RESUMEN
Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease driven by gain-of-function variants in activin receptor-like kinase 2 (ALK2), the most common variant being ALK2R206H. In FOP, ALK2 variants display increased and dysregulated signaling through the bone morphogenetic protein (BMP) pathway resulting in progressive and permanent replacement of skeletal muscle and connective tissues with heterotopic bone, ultimately leading to severe debilitation and premature death. Here, we describe the discovery of BLU-782 (IPN60130), a small-molecule ALK2R206H inhibitor developed for the treatment of FOP. A small-molecule library was screened in a biochemical ALK2 binding assay to identify potent ALK2 binding compounds. Iterative rounds of structure-guided drug design were used to optimize compounds for ALK2R206H binding, ALK2 selectivity, and other desirable pharmacokinetic properties. BLU-782 preferentially bound to ALK2R206H with high affinity, inhibiting signaling from ALK2R206H and other rare FOP variants in cells in vitro without affecting signaling of closely related homologs ALK1, ALK3, and ALK6. In vivo efficacy of BLU-782 was demonstrated using a conditional knock-in ALK2R206H mouse model, where prophylactic oral dosing reduced edema and prevented cartilage and heterotopic ossification (HO) in both muscle and bone injury models. BLU-782 treatment preserved the normal muscle-healing response in ALK2R206H mice. Delayed dosing revealed a short 2-day window after injury when BLU-782 treatment prevented HO in ALK2R206H mice, but dosing delays of 4 days or longer abrogated HO prevention. Together, these data suggest that BLU-782 may be a candidate for prevention of HO in FOP.
Asunto(s)
Modelos Animales de Enfermedad , Miositis Osificante , Osificación Heterotópica , Animales , Miositis Osificante/tratamiento farmacológico , Miositis Osificante/metabolismo , Osificación Heterotópica/tratamiento farmacológico , Osificación Heterotópica/metabolismo , Osificación Heterotópica/prevención & control , Ratones , Humanos , Receptores de Activinas Tipo II/metabolismo , Receptores de Activinas Tipo I/metabolismo , Receptores de Activinas Tipo I/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacosRESUMEN
Heterotopic ossification (HO) is most dramatically manifested in the rare and severely debilitating disease, fibrodysplasia ossificans progressiva (FOP), in which heterotopic bone progressively accumulates in skeletal muscles and associated soft tissues. The great majority of FOP cases are caused by a single amino acid substitution in the type 1 bone morphogenetic protein (BMP) receptor ACVR1, a mutation that imparts responsiveness to activin A. Although it is well-established that biological sex is a critical variable in a range of physiological and disease processes, the impact of sex on HO in animal models of FOP has not been explored. We show that female FOP mice exhibit both significantly greater and more variable HO responses after muscle injury. Additionally, the incidence of spontaneous HO was significantly greater in female mice. This sex dimorphism is not dependent on gonadally derived sex hormones, and reciprocal cell transplantations indicate that apparent differences in osteogenic activity are intrinsic to the sex of the transplanted cells. By circumventing the absolute requirement for activin A using an agonist of mutant ACVR1, we show that the female-specific response to muscle injury or BMP2 implantation is dependent on activin A. These data identify sex as a critical variable in basic and pre-clinical studies of FOP.
Asunto(s)
Miositis Osificante , Osificación Heterotópica , Femenino , Ratones , Animales , Masculino , Miositis Osificante/genética , Miositis Osificante/metabolismo , Osificación Heterotópica/genética , Osificación Heterotópica/metabolismo , Osteogénesis , Mutación , Huesos/metabolismoRESUMEN
Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease characterized by progressive and catastrophic heterotopic ossification (HO) of skeletal muscle and associated soft tissues. FOP is caused by dominantly acting mutations in the gene encoding the bone morphogenetic protein (BMP) type I receptor, ACVR1 (ALK2), the most prevalent of which results in an arginine to histidine substitution at position 206 (ACVR1[R206H]). The fundamental pathological consequence of FOP-causing ACVR1 receptor mutations is to enable activin A to initiate canonical BMP signaling in fibro-adipogenic progenitors (FAPs), which drives HO. We developed a monoclonal blocking antibody (JAB0505) against the extracellular domain of ACVR1 and tested its effect on HO in 2 independent FOP mouse models. Although JAB0505 inhibited BMP-dependent gene expression in wild-type and ACVR1(R206H)-overexpressing cell lines, JAB0505 treatment profoundly exacerbated injury-induced HO. JAB0505-treated mice exhibited multiple, distinct foci of heterotopic lesions, suggesting an atypically broad anatomical domain of FAP recruitment to endochondral ossification. This was accompanied by dysregulated FAP population growth and an abnormally sustained immunological reaction following muscle injury. JAB0505 drove injury-induced HO in the absence of activin A, indicating that JAB0505 has receptor agonist activity. These data raise serious safety and efficacy concerns for the use of bivalent anti-ACVR1 antibodies to treat patients with FOP.
Asunto(s)
Miositis Osificante , Osificación Heterotópica , Receptores de Activinas Tipo I/genética , Animales , Proteínas Morfogenéticas Óseas/genética , Humanos , Ratones , Mutación , Miositis Osificante/genética , Miositis Osificante/metabolismo , Osificación Heterotópica/patología , OsteogénesisRESUMEN
Eculizumab, a monoclonal antibody (mAb) directed against complement protein C5, is considered to be the current standard of care for patients with paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome. This study describes the generation and preclinical attributes of ALXN1210, a new long-acting anti-C5 mAb, obtained through select modifications to eculizumab to both largely abolish target-mediated drug disposition (TMDD) and increase recycling efficiency via the neonatal Fc receptor (FcRn). To attenuate the effect of TMDD on plasma terminal half-life (t1/2), histidine substitutions were engineered into the complementarity-determining regions of eculizumab to enhance the dissociation rate of the mAb:C5 complex in the acidic early endosome relative to the slightly basic pH of blood. Antibody variants with optimal pH-dependent binding to C5 exhibited little to no TMDD in mice in the presence of human C5. To further enhance the efficiency of FcRn-mediated recycling of the antibody, two additional substitutions were introduced to increase affinity for human FcRn. These substitutions yielded an additional doubling of the t½ of surrogate anti-mouse C5 antibodies with reduced TMDD in transgenic mice expressing the human FcRn. In conclusion, ALXN1210 is a promising new therapeutic candidate currently in clinical development for treatment of patients with PNH and atypical hemolytic uremic syndrome.
Asunto(s)
Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/farmacología , Complemento C5/antagonistas & inhibidores , Diseño de Fármacos , Animales , Anticuerpos Monoclonales/farmacocinética , Afinidad de Anticuerpos , Evaluación Preclínica de Medicamentos , Hemólisis/inmunología , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Cinética , Ratones , Ratones Transgénicos , Unión Proteica , Receptores Fc/genéticaRESUMEN
C3 glomerulopathy refers to renal disorders characterized by abnormal accumulation of C3 within the kidney, commonly along the glomerular basement membrane (GBM). C3 glomerulopathy is associated with complement alternative pathway dysregulation, which includes functional defects in complement regulator factor H (FH). There is no effective treatment for C3 glomerulopathy. We investigated the efficacy of a recombinant mouse protein composed of domains from complement receptor 2 (CR2) and FH (CR2-FH) in two models of C3 glomerulopathy with either preexisting or triggered C3 deposition along the GBM. FH-deficient mice spontaneously develop renal pathology associated with abnormal C3 accumulation along the GBM and secondary plasma C3 deficiency. CR2-FH partially restored plasma C3 levels in FH-deficient mice 2 hours after intravenous injection. CR2-FH specifically targeted glomerular C3 deposits, reduced the linear C3 reactivity assessed with anti-C3 and anti-C3b/iC3b/C3c antibodies, and prevented further spontaneous accumulation of C3 fragments along the GBM. Reduction in glomerular C3d and C9/C5b-9 reactivity was observed after daily administration of CR2-FH for 1 week. In a second mouse model with combined deficiency of FH and complement factor I, CR2-FH prevented de novo C3 deposition along the GBM. These data show that CR2-FH protects the GBM from both spontaneous and triggered C3 deposition in vivo and indicate that this approach should be tested in C3 glomerulopathy.
Asunto(s)
Complemento C3/antagonistas & inhibidores , Membrana Basal Glomerular , Enfermedades Renales/tratamiento farmacológico , Proteínas Recombinantes de Fusión/uso terapéutico , Animales , Complemento C3/metabolismo , Modelos Animales de Enfermedad , Membrana Basal Glomerular/metabolismo , Enfermedades Renales/metabolismo , Ratones , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
Diseases of ectopic calcification of the vascular wall range from lethal orphan diseases such as generalized arterial calcification of infancy (GACI), to common diseases such as hardening of the arteries associated with aging and calciphylaxis of chronic kidney disease (CKD). GACI is a lethal orphan disease in which infants calcify the internal elastic lamina of their medium and large arteries and expire of cardiac failure as neonates, while calciphylaxis of CKD is a ubiquitous vascular calcification in patients with renal failure. Both disorders are characterized by vascular Mönckeburg's sclerosis accompanied by decreased concentrations of plasma inorganic pyrophosphate (PPi). Here we demonstrate that subcutaneous administration of an ENPP1-Fc fusion protein prevents the mortality, vascular calcifications and sequela of disease in animal models of GACI, and is accompanied by a complete clinical and biomarker response. Our findings have implications for the treatment of rare and common diseases of ectopic vascular calcification.
Asunto(s)
Enfermedades del Recién Nacido/enzimología , Enfermedades del Recién Nacido/prevención & control , Hidrolasas Diéster Fosfóricas/metabolismo , Pirofosfatasas/metabolismo , Calcificación Vascular/enzimología , Calcificación Vascular/prevención & control , Animales , Arterias/enzimología , Arterias/patología , Modelos Animales de Enfermedad , Femenino , Humanos , Fragmentos Fc de Inmunoglobulinas/administración & dosificación , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/metabolismo , Inmunoglobulina G/genética , Inmunoglobulina G/metabolismo , Recién Nacido , Enfermedades del Recién Nacido/genética , Enfermedades del Recién Nacido/mortalidad , Masculino , Ratones Endogámicos C57BL , Hidrolasas Diéster Fosfóricas/administración & dosificación , Hidrolasas Diéster Fosfóricas/genética , Pirofosfatasas/administración & dosificación , Pirofosfatasas/genética , Calcificación Vascular/genética , Calcificación Vascular/mortalidadRESUMEN
Previous studies have shown that some respiratory virus infections leave local populations of tissue TRM cells in the lungs which disappear as heterosubtypic immunity declines. The location of these TRM cells and their contribution to the protective CTL response have not been clearly defined. Here, fluorescence microscopy is used to show that some CD103(+) TRM cells remain embedded in the walls of the large airways long after pulmonary immunization but are absent from systemically primed mice. Viral clearance from the lungs of the locally immunized mice precedes the development of a robust Teff response in the lungs. Whereas large numbers of virus-specific CTLs collect around the bronchial tree during viral clearance, there is little involvement of the remaining lung tissue. Much larger numbers of TEM cells enter the lungs of the systemically immunized animals but do not prevent extensive viral replication or damage to the alveoli. Together, these experiments show that virus-specific antibodies and TRM cells are both required for optimal heterosubtypic immunity, whereas circulating memory CD8 T cells do not substantially alter the course of disease.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Protección Cruzada/inmunología , Virus de la Influenza A/inmunología , Pulmón/inmunología , Pulmón/virología , Infecciones por Orthomyxoviridae/prevención & control , Infecciones por Orthomyxoviridae/virología , Animales , Anticuerpos Antivirales/inmunología , Antígenos CD/metabolismo , Proliferación Celular , Perros , Inmunización , Memoria Inmunológica , Cadenas alfa de Integrinas/metabolismo , Pulmón/patología , Activación de Linfocitos/inmunología , Células de Riñón Canino Madin Darby , Ratones , Infecciones por Orthomyxoviridae/inmunología , Ovalbúmina/inmunología , Receptor de Muerte Celular Programada 1/metabolismo , Especificidad de la Especie , Factores de TiempoRESUMEN
The initial engagement of the TCR through interaction with cognate peptide-MHC is a requisite for T cell activation and confers Ag specificity. Although this is a key event in T cell activation, the duration of these interactions may affect the proliferative capacity and differentiation of the activated cells. In this study, we developed a system to evaluate the temporal requirements for antigenic stimulation during an immune response in vivo. Using Abs that target specific Ags in the context of MHC, we were able to manipulate the duration of Ag availability to both CD4 and CD8 T cells during an active infection. During the primary immune response, the magnitude of the CD4 and CD8 T cell response was dependent on the duration of Ag availability. Both CD4 and CD8 T cells required sustained antigenic stimulation for maximal expansion. Memory cell differentiation was also dependent on the duration of Ag exposure, albeit to a lesser extent. However, memory development did not correlate with the magnitude of the primary response, suggesting that the requirements for continued expansion of T cells and memory differentiation are distinct. Finally, a shortened period of Ag exposure was sufficient to achieve optimal expansion of both CD4 and CD8 T cells during a recall response. It was also revealed that limiting exposure to Ag late during the response may enhance the CD4 T cell memory pool. Collectively, these data indicated that Ag remains a critical component of the T cell response after the initial APC-T cell interaction.
Asunto(s)
Antígenos Virales/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Memoria Inmunológica/inmunología , Activación de Linfocitos/inmunología , Animales , Presentación de Antígeno/inmunología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD8-positivos/citología , Diferenciación Celular/inmunología , Separación Celular , Citometría de Flujo , Ratones , Ratones Endogámicos C57BL , Virosis/inmunologíaRESUMEN
Cytotoxic T lymphocytes (CTLs) are important targets for vaccines against a wide variety of infections that enter the body via mucosal tissues. To induce effective immunity these vaccines must include the most protective epitopes and elicit rapid recall responses at the site of infection. Although live attenuated viruses are sometimes used to induce cellular immunity against recurrent influenza infections, the mechanisms that determine the magnitude of the response to individual viral components are very poorly defined. Heterosubtypic infections in C57BL/6 mice illustrate an additional level of complexity, when the antigen specificity of the response shifts dramatically between primary and secondary challenge. This model provides a unique opportunity to identify the mechanisms that regulate memory CD8(+) T-cell reactivation in vivo and control the specificity of the recall response by pathogen-specific CTL. We show that multiple factors contribute to the changing pattern of immunodominance during secondary infection, including the location of the memory CD8(+) T cells at the time of reinfection and their ability to directly recognize migratory CD103(+) DCs as they arrive in the lung draining LN.
Asunto(s)
Linfocitos T CD8-positivos/metabolismo , Células Dendríticas/metabolismo , Infecciones por Orthomyxoviridae/inmunología , Orthomyxoviridae/inmunología , Subgrupos de Linfocitos T/metabolismo , Animales , Antígenos CD/biosíntesis , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Linfocitos T CD8-positivos/virología , Movimiento Celular , Células Cultivadas , Células Dendríticas/inmunología , Células Dendríticas/patología , Células Dendríticas/virología , Humanos , Epítopos Inmunodominantes/metabolismo , Memoria Inmunológica , Vacunas contra la Influenza , Cadenas alfa de Integrinas/biosíntesis , Ganglios Linfáticos/patología , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Orthomyxoviridae/patogenicidad , Especificidad del Receptor de Antígeno de Linfocitos T , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/patología , Subgrupos de Linfocitos T/virologíaRESUMEN
Viral infections often gain access to the body of their host by exploiting areas of natural vulnerability, such as the semipermeable surfaces of mucosal tissues which are adapted for adsorption of nutrients and other diffusible molecules. Once the microbes have crossed the epithelial barrier, they can disperse to other tissues where eradication may not be possible. The best opportunity for successful immune intervention is immediately after infection while the pathogen is confined to a localized area of the body. Cytotoxic T lymphocytes (CTL) which reside at the site where the infection begins can make an important contribution to immunity by reducing early dissemination of the infection. Because the lungs provide easy access points for many pathogens to enter the body, they require protection from many complementary mechanisms, including pathogen-specific cytotoxic T cells. In this study we show that an enduring response to pathogen-derived peptide antigens facilitates sustained surveillance of the lungs by pathogen-specific CTL during the recovery from influenza virus infection. Our studies show that these processed peptide antigens reinforce expression of two homing receptors (CD69 and CD103) which help recently activated virus-specific CTL colonize the lungs during a mild inflammatory response. We suggest that this requirement for prolonged antigen presentation to reinforce local CTL responses in the lungs explains why protective cellular immunity quickly declines following influenza virus infection and other viral infections that enter the body via mucosal tissues.
Asunto(s)
Antígenos Virales/inmunología , Pulmón/inmunología , Pulmón/virología , Infecciones por Orthomyxoviridae/inmunología , Orthomyxoviridae/inmunología , Receptores de Antígenos/biosíntesis , Linfocitos T Citotóxicos/inmunología , Animales , Antígenos CD/biosíntesis , Antígenos CD/inmunología , Antígenos de Diferenciación de Linfocitos T/biosíntesis , Antígenos de Diferenciación de Linfocitos T/inmunología , Expresión Génica , Cadenas alfa de Integrinas/biosíntesis , Cadenas alfa de Integrinas/inmunología , Lectinas Tipo C/biosíntesis , Lectinas Tipo C/inmunología , Ratones , Orthomyxoviridae/patogenicidad , Infecciones por Orthomyxoviridae/virología , Receptores de Antígenos/inmunologíaRESUMEN
Previous studies in rodents and people have demonstrated that repeated tick exposure is associated with reduced Borrelia burgdorferi transmission but the mechanism of prevention remains unclear. We examined the acute histopathologic reactions to initial and repeated Ixodes scapularis bites in BALB/c mice and in people. Skin biopsies of BALB/c mice infested for the first time by I. scapularis nymphs revealed vascular dilatation and an accumulation of inflammatory cells adjacent to the bite site but absent at the site of tick attachment. Such changes would enhance tick-borne pathogen transmission. Mice reexposed to I. scapularis nymphs experienced a decrease in vascular dilatation and a marked increase in inflammatory cells at the site of tick attachment. Skin biopsies of people with attached I. scapularis nymphs revealed similar histologic patterns. These results indicate that cellular changes at the tick-dermal interface following I. scapularis attachment are likely to allow for successful transmission of tick-borne pathogens in non-tick-immune hosts and to inhibit tick-borne pathogen transmission in hosts that have developed tick immunity.
Asunto(s)
Mordeduras y Picaduras/complicaciones , Infestaciones Ectoparasitarias/patología , Ixodes , Animales , Modelos Animales de Enfermedad , Infestaciones Ectoparasitarias/transmisión , Infestaciones Ectoparasitarias/veterinaria , Humanos , Ratones , Ratones Endogámicos BALB C/parasitología , Enfermedades por Picaduras de Garrapatas/prevención & controlRESUMEN
Ticks introduce a variety of pharmacologically active molecules into their host during attachment and feeding in order to obtain a blood meal. People who are repeatedly exposed to ticks may develop an immune response to tick salivary proteins. Despite this response, people usually are unaware of having been bitten, especially if they are not repeatedly exposed to ticks. In order to develop a laboratory marker of tick exposure that would be useful in understanding the epidemiology of tick-borne infection and the immune response to tick bite, we developed an enzyme-linked immunosorbent assay (ELISA) to detect antibody to a recombinant form of calreticulin protein found in the salivary glands of Ixodes scapularis, a member of a complex of Ixodes ticks that serve as the vectors for Lyme disease, human babesiosis, and human granulocytic anaplasmosis. Using this assay, we tested sera obtained from C3H/HeN and BALB/c mice before and after experimental deer tick infestation. These mice developed antibody to Ixodes calreticulin antigen after infestation. We then used the same assay to test sera obtained from people before and after they experienced deer tick bite(s). People experiencing deer tick bite(s) developed Ixodes calreticulin-specific antibody responses that persisted for up to 17 months. This Ixodes recombinant calreticulin ELISA provides objective evidence of deer tick exposure in people.