RESUMEN
Fibrosis is involved in 45% of deaths in the United States, and no treatment exists to reverse the progression of lung or kidney fibrosis. Myofibroblasts are key to the progression and maintenance of fibrosis. We investigated features of cell adhesion necessary for monocytes to differentiate into myofibroblasts, seeking to identify pathways key to myofibroblast differentiation. Blocking antibodies against integrins α3, αM, and αMß2 de-differentiate myofibroblasts in vitro, lower the pro-fibrotic secretome of myofibroblasts, and treat lung fibrosis and inhibit kidney fibrosis in vivo. Decorin's collagen-binding peptide can be used to direct functionalized blocking antibodies (against integrins-α3, -αM, -αMß2) to both fibrotic lungs and fibrotic kidneys, reducing the dose of antibody necessary to treat fibrosis. This targeted immunotherapy blocking key integrins may be an effective therapeutic for the treatment of fibrosis.
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Fibrosis , Miofibroblastos , Fibrosis Pulmonar , Miofibroblastos/metabolismo , Miofibroblastos/patología , Animales , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patología , Humanos , Ratones , Anticuerpos Bloqueadores/farmacología , Diferenciación Celular , Integrina alfa3/metabolismo , Enfermedades Renales/metabolismo , Enfermedades Renales/patología , Riñón/patología , Riñón/metabolismoRESUMEN
OBJECTIVE: Glucagon-like peptide 1 (GLP-1) receptor agonists reduce food intake, producing remarkable weight loss in overweight and obese individuals. While much of this weight loss is fat mass, there is also a loss of lean mass, similar to other approaches that induce calorie deficit. Targeting signaling pathways that regulate skeletal muscle hypertrophy is a promising avenue to preserve lean mass and modulate body composition. Myostatin and Activin A are TGFß-like ligands that signal via the activin type II receptors (ActRII) to antagonize muscle growth. Pre-clinical and clinical studies demonstrate that ActRII blockade induces skeletal muscle hypertrophy and reduces fat mass. In this manuscript, we test the hypothesis that combined ActRII blockade and GLP-1 receptor agonism will preserve muscle mass, leading to improvements in skeletomuscular and metabolic function and enhanced fat loss. METHODS: In this study, we explore the therapeutic potential of bimagrumab, a monoclonal antibody against ActRII, to modify body composition alone and during weight loss induced by GLP-1 receptor agonist semaglutide in diet-induced obese mice. Mechanistically, we define the specific role of the anabolic kinase Akt in mediating the hypertrophic muscle effects of ActRII inhibition in vivo. RESULTS: Treatment of obese mice with bimagrumab induced a â¼10 % increase in lean mass while simultaneously decreasing fat mass. Daily treatment of obese mice with semaglutide potently decreased body weight; this included a significant decrease in both muscle and fat mass. Combination treatment with bimagrumab and semaglutide led to superior fat mass loss while simultaneously preserving lean mass despite reduced food intake. Treatment with both drugs was associated with improved metabolic outcomes, and increased lean mass was associated with improved exercise performance. Deletion of both Akt isoforms in skeletal muscle modestly reduced, but did not prevent, muscle hypertrophy driven by ActRII inhibition. CONCLUSIONS: Collectively, these data demonstrate that blockade of ActRII signaling improves body composition and metabolic parameters during calorie deficit driven by GLP-1 receptor agonism and demonstrate the existence of Akt-independent pathways supporting muscle hypertrophy in the absence of ActRII signaling.
Asunto(s)
Receptores de Activinas Tipo II , Anticuerpos Monoclonales Humanizados , Receptor del Péptido 1 Similar al Glucagón , Obesidad , Proteínas Proto-Oncogénicas c-akt , Pérdida de Peso , Animales , Ratones , Receptores de Activinas Tipo II/antagonistas & inhibidores , Receptores de Activinas Tipo II/metabolismo , Activinas/metabolismo , Anticuerpos Bloqueadores/metabolismo , Anticuerpos Bloqueadores/farmacología , Anticuerpos Bloqueadores/uso terapéutico , Receptor del Péptido 1 Similar al Glucagón/agonistas , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Hipertrofia/metabolismo , Ratones Obesos , Músculo Esquelético/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Anticuerpos Monoclonales Humanizados/administración & dosificación , Obesidad/tratamiento farmacológicoRESUMEN
Expansion and differentiation of antigen-experienced PD-1+TCF-1+ stem-like CD8+ T cells into effector cells is critical for the success of immunotherapies based on PD-1 blockade1-4. Hashimoto et al. have shown that, in chronic infections, administration of the cytokine interleukin (IL)-2 triggers an alternative differentiation path of stem-like T cells towards a distinct population of 'better effector' CD8+ T cells similar to those generated in an acute infection5. IL-2 binding to the IL-2 receptor α-chain (CD25) was essential in triggering this alternative differentiation path and expanding better effectors with distinct transcriptional and epigenetic profiles. However, constitutive expression of CD25 on regulatory T cells and some endothelial cells also contributes to unwanted systemic effects from IL-2 therapy. Therefore, engineered IL-2 receptor ß- and γ-chain (IL-2Rßγ)-biased agonists are currently being developed6-10. Here we show that IL-2Rßγ-biased agonists are unable to preferentially expand better effector T cells in cancer models and describe PD1-IL2v, a new immunocytokine that overcomes the need for CD25 binding by docking in cis to PD-1. Cis binding of PD1-IL2v to PD-1 and IL-2Rßγ on the same cell recovers the ability to differentiate stem-like CD8+ T cells into better effectors in the absence of CD25 binding in both chronic infection and cancer models and provides superior efficacy. By contrast, PD-1- or PD-L1-blocking antibodies alone, or their combination with clinically relevant doses of non-PD-1-targeted IL2v, cannot expand this unique subset of better effector T cells and instead lead to the accumulation of terminally differentiated, exhausted T cells. These findings provide the basis for the development of a new generation of PD-1 cis-targeted IL-2R agonists with enhanced therapeutic potential for the treatment of cancer and chronic infections.
Asunto(s)
Linfocitos T CD8-positivos , Receptor de Muerte Celular Programada 1 , Receptores de Interleucina-2 , Anticuerpos Bloqueadores/inmunología , Anticuerpos Bloqueadores/farmacología , Anticuerpos Bloqueadores/uso terapéutico , Antígeno B7-H1/antagonistas & inhibidores , Antígeno B7-H1/inmunología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Infecciones/tratamiento farmacológico , Infecciones/inmunología , Interleucina-2/inmunología , Interleucina-2/farmacología , Interleucina-2/uso terapéutico , Subunidad alfa del Receptor de Interleucina-2/agonistas , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptores de Interleucina-2/agonistasRESUMEN
Immune checkpoint inhibitors and vascular endothelial growth factor receptor tyrosine kinase inhibitors (VEGFR TKIs) are mainstream treatments for renal cell carcinoma (RCC). Both T cells and macrophages infiltrate the tumor microenvironment of RCC. CD47, an immune checkpoint of macrophages, transmits the "don't eat me" signal to macrophages. We propose a novel therapeutic strategy that activates the antitumor effect of macrophages. We found that CD47 was expressed in patients with RCC, and high CD47 expression was indicative of worse overall survival in datasets from The Cancer Genome Atlas. We observed that CD47-blocking antibodies enhanced the antitumor effect of macrophages against human RCC cell lines. Trogocytosis, rather than phagocytosis, occurred and was promoted by increased cell-to-cell contact between macrophages and RCC cells. Trogocytosis induced by CD47 blockade occurred in the presence of CD11b integrin signaling in macrophages and was augmented when RCC cells were exposed to VEGFR TKIs, except for sunitinib. In conclusion, this study presents evidence that anti-CD47 blocking antibodies improve the antitumor effect of macrophages in RCC. In combination with VEGFR TKIs, CD47 blockade is a potential therapeutic strategy for patients with RCC.
Asunto(s)
Carcinoma de Células Renales , Neoplasias Renales , Anticuerpos Bloqueadores/farmacología , Antígenos de Diferenciación/metabolismo , Antígeno CD47/metabolismo , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/metabolismo , Humanos , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/metabolismo , Macrófagos/metabolismo , Fagocitosis , Receptores Inmunológicos/metabolismo , Trogocitosis , Microambiente Tumoral , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Aims: Subarachnoid hemorrhage (SAH) is a devastating stroke subtype. Following SAH, erythrocyte lysis contributes to cell death and brain injuries. Blockage of the anti-phagocytic receptor Cluster of Differentiation 47 (CD47) enhances phagocyte clearance of erythrocytes, though it has not been well-studied post-SAH. The current study aims to determine whether anti-CD47 treatment can enhance blood clearance after experimental SAH. Methods: The prechiasmatic blood injection model of SAH was used in mice. Mice were either treated with the CD47-blocking antibody or IgG as control. The effect of the anti-CD47 antibody on blood clearance and neurological function following SAH was determined. Neuroinflammation and neuronal injury were compared between the treatment and control samples on day 1 and day 7 after SAH using flow cytometry, immunofluorescence, Fluoro-Jade C, and Nissl staining, RT-PCR, and Western blot analysis. Results: CD47-blocking antibody sped-up blood clearance after SAH, and resulted in less neuronal injury and neurological deficits than control samples. Microglia played a role in the anti-CD47 blockade. Following SAH Following SAH, CD47 antibody-treated mice had less neuroinflammation and lower levels of apoptosis compared to controls and both one and 7 days. Conclusions: CD47 antibody treatment has a neuroprotective effect following SAH, by increasing blood clearance rate and reducing brain injury. These findings suggest CD47 antibody treatment may improve SAH patient outcomes.
Asunto(s)
Lesiones Encefálicas , Fármacos Neuroprotectores , Hemorragia Subaracnoidea , Animales , Anticuerpos Bloqueadores/farmacología , Lesiones Encefálicas/tratamiento farmacológico , Lesiones Encefálicas/etiología , Antígeno CD47/metabolismo , Ratones , Microglía/metabolismo , Fármacos Neuroprotectores/farmacología , Hemorragia Subaracnoidea/tratamiento farmacológico , Hemorragia Subaracnoidea/metabolismoRESUMEN
Extracellular vesicles (EVs) and viruses share common features: size, structure, biogenesis and uptake. In order to generate EVs expressing the SARS-CoV-2 spike protein on their surface (S-EVs), we collected EVs from SARS-CoV-2 spike expressing human embryonic kidney (HEK-293T) cells by stable transfection with a vector coding for the S1 and S2 subunits. S-EVs were characterized using nanoparticle tracking analysis, ExoView and super-resolution microscopy. We obtained a population of EVs of 50 to 200 nm in size. Spike expressing EVs represented around 40% of the total EV population and co-expressed spike protein with tetraspanins on the surfaces of EVs. We subsequently used ACE2-positive endothelial and bronchial epithelial cells for assessing the internalization of labeled S-EVs using a cytofluorimetric analysis. Internalization of S-EVs was higher than that of control EVs from non-transfected cells. Moreover, S-EV uptake was significantly decreased by anti-ACE2 antibody pre-treatment. Furthermore, colchicine, a drug currently used in clinical trials, significantly reduced S-EV entry into the cells. S-EVs represent a simple, safe, and scalable model to study host-virus interactions and the mechanisms of novel therapeutic drugs.
Asunto(s)
COVID-19/metabolismo , Vesículas Extracelulares/metabolismo , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Enzima Convertidora de Angiotensina 2/inmunología , Anticuerpos Bloqueadores/farmacología , COVID-19/virología , Línea Celular , Células Cultivadas , Colchicina/farmacología , Citometría de Flujo/métodos , Células HEK293 , Interacciones Microbiota-Huesped/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/virología , Humanos , Microscopía Fluorescente/métodos , Unión Proteica/efectos de los fármacos , SARS-CoV-2/fisiologíaRESUMEN
Tumor-associated macrophages (TAM) are the most abundant immune cells in the tumor microenvironment. They consist of various subsets but primarily resemble the M2 macrophage phenotype. TAMs are known to promote tumor progression and are associated with poor clinical outcomes. CD47 on tumor cells and SIRPα on TAMs facilitate a "don't-eat-me" signal which prevents cancer cells from immune clearance. Therefore, blockade of the CD47-SIRPα interaction represents a promising strategy for tumor immunotherapy. Here, we present the results on ZL-1201, a differentiated and potent anti-CD47 antibody with improved hematologic safety profile compared with 5F9 benchmark. ZL-1201 enhanced phagocytosis in combination with standards of care (SoC) therapeutic antibodies in in vitro coculture systems using a panel of tumor models and differentiated macrophages, and these combinational effects are Fc dependent while potently enhancing M2 phagocytosis. In vivo xenograft studies showed that enhanced antitumor activities were seen in a variety of tumor models treated with ZL-1201 in combination with other therapeutic mAbs, and maximal antitumor activities were achieved in the presence of chemotherapy in addition to the combination of ZL-1201 with other mAbs. Moreover, tumor-infiltrating immune cells and cytokine analysis showed that ZL-1201 and chemotherapies remodel the tumor microenvironment, which increases antitumor immunity, leading to augmented antitumor efficacy when combined with mAbs. Significance: ZL-1201 is a novel anti-CD47 antibody that has improved hematologic safety profiles and combines with SoC, including mAbs and chemotherapies, to potently facilitate phagocytosis and antitumor efficacy.
Asunto(s)
Antineoplásicos , Macrófagos Asociados a Tumores , Humanos , Línea Celular Tumoral , Macrófagos , Fagocitosis , Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Anticuerpos Bloqueadores/farmacologíaRESUMEN
OBJECTIVE: Antibody blockade of the "do not eat me" signal CD47 (cluster of differentiation 47) enhances efferocytosis and reduces lesion size and necrotic core formation in murine atherosclerosis. TNF (Tumor necrosis factor)-α expression directly enhances CD47 expression, and elevated TNF-α is observed in the absence of the proefferocytosis receptor LRP1 (low-density lipoprotein receptor-related protein 1), a regulator of atherogenesis and inflammation. Thus, we tested the hypothesis that CD47 blockade requires the presence of macrophage LRP1 to enhance efferocytosis, temper TNF-α-dependent inflammation, and limit atherosclerosis. Approach and Results: Mice lacking systemic apoE (apoE-/-), alone or in combination with the loss of macrophage LRP1 (double knockout), were fed a Western-type diet for 12 weeks while receiving anti-CD47 antibody (anti-CD47) or IgG every other day. In apoE-/- mice, treatment with anti-CD47 reduced lesion size by 25.4%, decreased necrotic core area by 34.5%, and decreased the ratio of free:macrophage-associated apoptotic bodies by 47.6% compared with IgG controls (P<0.05), confirming previous reports. Double knockout mice treated with anti-CD47 showed no differences in lesion size, necrotic core area, or the ratio of free:macrophage-associated apoptotic bodies compared with IgG controls. In vitro efferocytosis was 30% higher when apoE-/- phagocytes were incubated with anti-CD47 compared with IgG controls (P<0.05); however, anti-CD47 had no effect on efferocytosis in double knockout phagocytes. Analyses of mRNA and protein showed increased CD47 expression in anti-inflammatory IL (interleukin)-4 treated LRP1-/- macrophages compared with wild type, but no differences were observed in inflammatory lipopolysaccharide-treated macrophages. CONCLUSIONS: The proefferocytosis receptor LRP1 in macrophages is necessary for anti-CD47 blockade to enhance efferocytosis, limit atherogenesis, and decrease necrotic core formation in the apoE-/- model of atherosclerosis.
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Antiinflamatorios/farmacología , Anticuerpos Bloqueadores/farmacología , Aorta/efectos de los fármacos , Enfermedades de la Aorta/prevención & control , Aterosclerosis/prevención & control , Antígeno CD47/antagonistas & inhibidores , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Macrófagos/efectos de los fármacos , Fagocitosis/efectos de los fármacos , Animales , Aorta/inmunología , Aorta/metabolismo , Aorta/patología , Enfermedades de la Aorta/inmunología , Enfermedades de la Aorta/metabolismo , Enfermedades de la Aorta/patología , Aterosclerosis/inmunología , Aterosclerosis/metabolismo , Aterosclerosis/patología , Antígeno CD47/inmunología , Antígeno CD47/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados para ApoE , Necrosis , Placa Aterosclerótica , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
BACKGROUND AND OBJECTIVES: To evaluate the pathophysiology of neuromyelitis optica spectrum disorder (NMOSD) and the therapeutic mechanism and levels of interleukin-6 (IL-6) blockade (satralizumab), especially with respect to blood-brain barrier (BBB) disruption with the new in vitro and ex vivo human BBB models and in vivo model. METHODS: We constructed new static in vitro and flow-based ex vivo models for evaluating continued barrier function, leukocyte transmigration, and intracerebral transferability of neuromyelitis optica-immunoglobulin G (NMO-IgG) and satralizumab across the BBB using the newly established triple coculture system that are specialized to closely mimic endothelial cell contact of pericytes and endfeet of astrocytes. In the in vivo study, we assessed the effects of an anti-IL-6 receptor antibody for mice (MR16-1) on in vivo BBB disruption in mice with experimental autoimmune encephalomyelitis in which IL-6 concentration in the spinal cord dramatically increases. RESULTS: In vitro and ex vivo experiments demonstrated that NMO-IgG increased intracerebral transferability of satralizumab and NMO-IgG and that satralizumab suppressed the NMO-IgG-induced transmigration of T cells and barrier dysfunction. In the in vivo study, the blockade of IL-6 signaling suppressed the migration of T cells into the spinal cord and prevented the increased BBB permeability. DISCUSSION: These results suggest that (1) our triple-cultured in vitro and in ex vivo BBB models are ideal for evaluating barrier function, leukocyte transmigration, and intracerebral transferability; (2) NMO-IgG increased the intracerebral transferability of NMO-IgG via decreasing barrier function and induced secretion of IL-6 from astrocytes causing more dysfunction of the barrier and disrupting controlled cellular infiltration; and (3) satralizumab, which can pass through the BBB in the presence of NMO-IgG, suppresses the BBB dysfunction and the infiltration of inflammatory cells, leading to prevention of onset of NMOSD.
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Anticuerpos Bloqueadores/farmacología , Autoanticuerpos/farmacología , Barrera Hematoencefálica , Encefalomielitis Autoinmune Experimental/inmunología , Interleucina-6/inmunología , Neuromielitis Óptica , Animales , Anticuerpos Monoclonales Humanizados/farmacología , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/fisiopatología , Células Cultivadas , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Femenino , Humanos , Inmunoglobulina G , Ratones , Ratones Endogámicos C57BL , Neuromielitis Óptica/inmunología , Neuromielitis Óptica/prevención & controlRESUMEN
BACKGROUND AND OBJECTIVES: Costimulation by CD40 and its ligand CD40L (CD154) is important for the functional differentiation of T cells. Preclinical studies have recognized the importance of this costimulatory interaction in the pathogenesis of experimental models of multiple sclerosis (MS). To determine safety, pharmacokinetics, and immune effect of a humanized monoclonal antibody (mAb) against CD40 ligand (toralizumab/IDEC-131) in patients with relapsing-remitting MS (RRMS). METHODS: This single-institution open-label dose-escalation study (phase I) enrolled 12 patients with RRMS to receive 4 doses of 1, 5, 10, or 15 mg/kg of humanized αCD40L (toralizumab) IV infusion every other week. Patients were followed up to 18 weeks, annually, and finally at 5 years. In addition to safety and pharmacokinetics, other secondary and exploratory measurements are immune effects, clinical, MRI, laboratory, and neuropsychological evaluations. RESULTS: Fifteen adverse events, all of mild to moderate severity, were considered to be of possible or of unknown relationship to treatment. No serious adverse events, including thromboembolic events, occurred during the 18-week defined study period. Annual and long-term follow-up at 5 years revealed no delayed toxicity. Pharmacokinetics were nonlinear between the 5 and 10 mg/kg dose groups. The serum half-life of toralizumab was consistent between the dose groups with a mean of 15.3 days (SD = 1.9). Flow cytometry revealed no depletion of lymphocyte subsets. An increase in the CD25+/CD3+ and CD25+/CD4+ ratio and a shift toward an anti-inflammatory cytokine response were seen after treatment. DISCUSSION: Our study suggests that blocking CD40L is safe and well tolerated in patients with RRMS while increasing CD25 + T cells and anti-inflammatory cytokine profile. These findings support further studies to assess the efficacy of blocking CD40L as a potential treatment of RRMS. CLASSIFICATION OF EVIDENCE: This study provides Class IV evidence on the safety, pharmacokinetics, and immune effects of an mAb to CD40L in patients with RRMS.
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Anticuerpos Bloqueadores/farmacología , Anticuerpos Monoclonales Humanizados/farmacología , Factores Inmunológicos/farmacología , Esclerosis Múltiple Recurrente-Remitente/tratamiento farmacológico , Esclerosis Múltiple Recurrente-Remitente/inmunología , Adulto , Anticuerpos Bloqueadores/administración & dosificación , Anticuerpos Bloqueadores/efectos adversos , Anticuerpos Monoclonales Humanizados/administración & dosificación , Anticuerpos Monoclonales Humanizados/efectos adversos , Anticuerpos Monoclonales Humanizados/farmacocinética , Ligando de CD40 , Femenino , Estudios de Seguimiento , Humanos , Factores Inmunológicos/administración & dosificación , Factores Inmunológicos/efectos adversos , Factores Inmunológicos/farmacocinética , Masculino , Persona de Mediana Edad , Evaluación de Resultado en la Atención de SaludRESUMEN
CD47 is the only 5-transmembrane (5-TM) spanning receptor of the immune system. Its extracellular domain (ECD) is a cell surface marker of self that binds SIRPα and inhibits macrophage phagocytosis, and cancer immuno-therapy approaches in clinical trials are focused on blocking CD47/SIRPα interaction. We present the crystal structure of full length CD47 bound to the function-blocking antibody B6H12. CD47 ECD is tethered to the TM domain via a six-residue peptide linker (114RVVSWF119) that forms an extended loop (SWF loop), with the fundamental role of inserting the side chains of W118 and F119 into the core of CD47 extracellular loop region (ECLR). Using hydrogen-deuterium exchange and molecular dynamics simulations we show that CD47's ECLR architecture, comprised of two extracellular loops and the SWF loop, creates a molecular environment stabilizing the ECD for presentation on the cell surface. These findings provide insights into CD47 immune recognition, signaling and therapeutic intervention.
Asunto(s)
Biomarcadores , Antígeno CD47/química , Antígeno CD47/metabolismo , Proteínas Portadoras/metabolismo , Receptores Inmunológicos/metabolismo , Anticuerpos Bloqueadores/química , Anticuerpos Bloqueadores/farmacología , Antígenos de Diferenciación/inmunología , Sitios de Unión , Antígeno CD47/efectos de los fármacos , Antígeno CD47/genética , Humanos , Macrófagos/metabolismo , Modelos Moleculares , Fagocitosis/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
Cancer immunotherapies often modulate macrophage effector function by introducing either targeting antibodies that activate Fcγ receptors (FcγRs) or blocking antibodies that disrupt inhibitory SIRPα-CD47 engagement. However, how these competing signals are integrated is poorly understood, raising questions about how to effectively titrate immune responses. Here, we find that macrophage phagocytic decisions are regulated by the ratio of activating ligand to inhibitory ligand over a broad range of absolute molecular densities. Using both endogenous and chimeric receptors, we show that activating:inhibitory ligand ratios of at least 10:1 are required to promote phagocytosis of model antibody-opsonized CD47-inhibited targets and that lowering that ratio reduces FcγR phosphorylation because of inhibitory phosphatases recruited to CD47-bound SIRPα. We demonstrate that ratiometric signaling is critical for phagocytosis of tumor cells and can be modified by blocking SIRPα, indicating that balancing targeting and blocking antibodies may be important for controlling macrophage phagocytosis in cancer immunotherapy.
Asunto(s)
Anticuerpos Bloqueadores/farmacología , Antígeno CD47/inmunología , Fagocitosis/efectos de los fármacos , Receptores de IgG/metabolismo , Animales , Anticuerpos/farmacología , Proteínas Portadoras , Neoplasias/patología , Fagocitosis/inmunología , Fosforilación/fisiologíaRESUMEN
Migration of mature dendritic cells (DCs) to lymph nodes is critical for the initiation of adaptive immunity. CCR7, a G-protein-coupled receptor for CCL19/21 chemokines, is known to be essential for chemotaxis of mature DCs, but the molecular mechanism linking inflammation to chemotaxis remains unclear. We previously demonstrated that fascin1, an actin-bundling protein, increases chemotaxis of mature mouse DCs. In this article, we demonstrated that fascin1 enhanced IL-6 secretion and signaling of mature mouse DCs. Furthermore, we demonstrated that IL-6 signaling is required for chemotaxis. Blockage of IL-6 signaling in wild-type DCs with an anti-IL-6 receptor α (IL-6Rα) Ab inhibited chemotaxis toward CCL19. Likewise, knockout of IL-6Rα inhibited chemotaxis of bone marrow-derived DCs. The addition of soluble IL-6Rα and IL-6 rescued chemotaxis of IL-6Rα knockout bone marrow-derived DCs, underscoring the role of IL-6 signaling in chemotaxis. We found that IL-6 signaling is required for internalization of CCR7, the initial step of CCR7 recycling. CCR7 recycling is essential for CCR7-mediated chemotaxis, explaining why IL-6 signaling is required for chemotaxis of mature DCs. Our results have identified IL-6 signaling as a new regulatory pathway for CCR7/CCL19-mediated chemotaxis and suggest that rapid migration of mature DCs to lymph nodes depends on inflammation-associated IL-6 signaling.
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Antígenos de Diferenciación/metabolismo , Células Dendríticas/inmunología , Interleucina-6/metabolismo , Proteínas de Microfilamentos/metabolismo , Receptores CCR7/metabolismo , Receptores Odorantes/metabolismo , Animales , Anticuerpos Bloqueadores/farmacología , Antígenos de Diferenciación/genética , Diferenciación Celular , Células Cultivadas , Quimiotaxis , Regulación de la Expresión Génica , Ratones , Ratones Noqueados , Proteínas de Microfilamentos/genética , Receptores de Interleucina-6/genética , Receptores de Interleucina-6/inmunología , Receptores de Interleucina-6/metabolismo , Receptores Odorantes/genética , Transducción de SeñalRESUMEN
Fel d1 is an important allergen produced by cats that causes IgE reactions in up to 95% of cat-allergic adults. Immunotherapy to reduce human allergy to cats has demonstrated that people have the capacity to produce allergen-specific neutralizing antibodies that block IgE-mediated allergic responses. We wished to determine if "blocking" antibodies could be used to reduce the IgE binding ability of cat allergens prior to their exposure to humans. Here, we describe the characterization of Fel d1-specific antibodies. We demonstrated the efficacy of a rabbit polyclonal and an allergen-specific chicken IgY to bind to Fel d1 in cat saliva and block Fel d1-IgE binding and IgE-mediated basophil degranulation. Fel d1 blocking antibodies offer a new and exciting approach to the neutralization of cat allergens.
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Alérgenos/inmunología , Anticuerpos Bloqueadores/farmacología , Glicoproteínas/antagonistas & inhibidores , Hipersensibilidad Inmediata/prevención & control , Mascotas/inmunología , Animales , Anticuerpos Bloqueadores/aislamiento & purificación , Anticuerpos Bloqueadores/uso terapéutico , Basófilos/efectos de los fármacos , Basófilos/inmunología , Gatos , Degranulación de la Célula/efectos de los fármacos , Línea Celular Tumoral , Pollos , Glicoproteínas/inmunología , Humanos , Hipersensibilidad Inmediata/inmunología , Inmunoglobulina E/inmunología , Inmunoglobulina E/metabolismo , Conejos , Ratas , Saliva/inmunologíaRESUMEN
Lymph node (LN) is a key tissue in the pathophysiology of mature blood cancers, especially for chronic lymphocytic leukemia (CLL). Within the multiple de-regulated pathways affecting CLL homeostasis, the CC-chemokine receptor 7 (CCR7) grants homing of CLL cells into the LN where protective environments foster tumor progression. To cover the lack of specific therapies targeting the CCR7-dependence of CLL to enter into the LN, and aiming to displace the disease from LN, we generated CAP-100, an antibody that specifically binds to hCCR7 and neutralizes its ligand-binding site and signaling. In various in vitro and in vivo preclinical models CAP-100 strongly inhibited CCR7-induced migration, extravasation, homing, and survival in CLL samples. Moreover, it triggered potent tumor cell killing, mediated by host immune mechanisms, and was effective in xenograft models of high-risk disease. Additionally, CAP-100 showed a favorable toxicity profile on relevant hematopoietic subsets. Our results validated CAP-100 as a novel therapeutic tool to prevent the access of CLL cells, and other neoplasia with nodal-dependence, into the LN niches, thus hitting a central hub in the pathogenesis of cancer. The first-in-human clinical trial (NCT04704323), which will evaluate this novel therapeutic approach in CLL patients, is pending.
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Anticuerpos Bloqueadores/farmacología , Quimiotaxis de Leucocito/efectos de los fármacos , Leucemia Linfocítica Crónica de Células B/patología , Metástasis Linfática/patología , Receptores CCR7/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Humanos , Inmunoglobulina G/farmacología , Macaca fascicularis , RatonesAsunto(s)
Anticuerpos Bloqueadores/farmacología , Variación Biológica Poblacional , Inhibidores de Puntos de Control Inmunológico/farmacología , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Animales , Modelos Animales de Enfermedad , Humanos , Melanoma Experimental , Ratones , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND Enzymatically inactive chitinase-like protein CHI3L1 is overexpressed in diffuse large B cell lymphoma (DLBCL) patients with PD-L1 imbalance and promotes tumor progression in the microenvironment. Based on this, we investigated how CHI3L1 acts on the proliferation and apoptosis of DLBCL and whether there is a synergy of CHI3L1 in combination with anti-PD-L1 antibodies in vivo. MATERIAL AND METHODS CHI3L1 was detected by quantitative real-time PCR (RT-PCR) and western blot (WB) in B-lymphoma cell lines. CHI3L1 interference plasmids were constructed, and the levels of proliferation, cell cycle, apoptosis, and cell survival were examined in vitro in B-lymphoma cell lines and in vivo in a murine xenograft model by RT-PCR, WB, CCK-8, and flow cytometry. RESULTS CHI3L1 was significantly expressed in SU-DHL-4 cells. CHI3L1-interfered RNA ShRNA-CHI3L1-1 was chosen to be used in the next experiment because it had a better interference effect. Dampened cell proliferation level, arrested cell cycle, reduced protein expressions of cyclin D1 and cyclin D2, and promoted cell apoptosis level were observed after SU-DHL-4 was transfected with ShRNA-CHI3L1-1. Furthermore, we also noticed increased expression of Bcl-2, decreased expressions of bax, cleaved caspase 3 and cleaved PARP, promoted cell survival-related protein p53, and reduced survivin. CONCLUSIONS This study demonstrated that knockdown of CHI3L1 inhibits cancer cell proliferation by regulating cell cycles, promotes cancer cell apoptosis, and enhances the pro-apoptotic effect of anti-PD-L1 antibody both in vivo and in vitro in DLBCL.
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Anticuerpos Bloqueadores/farmacología , Linfocitos B/patología , Antígeno B7-H1/metabolismo , Proteína 1 Similar a Quitinasa-3/metabolismo , Linfoma de Células B Grandes Difuso/metabolismo , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Proteína 1 Similar a Quitinasa-3/genética , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Tolerancia Inmunológica , Linfoma de Células B Grandes Difuso/genética , Mutación/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ARN Interferente Pequeño/genética , Microambiente Tumoral , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Treatment of relapsed/resistant acute myeloid leukaemia (AML) remains a significant area of unmet patient need, the outlook for most patients remaining extremely poor. A promising approach is to augment the anti-tumour immune response in these patients; most cancers do not activate immune effector cells because they express immunosuppressive ligands. We have previously shown that CD200 (an immunosuppressive ligand) is overexpressed in AML and confers an inferior overall survival compared to CD200low/neg patients. Here we show that a fully human anti-CD200 antibody (TTI-CD200) can block the interaction of CD200 with its receptor and restore AML immune responses in vitro and in vivo.
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Anticuerpos Bloqueadores/inmunología , Antígenos CD/inmunología , Antineoplásicos Inmunológicos/uso terapéutico , Inmunidad/inmunología , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/terapia , Animales , Anticuerpos Bloqueadores/farmacología , Antígenos CD/efectos de los fármacos , Estudios de Casos y Controles , Células Asesinas Inducidas por Citocinas/inmunología , Humanos , Inmunidad/efectos de los fármacos , Terapia de Inmunosupresión/métodos , Leucemia Mieloide Aguda/mortalidad , Ligandos , Ratones , Modelos Animales , Prevención Secundaria/métodos , Trasplante Heterólogo/métodosRESUMEN
Ganglioside GD3, a major ganglioside species in neural stem cells, plays a crucial role in maintenance of the self-renewal capacity of these cells. However, its bioactivity in postnatally differentiated neurons in the neurogenic regions of adult brains has not been elucidated. Here, we describe for the first time that deletion of GD3 not only impairs neurotrophin-induced stem cell proliferation, but also alters the dendritic structure as well as the number of synapses of nascent neurons in the dentate gyrus of adult brain. When examining the behavioral phenotypes, GD3 synthase-knockout (GD3S-KO) mice displayed impairment in hippocampus-dependent memory function. To further gain insight into its cellular function, we examined GD3-binding partners from mouse brain extract using a GD3-specific monoclonal antibody, R24, followed by LC-MS/MS analysis and identified a mitochondrial fission protein, the dynamin-related protein-1 (Drp1), as a novel GD3-binding protein. Biochemical and imaging analyses revealed mitochondrial fragmentation in GD3-depleted dentate gyrus neurons, suggesting that GD3 is essential for the mitochondrial Drp1 turnover that is required for efficient mitochondrial fission. These results suggest that GD3 is required for proper dendritic and spine maturation of newborn neurons in adult brain through the regulation of mitochondrial dynamics.
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Dendritas/fisiología , Gangliósidos/fisiología , Hipocampo/crecimiento & desarrollo , Hipocampo/fisiología , Mitocondrias/fisiología , Células-Madre Neurales/fisiología , Neuronas/fisiología , Animales , Anticuerpos Bloqueadores/farmacología , Anticuerpos Monoclonales , Conducta Animal , Cognición , Espinas Dendríticas/fisiología , Dinaminas/genética , Dinaminas/fisiología , Gangliósidos/antagonistas & inhibidores , Gangliósidos/genética , Trastornos de la Memoria/genética , Trastornos de la Memoria/psicología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/ultraestructura , Dinámicas MitocondrialesRESUMEN
Amyloid-ß (Aß) deposits are a relatively late consequence of Aß aggregation in Alzheimer's disease. When pathogenic Aß seeds begin to form, propagate and spread is not known, nor are they biochemically defined. We tested various antibodies for their ability to neutralize Aß seeds before Aß deposition becomes detectable in Aß precursor protein-transgenic mice. We also characterized the different antibody recognition profiles using immunoprecipitation of size-fractionated, native, mouse and human brain-derived Aß assemblies. At least one antibody, aducanumab, after acute administration at the pre-amyloid stage, led to a significant reduction of Aß deposition and downstream pathologies 6 months later. This demonstrates that therapeutically targetable pathogenic Aß seeds already exist during the lag phase of protein aggregation in the brain. Thus, the preclinical phase of Alzheimer's disease-currently defined as Aß deposition without clinical symptoms-may be a relatively late manifestation of a much earlier pathogenic seed formation and propagation that currently escapes detection in vivo.