RESUMEN
Multiple sclerosis (MS), a demyelinating autoimmune disease of the central nervous system (CNS), predominately affects females compared to males. Tumor necrosis factor (TNF), a pro-inflammatory cytokine, signaling through TNF receptor 1 contributes to inflammatory disease pathogenesis. In contrast, TNF receptor 2 signaling is neuroprotective. Current anti-TNF MS therapies are shown to be detrimental to patients due to pleiotropic effects on both pro- and anti-inflammatory functions. Using a non-pertussis toxin (nPTX) experimental autoimmune encephalomyelitis (EAE) model in C57BL/6 mice, we systemically administered a TNFR2 agonist (p53-sc-mTNFR2) to investigate behavioral and pathophysiological changes in both female and male mice. Our data shows that TNFR2 activation alleviates motor and sensory symptoms in females. However, in males, the agonist only alleviates sensory symptoms and not motor. nPTX EAE induction in TNFR2 global knockout mice caused exacerbated motor symptoms in females along with an earlier day of onset, but not in males. Our data demonstrates that TNFR2 agonist efficacy is sex-specific for alleviation of motor symptoms, however, it effectively reduces mechanical hypersensitivity in both females and males. Altogether, these data support the therapeutic promise TNFR2 agonism holds as an MS therapeutic and, more broadly, to treat central neuropathic pain.
Asunto(s)
Encefalomielitis Autoinmune Experimental , Esclerosis Múltiple , Humanos , Masculino , Femenino , Ratones , Animales , Receptores Tipo II del Factor de Necrosis Tumoral/agonistas , Receptores Tipo II del Factor de Necrosis Tumoral/metabolismo , Receptores Tipo II del Factor de Necrosis Tumoral/uso terapéutico , Inhibidores del Factor de Necrosis Tumoral/uso terapéutico , Ratones Endogámicos C57BL , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/metabolismo , Proteínas de la Mielina , Factor de Necrosis Tumoral alfa/metabolismo , Ratones NoqueadosRESUMEN
Tumor necrosis factor alpha (TNF-α) and its key role in modulating immune responses has been widely recognized as a therapeutic target for inflammatory and neurodegenerative diseases. Even though inhibition of TNF-α is beneficial for the treatment of certain inflammatory diseases, total neutralization of TNF-α largely failed in the treatment of neurodegenerative diseases. TNF-α exerts distinct functions depending on interaction with its two TNF receptors, whereby TNF receptor 1 (TNFR1) is associated with neuroinflammation and apoptosis and TNF receptor 2 (TNFR2) with neuroprotection and immune regulation. Here, we investigated the effect of administering the TNFR1-specific antagonist Atrosimab, as strategy to block TNFR1 signaling while maintaining TNFR2 signaling unaltered, in an acute mouse model for neurodegeneration. In this model, a NMDA-induced lesion that mimics various hallmarks of neurodegenerative diseases, such as memory loss and cell death, was created in the nucleus basalis magnocellularis and Atrosimab or control protein was administered centrally. We showed that Atrosimab attenuated cognitive impairments and reduced neuroinflammation and neuronal cell death. Our results demonstrate that Atrosimab is effective in ameliorating disease symptoms in an acute neurodegenerative mouse model. Altogether, our study indicates that Atrosimab may be a promising candidate for the development of a therapeutic strategy for the treatment of neurodegenerative diseases.
Asunto(s)
Enfermedades Neurodegenerativas , Receptores Tipo II del Factor de Necrosis Tumoral , Receptores Tipo I de Factores de Necrosis Tumoral , Animales , Ratones , Modelos Animales de Enfermedad , Trastornos de la Memoria/tratamiento farmacológico , Enfermedades Neuroinflamatorias , Receptores Tipo I de Factores de Necrosis Tumoral/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa , Enfermedades Neurodegenerativas/tratamiento farmacológicoRESUMEN
TNF signaling is an essential regulator of cellular homeostasis. Through its two receptors TNFR1 and TNFR2, soluble versus membrane-bound TNF enable cell death or survival in a variety of cell types. TNF-TNFRs signaling orchestrates important biological functions such as inflammation, neuronal activity as well as tissue de- and regeneration. TNF-TNFRs signaling is a therapeutic target for neurodegenerative diseases such as multiple sclerosis (MS) and Alzheimer's disease (AD), but animal and clinical studies yielded conflicting findings. Here, we ask whether a sequential modulation of TNFR1 and TNFR2 signaling is beneficial in experimental autoimmune encephalomyelitis (EAE), an experimental mouse model that recapitulates inflammatory and demyelinating aspects of MS. To this end, human TNFR1 antagonist and TNFR2 agonist were administered peripherally at different stages of disease development in TNFR-humanized mice. We found that stimulating TNFR2 before onset of symptoms leads to improved response to anti-TNFR1 therapeutic treatment. This sequential treatment was more effective in decreasing paralysis symptoms and demyelination, when compared to single treatments. Interestingly, the frequency of the different immune cell subsets is unaffected by TNFR modulation. Nevertheless, treatment with only a TNFR1 antagonist increases T-cell infiltration in the central nervous system (CNS) and B-cell cuffing at the perivascular sites, whereas a TNFR2 agonist promotes Treg CNS accumulation. Our findings highlight the complicated nature of TNF signaling which requires a timely balance of selective activation and inhibition of TNFRs in order to exert therapeutic effects in the context of CNS autoimmunity.
Asunto(s)
Encefalomielitis Autoinmune Experimental , Esclerosis Múltiple , Receptores Tipo II del Factor de Necrosis Tumoral , Receptores Tipo I de Factores de Necrosis Tumoral , Animales , Humanos , Ratones , Sistema Nervioso Central/metabolismo , Encefalomielitis Autoinmune Experimental/metabolismo , Inflamación , Esclerosis Múltiple/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/agonistas , Receptores Tipo II del Factor de Necrosis Tumoral/agonistas , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
BACKGROUND: Tumour necrosis factor (TNF) is a pleiotropic cytokine and master regulator of the immune system. It acts through two receptors resulting in often opposing biological effects, which may explain the lack of therapeutic potential obtained so far in multiple sclerosis (MS) with non-receptor-specific anti-TNF therapeutics. Under neuroinflammatory conditions, such as MS, TNF receptor-1 (TNFR1) is believed to mediate the pro-inflammatory activities associated with TNF, whereas TNF receptor-2 (TNFR2) may instead induce anti-inflammatory effects as well as promote remyelination and neuroprotection. In this study, we have investigated the therapeutic potential of blocking TNFR1 whilst simultaneously stimulating TNFR2 in a mouse model of MS. METHODS: Experimental autoimmune encephalomyelitis (EAE) was induced with myelin oligodendrocyte glycoprotein (MOG35-55) in humanized TNFR1 knock-in mice. These were treated with a human-specific TNFR1-selective antagonistic antibody (H398) and a mouse-specific TNFR2 agonist (EHD2-sc-mTNFR2), both in combination and individually. Histopathological analysis of spinal cords was performed to investigate demyelination and inflammatory infiltration, as well as axonal and neuronal degeneration. Retinas were examined for any protective effects on retinal ganglion cell (RGC) degeneration and neuroprotective signalling pathways analysed by Western blotting. RESULTS: TNFR modulation successfully ameliorated symptoms of EAE and reduced demyelination, inflammatory infiltration and axonal degeneration. Furthermore, the combinatorial approach of blocking TNFR1 and stimulating TNFR2 signalling increased RGC survival and promoted the phosphorylation of Akt and NF-κB, both known to mediate neuroprotection. CONCLUSION: These results further support the potential of regulating the balance of TNFR signalling, through the co-modulation of TNFR1 and TNFR2 activity, as a novel therapeutic approach in treating inflammatory demyelinating disease.
Asunto(s)
Encefalomielitis Autoinmune Experimental , Esclerosis Múltiple , Ratones , Humanos , Animales , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Receptores Tipo II del Factor de Necrosis Tumoral/genética , Receptores Tipo II del Factor de Necrosis Tumoral/metabolismo , Inhibidores del Factor de Necrosis Tumoral , Encefalomielitis Autoinmune Experimental/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Anticuerpos/uso terapéuticoRESUMEN
The cross-talk between thymocytes and thymic stromal cells is fundamental for T cell development. In humans, intrathymic development of dendritic cells (DCs) is evident but its physiological significance is unknown. Here we showed that DC-biased precursors depended on the expression of the transcription factor IRF8 to express the membrane-bound precursor form of the cytokine TNF (tmTNF) to promote differentiation of thymus seeding hematopoietic progenitors into T-lineage specified precursors through activation of the TNF receptor (TNFR)-2 instead of TNFR1. In vitro recapitulation of TNFR2 signaling by providing low-density tmTNF or a selective TNFR2 agonist enhanced the generation of human T cell precursors. Our study shows that, in addition to mediating thymocyte selection and maturation, DCs function as hematopoietic stromal support for the early stages of human T cell development and provide proof of concept that selective targeting of TNFR2 can enhance the in vitro generation of T cell precursors for clinical application.
Asunto(s)
Células Dendríticas , Receptores Tipo II del Factor de Necrosis Tumoral , Humanos , Diferenciación Celular , Linaje de la Célula , Factores Reguladores del Interferón/metabolismo , Receptores Tipo II del Factor de Necrosis Tumoral/metabolismo , Timo/metabolismo , Factores de Necrosis Tumoral/metabolismoRESUMEN
Chronic nonresolving inflammatory syndrome is a major disease feature in myeloproliferative neoplasms (MPNs). Systemic inflammation promotes the growth of the JAK2-V617F+ hematopoietic stem cell clone and is associated with constitutive symptoms (eg, fever, cachexia, and fatigue). Therefore, it is being discussed whether anti-inflammatory therapy, in addition to the well-established JAK inhibitor therapy, may be beneficial in the control of constitutive symptoms. Moreover, effective control of the inflammatory microenvironment may contribute to prevent transformation into secondary myelofibrosis and acute leukemia. Given the pivotal role of tumor necrosis factor α (TNF-α) in MPN and the distinct roles of TNF-α receptor 1 (TNFR1) and TNFR2 in inflammation, we investigated the therapeutic effects of αTNFR1 and αTNFR2 antibody treatment in MPN-like disease using the JAK2+/VF knock-in mouse model. Peripheral blood counts, bone marrow/spleen histopathology, and inflammatory cytokine levels in serum were investigated. αTNFR2 antibody treatment decreased white blood cells and modulated the serum levels of several cytokines [CXCL2, CXCL5, interleukin-12(p40)], as well as of macrophage colony-stimulating factor, but they lacked efficacy to ameliorate hematocrit and splenomegaly. αTNFR1 antibody treatment resulted in the mild suppression of elevated hematocrit of -10.7% and attenuated splenomegaly (22% reduction in spleen weight). In conclusion, our studies show that TNFR1 and TNFR2 play different roles in the biology of JAK2-V617F-induced disease that may be of relevance in future therapeutic settings.
Asunto(s)
Trastornos Mieloproliferativos , Mielofibrosis Primaria , Animales , Antiinflamatorios , Ratones , Trastornos Mieloproliferativos/tratamiento farmacológico , Trastornos Mieloproliferativos/genética , Mielofibrosis Primaria/tratamiento farmacológico , Mielofibrosis Primaria/genética , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Receptores Tipo II del Factor de Necrosis Tumoral/genéticaRESUMEN
Therapeutics that block tumor necrosis factor (TNF), and thus activation of TNF receptor 1 (TNFR1) and TNFR2, are clinically used to treat inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease and psoriasis. However, TNFR1 and TNFR2 work antithetically to balance immune responses involved in inflammatory diseases. In particular, TNFR1 promotes inflammation and tissue degeneration, whereas TNFR2 contributes to immune modulation and tissue regeneration. We, therefore, have developed the monovalent antagonistic anti-TNFR1 antibody derivative Atrosimab to selectively block TNFR1 signaling, while leaving TNFR2 signaling unaffected. Here, we describe that Atrosimab is highly stable at different storage temperatures and demonstrate its therapeutic efficacy in mouse models of acute and chronic inflammation, including experimental arthritis, non-alcoholic steatohepatitis (NASH) and experimental autoimmune encephalomyelitis (EAE). Our data support the hypothesis that it is sufficient to block TNFR1 signaling, while leaving immune modulatory and regenerative responses via TNFR2 intact, to induce therapeutic effects. Collectively, we demonstrate the therapeutic potential of the human TNFR1 antagonist Atrosimab for treatment of chronic inflammatory diseases.
Asunto(s)
Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Factores Inmunológicos/farmacología , Receptores Tipo I de Factores de Necrosis Tumoral/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Animales , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/inmunología , Humanos , Ratones , Ratones Transgénicos , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis Tumoral/inmunología , Transducción de Señal/genética , Transducción de Señal/inmunologíaRESUMEN
Antibody-based therapies hold promise for a safe and efficient treatment of cancer. The identification of target tumor cells through a specific antigen enriched on their surface and the subsequent delivery of the therapeutic agent only to those cells requires, besides the efficacy of the therapeutic agent itself, the identification of an antigen enriched on the surface of tumor cells, the generation of high affinity antibodies against that antigen. We have generated single-domain antibodies (nanobodies) against the voltage-gated potassium channel Kv10.1, which outside of the brain is detectable almost exclusively in tumor cells. The nanobody with highest affinity was fused to an improved form of the tumor necrosis factor-related apoptosis inducing ligand TRAIL, to target this cytokine to the surface of tumor cells. The resulting construct, VHH-D9-scTRAIL, shows rapid and strong apoptosis induction in different tumor models in cell culture. The construct combines two sources of specificity, the expression of the antigen restricted to tumor cells and the tumor selectivity of TRAIL. Such specificity combined with the high affinity obtained through nanobodies make the novel agent a promising concept for cancer therapy.
RESUMEN
Tumor necrosis factor (TNF) is a central regulator of immunity. Due to its dominant pro-inflammatory effects, drugs that neutralize TNF were developed and are clinically used to treat inflammatory and autoimmune diseases, such as rheumatoid arthritis, inflammatory bowel disease and psoriasis. However, despite their clinical success the use of anti-TNF drugs is limited, in part due to unwanted, severe side effects and in some diseases its use even is contraindicative. With gaining knowledge about the signaling mechanisms of TNF and the differential role of the two TNF receptors (TNFR), alternative therapeutic concepts based on receptor selective intervention have led to the development of novel protein therapeutics targeting TNFR1 with antagonists and TNFR2 with agonists. These antibodies and bio-engineered ligands are currently in preclinical and early clinical stages of development. Preclinical data obtained in different disease models show that selective targeting of TNFRs has therapeutic potential and may be superior to global TNF blockade in several disease indications.
RESUMEN
Non-alcoholic fatty liver disease (NAFLD) shows an increasing prevalence and is associated with the development of liver fibrosis and cirrhosis as the major risk factors of liver-related mortality in this disease. The therapeutic possibilities are limited and restricted to life style intervention, since specific drugs for NAFLD are unavailable so far. TNFα has been implicated as a major pathogenic driver of NAFLD. TNFα-mediated liver injury occurs mainly via TNF-receptor-1 (TNFR1) signaling, whereas TNFR2 mediates protective pathways. In this study, we analyzed the therapeutic effects of a novel antibody, which selectively inhibits TNFR1 while retaining protective TNFR2 signaling in a high-fat diet (HFD) mouse model of NAFLD. Mice were fed with HFD for 32 weeks and treated with anti-TNFR1-antibody or control-antibody for the last 8 weeks. We then investigated the mechanisms of TNFR1 inhibition on liver steatosis, inflammatory liver injury, insulin resistance and fibrosis. Compared to control-antibody treatment, TNFR1 inhibition significantly reduced liver steatosis and triglyceride content, which was accompanied by reduced expression and activation of the transcription factor SREBP1 and downstream target genes of lipogenesis. Furthermore, inhibition of TNFR1 resulted in reduced activation of the MAP kinase MKK7 and its downstream target JNK, which was associated with significant improvement of insulin resistance. Apoptotic liver injury, NAFLD activity and alanine aminotransferase (ALT) levels, as well as liver fibrosis significantly decreased by anti-TNFR1 compared to control-antibody treatment. Thus, our results suggest selective TNFR1 inhibition as a promising approach for NAFLD treatment.
Asunto(s)
Hígado Graso/terapia , Cirrosis Hepática/terapia , Enfermedad del Hígado Graso no Alcohólico/terapia , Factor 1 Asociado a Receptor de TNF/antagonistas & inhibidores , Animales , Hígado Graso/genética , Humanos , Cirrosis Hepática/genética , Masculino , Ratones , Enfermedad del Hígado Graso no Alcohólico/genéticaRESUMEN
PURPOSE: The tumor microenvironment plays a key role in cancer development and progression and is involved in resistance to chemo- and immunotherapy. Cancer-associated fibroblast expressing fibroblast-activating protein α (FAPα) is one of the predominant stroma cell types and is involved in resistance to immunotherapy. EXPERIMENTAL DESIGN: We generated OMTX705, a novel antibody-drug conjugate from a humanized anti-FAP antibody linked to a new cytolysin. Here, we studied its antineoplastic activity in vitro and in preclinical mouse models alone and in combination with chemotherapy as well as immunotherapy in PD-1-resistant tumors. RESULTS: In Avatar models, OMTX705 showed a 100% tumor growth inhibition and prolonged tumor regressions as single agent and in combination with chemotherapy. Treatment rechallenge following treatment discontinuation induced additional tumor regression, suggesting lack of treatment resistance. In a mouse model with a humanized immune system resistant to PD-1 inhibition, OMTX705 increased tumor infiltration by CD8+ T cells, induced complete regressions, and delayed tumor recurrence. CONCLUSIONS: These data suggest that FAP targeting with OMTX705 represents a novel and potent strategy for cancer treatment, including tumors resistant to immunotherapy, and support its clinical development.
Asunto(s)
Antineoplásicos/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Inmunoconjugados/farmacología , Proteínas de la Membrana/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales Humanizados/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Endopeptidasas , Humanos , Inmunomodulación/efectos de los fármacos , Ratones , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Tumor necrosis factor receptor 2 (TNFR2) is a transmembrane receptor that is linked to immune modulation and tissue regeneration. Here, we show that TNFR2 essentially promotes long-term pain resolution independently of sex. Genetic deletion of TNFR2 resulted in impaired neuronal regeneration and chronic nonresolving pain after chronic constriction injury (CCI). Further, pharmacological activation of TNFR2 using the TNFR2 agonist EHD2-sc-mTNFR2 in mice with chronic neuropathic pain promoted long-lasting pain recovery. TNFR2 agonist treatment reduced neuronal injury, alleviated peripheral and central inflammation, and promoted repolarization of central nervous system (CNS)-infiltrating myeloid cells into an antiinflammatory/reparative phenotype. Depletion of regulatory T cells (Tregs) delayed spontaneous pain recovery and abolished the therapeutic effect of EHD2-sc-mTNFR2 This study therefore reveals a function of TNFR2 in neuropathic pain recovery and demonstrates that both TNFR2 signaling and Tregs are essential for pain recovery after CCI. Therefore, therapeutic strategies based on the concept of enhancing TNFR2 signaling could be developed into a nonopioid therapy for the treatment of chronic neuropathic pain.
Asunto(s)
Dolor Crónico/inmunología , Neuralgia/inmunología , Receptores Tipo II del Factor de Necrosis Tumoral/inmunología , Transducción de Señal/inmunología , Linfocitos T Reguladores/inmunología , Animales , Dolor Crónico/genética , Dolor Crónico/patología , Dolor Crónico/terapia , Femenino , Inflamación/genética , Inflamación/inmunología , Inflamación/patología , Depleción Linfocítica , Masculino , Ratones , Ratones Noqueados , Neuralgia/genética , Neuralgia/patología , Neuralgia/terapia , Receptores Tipo II del Factor de Necrosis Tumoral/genética , Transducción de Señal/genética , Linfocitos T Reguladores/patologíaRESUMEN
Tumor necrosis factor receptor 2 (TNFR2) is a transmembrane receptor that promotes immune modulation and tissue regeneration and is recognized as a potential therapeutic target for multiple sclerosis (MS). However, TNFR2 also contributes to T effector cell function and macrophage-TNFR2 recently was shown to promote disease development in the experimental autoimmune encephalomyelitis (EAE) model of MS. We here demonstrate that systemic administration of a TNFR2 agonist alleviates peripheral and central inflammation, and reduces demyelination and neurodegeneration, indicating that protective signals induced by TNFR2 exceed potential pathogenic TNFR2-dependent responses. Our behavioral data show that systemic treatment of female EAE mice with a TNFR2 agonist is therapeutic on motor symptoms and promotes long-term recovery from neuropathic pain. Mechanistically, our data indicate that TNFR2 agonist treatment follows a dual mode of action and promotes both suppression of CNS autoimmunity and remyelination. Strategies based on the concept of exogenous activation of TNFR2 therefore hold great promise as a new therapeutic approach to treat motor and sensory disease in MS as well as other inflammatory diseases or neuropathic pain conditions.
Asunto(s)
Esclerosis Múltiple/metabolismo , Receptores Tipo II del Factor de Necrosis Tumoral/agonistas , Receptores Tipo II del Factor de Necrosis Tumoral/metabolismo , Animales , Autoinmunidad/inmunología , Enfermedades Desmielinizantes/metabolismo , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Femenino , Inflamación/patología , Macrófagos/patología , Ratones , Ratones Endogámicos C57BL , Esclerosis Múltiple/patología , Neuralgia/patología , Enfermedades Neurodegenerativas/metabolismo , Médula Espinal/patología , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
The development of alternative therapeutic strategies to tumor necrosis factor (TNF)-blocking antibodies for the treatment of inflammatory diseases has generated increasing interest. In particular, selective inhibition of TNF receptor 1 (TNFR1) promises a more precise intervention, tackling only the pro-inflammatory responses mediated by TNF while leaving regenerative and pro-survival signals transduced by TNFR2 untouched. We recently generated a monovalent anti-TNFR1 antibody fragment (Fab 13.7) as an efficient inhibitor of TNFR1. To improve the pharmacokinetic properties of Fab 13.7, the variable domains of the heavy and light chains were fused to the N-termini of newly generated heterodimerizing Fc chains. This novel Fc heterodimerization technology, designated "Fc-one/kappa" (Fc1κ) is based on interspersed constant Ig domains substituting the CH3 domains of a γ1 Fc. The interspersed immunoglobulin (Ig) domains originate from the per se heterodimerizing constant CH1 and CLκ domains and contain sequence stretches of an IgG1 CH3 domain, destined to enable interaction with the neonatal Fc receptor, and thus promote extended serum half-life. The resulting monovalent Fv-Fc1κ fusion protein (Atrosimab) retained strong binding to TNFR1 as determined by enzyme-linked immunosorbent assay and quartz crystal microbalance, and potently inhibited TNF-induced activation of TNFR1. Atrosimab lacks agonistic activity for TNFR1 on its own and in the presence of anti-human IgG antibodies and displays clearly improved pharmacokinetic properties.
Asunto(s)
Anticuerpos Monoclonales/genética , Anticuerpos Neutralizantes/genética , Fragmentos Fc de Inmunoglobulinas/genética , Inmunoterapia/métodos , Inflamación/terapia , Ingeniería de Proteínas/métodos , Proteínas Recombinantes de Fusión/metabolismo , Animales , Anticuerpos Monoclonales/metabolismo , Anticuerpos Neutralizantes/metabolismo , Dimerización , Ensayo de Inmunoadsorción Enzimática , Humanos , Inflamación/inmunología , Ratones , Ratones Endogámicos C57BL , Receptores Tipo I de Factores de Necrosis Tumoral/inmunología , Proteínas Recombinantes de Fusión/genética , Factor de Necrosis Tumoral alfa/antagonistas & inhibidoresRESUMEN
AIM: The activation of the TNFR2 receptor is beneficial in several pathologies of the central nervous system, and this study examines whether it can ameliorate the recovery process following spinal cord injury. METHODS: EHD2-sc-mTNFR2 , an agonist specific for TNFR2, was used to treat neurons exposed to high levels of glutamate in vitro. In vivo, it was infused directly to the spinal cord via osmotic pumps immediately after a contusion to the cord at the T9 level. Locomotion behavior was assessed for 6 weeks, and the tissue was analyzed (lesion size, RNA and protein expression, cell death) after injury. Somatosensory evoked potentials were also measured in response to hindlimb stimulation. RESULTS: The activation of TNFR2 protected neurons from glutamate-mediated excitotoxicity through the activation of phosphoinositide-3 kinase gamma in vitro and improved the locomotion of animals following spinal cord injury. The extent of the injury was not affected by infusing EHD2-sc-mTNFR2 , but higher levels of neurofilament H and 2', 3'-cyclic-nucleotide 3'-phosphodiesterase were observed 6 weeks after the injury. Finally, the activation of TNFR2 after injury increased the neural response recorded in the cortex following hindlimb stimulation. CONCLUSION: The activation of TNFR2 in the spinal cord following contusive injury leads to enhanced locomotion and better cortical responses to hindlimb stimulation.
Asunto(s)
Contusiones/tratamiento farmacológico , Receptores Tipo II del Factor de Necrosis Tumoral/agonistas , Traumatismos de la Médula Espinal/tratamiento farmacológico , Animales , Células Cultivadas , Fosfatidilinositol 3-Quinasa Clase Ib/fisiología , Citocinas/análisis , Femenino , Ratones , Ratones Endogámicos C57BL , Receptores Tipo II del Factor de Necrosis Tumoral/fisiología , Médula Espinal/efectos de los fármacos , Traumatismos de la Médula Espinal/inmunologíaRESUMEN
Selective inhibition of tumor necrosis factor (TNF) signaling through the proinflammatory axis of TNF-receptor 1 (TNFR1) while leaving pro-survival and regeneration-promoting signals via TNFR2 unaffected is a promising strategy to circumvent limitations of complete inhibition of TNF action by the approved anti-TNF drugs. A previously developed humanized antagonistic TNFR1-specific antibody, ATROSAB, showed potent inhibition of TNFR1-mediated cellular responses. Because the parental mouse antibody H398 possesses even stronger inhibitory potential, we scrutinized the specific binding parameters of the two molecules and revealed a faster dissociation of ATROSAB compared to H398. Applying affinity maturation and re-engineering of humanized variable domains, we generated a monovalent Fab derivative (13.7) of ATROSAB that exhibited increased binding to TNFR1 and superior inhibition of TNF-mediated TNFR1 activation, while lacking any agonistic activity even in the presence of cross-linking antibodies. In order to improve its pharmacokinetic properties, several Fab13.7-derived molecules were generated, including a PEGylated Fab, a mouse serum albumin fusion protein, a half-IgG with a dimerization-deficient Fc, and a newly designed Fv-Fc format, employing the knobs-into-holes technology. Among these derivatives, the Fv13.7-Fc displayed the best combination of improved pharmacokinetic properties and antagonistic activity, thus representing a promising candidate for further clinical development.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Receptores Tipo I de Factores de Necrosis Tumoral/antagonistas & inhibidores , Anticuerpos de Cadena Única/inmunología , Animales , Anticuerpos Monoclonales Humanizados/inmunología , Anticuerpos Neutralizantes/inmunología , Afinidad de Anticuerpos , Humanos , Ratones , Ingeniería de Proteínas/métodosRESUMEN
PURPOSE: Endoglin (ENG; CD105) is a coreceptor of the TGFß family that is highly expressed in proliferating endothelial cells. Often coopted by cancer cells, ENG can lead to neo-angiogenesis and vasculogenic mimicry in aggressive malignancies. It exists both as a transmembrane cell surface protein, where it primarily interacts with TGFß, and as a soluble matricellular protein (sENG) when cleaved by matrix metalloproteinase 14 (MMP14). High ENG expression has been associated with poor prognosis in Ewing sarcoma, an aggressive bone cancer that primarily occurs in adolescents and young adults. However, the therapeutic value of ENG targeting has not been fully explored in this disease. EXPERIMENTAL DESIGN: We characterized the expression pattern of transmembrane ENG, sENG, and MMP14 in preclinical and clinical samples. Subsequently, the antineoplastic potential of two novel ENG-targeting monoclonal antibody-drug conjugates (ADC), OMTX503 and OMTX703, which differed only by their drug payload (nigrin-b A chain and cytolysin, respectively), was assessed in cell lines and preclinical animal models of Ewing sarcoma. RESULTS: Both ADCs suppressed cell proliferation in proportion to the endogenous levels of ENG observed in vitro. Moreover, the ADCs significantly delayed tumor growth in Ewing sarcoma cell line-derived xenografts and patient-derived xenografts in a dose-dependent manner. CONCLUSIONS: Taken together, these studies demonstrate potent preclinical activity of first-in-class anti-ENG ADCs as a nascent strategy to eradicate Ewing sarcoma.
Asunto(s)
Antineoplásicos Inmunológicos/farmacología , Neoplasias Óseas/metabolismo , Endoglina/antagonistas & inhibidores , Inmunoconjugados/farmacología , Sarcoma de Ewing/metabolismo , Animales , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/patología , Línea Celular , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Expresión Génica , Humanos , Metaloproteinasa 14 de la Matriz/genética , Metaloproteinasa 14 de la Matriz/metabolismo , Ratones , Terapia Molecular Dirigida , Medicina de Precisión , Sarcoma de Ewing/tratamiento farmacológico , Sarcoma de Ewing/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Autoimmune diseases are caused by uncontrolled endogenous immune responses against healthy cells. They may develop due to an impaired function of regulatory T cells (Tregs), which normally suppress self-specific effector immune cells. Interleukin 2 (IL-2) and tumor necrosis factor (TNF) have been identified as key players that promote expansion, function, and stability of Tregs. In vivo, both low-dose IL-2 therapy and TNF receptor 2 (TNFR2) agonism were shown to expand Tregs and alleviate autoimmunity. We here designed a novel dimeric dual-acting fusion cytokine, where mouse IL-2 is genetically linked to a TNFR2-selective single-chain TNF mutein (IL2-EHD2-sc-mTNFR2). IL2-EHD2-sc-mTNFR2 showed high affinity to TNFR2 and efficiently activated IL-2 and TNFR2-selective signaling pathways. Further, IL2-EHD2-sc-mTNFR2 promoted superior Treg expansion, with both the IL-2 and the TNFR2 agonist (sc-mTNFR2) component necessary for this biological response. Ultimately, we propose that IL2-EHD2-sc-mTNFR2 is a dual-acting cytokine that efficiently promotes Treg expansion and might have a superior therapeutic window than conventional IL-2-based drugs.
RESUMEN
Tumour necrosis factor (TNF) signalling is mediated via two receptors, TNF-receptor 1 (TNFR1) and TNF-receptor 2 (TNFR2), which work antithetically to balance CNS immune responses involved in autoimmune diseases such as multiple sclerosis. To determine the therapeutic potential of selectively inhibiting TNFR1 in mice with experimental autoimmune encephalomyelitis, we used chimeric human/mouse TNFR1 knock-in mice allowing the evaluation of antagonistic anti-human TNFR1 antibody efficacy. Treatment of mice after onset of disease with ATROSAB resulted in a robust amelioration of disease severity, correlating with reduced central nervous system immune cell infiltration. Long-term efficacy of treatment was achieved by treatment with the parental mouse anti-human TNFR1 antibody, H398, and extended by subsequent re-treatment of mice following relapse. Our data support the hypothesis that anti-TNFR1 therapy restricts immune cell infiltration across the blood-brain barrier through the down-regulation of TNF-induced adhesion molecules, rather than altering immune cell composition or activity. Collectively, we demonstrate the potential for anti-human TNFR1 therapies to effectively modulate immune responses in autoimmune disease.
Asunto(s)
Anticuerpos Monoclonales de Origen Murino/farmacología , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Esclerosis Múltiple/tratamiento farmacológico , Receptores Tipo I de Factores de Necrosis Tumoral/antagonistas & inhibidores , Animales , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Femenino , Humanos , Ratones , Ratones Transgénicos , Esclerosis Múltiple/genética , Esclerosis Múltiple/metabolismo , Esclerosis Múltiple/patología , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismoRESUMEN
Co-stimulatory signals induced by ligands of the tumor necrosis factor superfamily (TNFSF) play a central role in T cell activation and have emerged as a promising strategy in cancer immunotherapy. Here, we established a novel class of bifunctional co-stimulatory fusion proteins with the aim to boost T cell activation at the level of T cell - antigen-presenting cell (APC) interaction. These novel dual-acting cytokine fusion proteins were created by connecting two different homotrimeric TNFSF ligands to form homotrimeric bifunctional molecules (Duokines) or by connecting single-chain derivatives of two different homotrimeric TNFSF with a single, flexible linker (single-chain Duokines, scDuokines). By linking the TNFSF ligands 4-1BBL, OX40L and CD27L in all possible combinations, cis-acting Duokines were generated that act on the same or adjacent T cells, while combining CD40L with 4-1BBL, OX40L and CD27L resulted in trans-acting Duokines acting simultaneously on APCs and T cells. In vitro, co-stimulation of T cells was seen for cis- and trans-acting Duokines and scDuokines in an antigen-independent as well as antigen-specific setting. Trans-acting molecules furthermore activated B cells, which represent a subclass of APCs. In a pilot experiment using the syngeneic B16-FAP mouse tumor model scDuokines displayed antitumoral activity in vivo in combination with a primary T cell-activating bispecific antibody, evident from reduced number of lung metastasis compared to the antibody-only treated group. Our data show that the bifunctional, co-stimulatory duokines are capable to enhance T cell-mediated anti-tumor immune responses, suggesting that they can serve as a new class of immuno-stimulatory molecules for use in cancer immunotherapy strategies.