , Fagocitosis , Receptores de IgG , Linfocitos T ReguladoresRESUMEN
Purpose: Diabetic macular edema (DME) is the leading cause of vision loss and blindness among working-age adults. Although current intravitreal anti-vascular endothelial growth factor (VEGF) therapies improve vision for many patients with DME, approximately half do not achieve the visual acuity required to drive. We therefore sought additional approaches to resolve edema and improve vision for these patients. Methods: We explored direct agonists of Tie2, a receptor known to stabilize vasculature and prevent leakage. We identified a multivalent PEG-Fab conjugate, Tie2.1-hexamer, that oligomerizes Tie2 and drives receptor activation and characterized its activities in vitro and in vivo. Results: Tie2.1-hexamer normalized and stabilized intercellular junctions of stressed endothelial cell monolayers in vitro, suppressed vascular leak in mice under conditions where anti-VEGF alone was ineffective, and demonstrated extended ocular exposure and robust pharmacodynamic responses in non-human primates. Conclusions: Tie2.1-hexamer directly activates the Tie2 pathway, reduces vascular leak, and is persistent within the vitreal humor. Translational Relevance: Our study presents a promising potential therapeutic for the treatment of DME.
Asunto(s)
Diabetes Mellitus , Retinopatía Diabética , Edema Macular , Ratones , Animales , Edema Macular/tratamiento farmacológico , Edema Macular/etiología , Retinopatía Diabética/tratamiento farmacológico , Factores de Crecimiento Endotelial/uso terapéutico , Agudeza Visual , Trastornos de la Visión/complicaciones , Trastornos de la Visión/tratamiento farmacológico , Ceguera/complicacionesRESUMEN
Phagocytosis plays important roles both in homeostasis and under pathological conditions. Fcγ receptor-mediated phagocytosis has been exploited as an integral mechanism for antibody-based therapies. Unlike Fcγ receptor-mediated phagocytosis, MerTK-mediated phagocytic clearance is immunologically silent. Here, we describe a bispecific antibody approach to harness MerTK for targeted clearance without inducing proinflammatory cytokine release associated with Fcγ receptor engagement. We generated bispecific antibodies targeting live B cells or amyloid beta aggregates to demonstrate the feasibility and versatility of this new approach.
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Péptidos beta-Amiloides/inmunología , Anticuerpos Biespecíficos/metabolismo , Linfocitos B/metabolismo , Macrófagos/metabolismo , Microglía/metabolismo , Tirosina Quinasa c-Mer/agonistas , Animales , Anticuerpos Biespecíficos/genética , Antígenos CD20/inmunología , Antígenos CD20/metabolismo , Linfocitos B/inmunología , Células Cultivadas , Citocinas/metabolismo , Humanos , Tolerancia Inmunológica , Mediadores de Inflamación/metabolismo , Macrófagos/inmunología , Ratones , Ratones Noqueados , Terapia Molecular Dirigida , Fagocitosis , Receptores de IgG/metabolismo , Tirosina Quinasa c-Mer/genética , Tirosina Quinasa c-Mer/inmunologíaRESUMEN
Inhibiting the programmed death-1 (PD-1) pathway is one of the most effective approaches to cancer immunotherapy, but its mechanistic basis remains incompletely understood. Binding of PD-1 to its ligand PD-L1 suppresses T-cell function in part by inhibiting CD28 signaling. Tumor cells and infiltrating myeloid cells can express PD-L1, with myeloid cells being of particular interest as they also express B7-1, a ligand for CD28 and PD-L1. Here we demonstrate that dendritic cells (DCs) represent a critical source of PD-L1, despite being vastly outnumbered by PD-L1+ macrophages. Deletion of PD-L1 in DCs, but not macrophages, greatly restricted tumor growth and led to enhanced antitumor CD8+ T-cell responses. Our data identify a unique role for DCs in the PD-L1-PD-1 regulatory axis and have implications for understanding the therapeutic mechanism of checkpoint blockade, which has long been assumed to reflect the reversal of T-cell exhaustion induced by PD-L1+ tumor cells.
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Antígeno B7-H1 , Neoplasias , Antígeno B7-H1/genética , Antígenos CD28/metabolismo , Células Dendríticas , Humanos , Ligandos , Neoplasias/genética , Receptor de Muerte Celular Programada 1/genéticaRESUMEN
Treatment of ocular disease is hindered by the presence of the blood-retinal barrier, which restricts access of systemic drugs to the eye. Intravitreal injections bypass this barrier, delivering high concentrations of drug to the targeted tissue. However, the recommended dosing interval for approved biologics is typically 6-12 weeks, and frequent travel to the physician's office poses a substantial burden for elderly patients with poor vision. Real-world data suggest that many patients are under-treated. Here, we investigate IgMs as a novel platform for treating ocular disease. We show that IgMs are well-suited to ocular administration due to moderate viscosity, long ocular exposure, and rapid systemic clearance. The complement-dependent cytotoxicity of IgMs can be readily removed with a P436G mutation, reducing safety liabilities. Furthermore, dodecavalent binding of IgM hexamers can potently activate pathways implicated in the treatment of progressive blindness, including the Tie2 receptor tyrosine kinase signaling pathway for the treatment of diabetic macular edema, or the death receptor 4 tumor necrosis family receptor pathway for the treatment of wet age-related macular degeneration. Collectively, these data demonstrate the promise of IgMs as therapeutic agonists for treating progressive blindness.
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Sistemas de Liberación de Medicamentos , Inmunoglobulina M/farmacología , Degeneración Macular , Cuerpo Vítreo/metabolismo , Animales , Células CHO , Cricetulus , Humanos , Inyecciones Intravítreas , Degeneración Macular/tratamiento farmacológico , Degeneración Macular/metabolismo , RatasRESUMEN
Purpose: Investigate a significant, dose-related increase in IOP, leading to glaucomatous damage to the neuroretina and optic nerve following intravitreal (ITV) administration of a bispecific F(ab')2 [anti-VEGF/Angiopoietins [ANGPT]F(ab')2] molecule in adult monkeys. Methods: ITV ocular tolerability and investigation of anti-VEGF/ANGPT F(ab')2 (blocking both ANGPT1 and ANGPT2) was done in monkeys; mechanistic studies were done in neonatal mice. Results: Following the second ITV dose of anti-VEGF/ANGPT F(ab')2, all 1.5- and 4-mg/eye treated monkeys developed elevated IOP, which eventually was associated with optic disc cupping and thinning of the neuroretinal rim. Histopathologic examination showed nonreversible axonal degeneration in the optic nerves of animals administered 1.5 mg/eye and higher that was considered secondary to high IOP. Anti-ANGPT Fab also caused elevated IOP in monkeys, but anti-VEGF Fab did not contribute to the IOP increase. In addition, an anti-ANGPT2-selective antibody did not change IOP. In mice simultaneous blockade of ANGPT1 and ANGPT2 impaired the expansion and formation of Schlemm's canal (SC) vessels, similar to genetic ablation of Angpt1/Angpt2 and their receptor TIE2. As previously reported, blocking ANGPT2 alone did not affect SC formation in mice. Conclusions: Dual inhibition of ANGPT1/ANGPT2, but not ANGPT2 alone, leads to increased IOP and glaucomatous damage in monkeys. This confirms a role for TIE2/ANGPT signaling in the control of IOP in adults, a finding initially identified in transgenic mice. Dual pharmacologic inhibition of ANGPT1/ANGPT2 may affect aqueous drainage and homeostasis in adult monkeys and may be useful in developing novel models of glaucoma.