RESUMEN
ABSTRACT: Iron plays a major role in the deterioration of ß-thalassemia. Indeed, the high levels of transferrin saturation and iron delivered to erythroid progenitors are associated with production of α-globin precipitates that negatively affect erythropoiesis. Matriptase-2/TMPRSS6, a membrane-bound serine protease expressed in hepatocytes, negatively modulates hepcidin production and thus is a key target to prevent iron overload in ß-thalassemia. To address safety concerns raised by the suppression of Tmprss6 by antisense oligonucleotides or small interfering RNA, we tested a fully human anti-matriptase-2 antibody, RLYB331, which blocks the protease activity of matriptase-2. When administered weekly to Hbbth3/+ mice, RLYB331 induced hepcidin expression, reduced iron loading, prevented the formation of toxic α-chain/heme aggregates, reduced ros oxygen species formation, and improved reticulocytosis and splenomegaly. To increase the effectiveness of RLYB331 in ß-thalassemia treatment even further, we administered RLYB331 in combination with RAP-536L, a ligand-trapping protein that contains the extracellular domain of activin receptor type IIB and alleviates anemia by promoting differentiation of late-stage erythroid precursors. RAP-536L alone did not prevent iron overload but significantly reduced apoptosis in the erythroid populations of the bone marrow, normalized red blood cell counts, and improved hemoglobin and hematocrit levels. Interestingly, the association of RLYB331 with RAP-536L entirely reversed the ß-thalassemia phenotype in Hbbth3/+ mice and simultaneously corrected iron overload, ineffective erythropoiesis, splenomegaly, and hematological parameters, suggesting that a multifunctional molecule consisting of the fusion of RLYB331 with luspatercept (human version of RAP-536L) would allow administration of a single medication addressing simultaneously the different pathophysiological aspects of ß-thalassemia.
Asunto(s)
Sobrecarga de Hierro , Proteínas de la Membrana , Serina Endopeptidasas , Talasemia beta , Ratones , Humanos , Animales , Hepcidinas , Talasemia beta/genética , Esplenomegalia , Sobrecarga de Hierro/tratamiento farmacológico , Hierro/metabolismoRESUMEN
As the number of antibody drugs being approved and marketed increases, our knowledge of what makes potential drug candidates a successful product has increased tremendously. One of the critical parameters that have become clear in the field is the importance of mAb "developability." Efforts are being increasingly focused on simultaneously selecting molecules that exhibit both desirable biological potencies and manufacturability attributes. In the current study mutations to improve the developability profile of a problematic antibody that inconsistently precipitates in a batch scale-dependent fashion using a standard platform purification process are described. Initial bioinformatic analysis showed the molecule has no obvious sequence or structural liabilities that might lead it to precipitate. Subsequent analysis of the molecule revealed the presence of two unusual positively charged mutations on the light chain at the interface of VH and VL domains, which were hypothesized to be the primary contributor to molecule precipitation during process development. To investigate this hypothesis, straightforward reversion to the germline of these residues was carried out. The resulting mutants have improved expression titers and recovered stability within a forced precipitation assay, without any change to biological activity. Given the time pressures of drug development in industry, process optimization of the lead molecule was carried out in parallel to the "retrospective" mutagenesis approach. Bespoke process optimization for large-scale manufacturing was successful. However, we propose that such context-dependent sequence liabilities should be included in the arsenal of in silico developability screening early in development; particularly since this specific issue can be efficiently mitigated without the requirement for extensive screening of lead molecule variants.
Asunto(s)
Anticuerpos Monoclonales , Ingeniería de Proteínas , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/genética , Línea Celular , Humanos , SolubilidadRESUMEN
The immunosuppressive tumor microenvironment constitutes a significant hurdle to immune checkpoint inhibitor responses. Both soluble factors and specialized immune cells, such as regulatory T cells (Treg), are key components of active intratumoral immunosuppression. Inducible costimulatory receptor (ICOS) can be highly expressed in the tumor microenvironment, especially on immunosuppressive Treg, suggesting that it represents a relevant target for preferential depletion of these cells. Here, we performed immune profiling of samples from tumor-bearing mice and patients with cancer to demonstrate differential expression of ICOS in immune T-cell subsets in different tissues. ICOS expression was higher on intratumoral Treg than on effector CD8 T cells. In addition, by immunizing an Icos knockout transgenic mouse line expressing antibodies with human variable domains, we selected a fully human IgG1 antibody called KY1044 that bound ICOS from different species. We showed that KY1044 induced sustained depletion of ICOShigh T cells but was also associated with increased secretion of proinflammatory cytokines from ICOSlow effector T cells (Teff). In syngeneic mouse tumor models, KY1044 depleted ICOShigh Treg and increased the intratumoral TEff:Treg ratio, resulting in increased secretion of IFNγ and TNFα by TEff cells. KY1044 demonstrated monotherapy antitumor efficacy and improved anti-PD-L1 efficacy. In summary, we demonstrated that using KY1044, one can exploit the differential expression of ICOS on T-cell subtypes to improve the intratumoral immune contexture and restore an antitumor immune response.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Proteína Coestimuladora de Linfocitos T Inducibles/metabolismo , Linfocitos T Reguladores/inmunología , Microambiente Tumoral/efectos de los fármacos , Animales , Anticuerpos Monoclonales/inmunología , Antígeno B7-H1/inmunología , Línea Celular Tumoral , Citocinas/metabolismo , Femenino , Humanos , Tolerancia Inmunológica/efectos de los fármacos , Proteína Coestimuladora de Linfocitos T Inducibles/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BLRESUMEN
Recombinant erythropoietin (EPO) and iron substitution are a standard of care for treatment of anemias associated with chronic inflammation, including anemia of chronic kidney disease. A black box warning for EPO therapy and concerns about negative side effects related to high-dose iron supplementation as well as the significant proportion of patients becoming EPO resistant over time explains the medical need to define novel strategies to ameliorate anemia of chronic disease (ACD). As hepcidin is central to the iron-restrictive phenotype in ACD, therapeutic approaches targeting hepcidin were recently developed. We herein report the therapeutic effects of a fully human anti-BMP6 antibody (KY1070) either as monotherapy or in combination with Darbepoetin alfa on iron metabolism and anemia resolution in 2 different, well-established, and clinically relevant rodent models of ACD. In addition to counteracting hepcidin-driven iron limitation for erythropoiesis, we found that the combination of KY1070 and recombinant human EPO improved the erythroid response compared with either monotherapy in a qualitative and quantitative manner. Consequently, the combination of KY1070 and Darbepoetin alfa resulted in an EPO-sparing effect. Moreover, we found that suppression of hepcidin via KY1070 modulates ferroportin expression on erythroid precursor cells, thereby lowering potentially toxic-free intracellular iron levels and by accelerating erythroid output as reflected by increased maturation of erythrocyte progenitors. In summary, we conclude that treatment of ACD, as a highly complex disease, becomes more effective by a multifactorial therapeutic approach upon mobilization of endogenous iron deposits and stimulation of erythropoiesis.
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Anemia/terapia , Anticuerpos Monoclonales/uso terapéutico , Proteína Morfogenética Ósea 6/antagonistas & inhibidores , Darbepoetina alfa/uso terapéutico , Anemia/tratamiento farmacológico , Anemia/etiología , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/inmunología , Artritis/inducido químicamente , Artritis/complicaciones , Médula Ósea/metabolismo , Proteína Morfogenética Ósea 6/inmunología , Proteínas de Transporte de Catión/metabolismo , Citocinas/sangre , Darbepoetina alfa/administración & dosificación , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Eritropoyetina/farmacología , Eritropoyetina/uso terapéutico , Células Hep G2 , Humanos , Hierro/metabolismo , Ratones , Proteínas Musculares/sangre , Polisacáridos Bacterianos/toxicidad , Distribución Aleatoria , Proteínas Recombinantes/inmunología , Insuficiencia Renal Crónica/complicacionesRESUMEN
Pulmonary arterial hypertension (PAH) is a rare but fatal disease. Current treatments increase life expectancy but have limited impact on the progressive pulmonary vascular remodelling that drives PAH. Osteoprotegerin (OPG) is increased within serum and lesions of patients with idiopathic PAH and is a mitogen and migratory stimulus for pulmonary artery smooth muscle cells (PASMCs). Here, we report that the pro-proliferative and migratory phenotype in PASMCs stimulated with OPG is mediated via the Fas receptor and that treatment with a human antibody targeting OPG can attenuate pulmonary vascular remodelling associated with PAH in multiple rodent models of early and late treatment. We also demonstrate that the therapeutic efficacy of the anti-OPG antibody approach in the presence of standard of care vasodilator therapy is mediated by a reduction in pulmonary vascular remodelling. Targeting OPG with a therapeutic antibody is a potential treatment strategy in PAH.
Asunto(s)
Anticuerpos/administración & dosificación , Hipertensión Pulmonar Primaria Familiar/tratamiento farmacológico , Osteoprotegerina/metabolismo , Animales , Movimiento Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Hipertensión Pulmonar Primaria Familiar/genética , Hipertensión Pulmonar Primaria Familiar/metabolismo , Hipertensión Pulmonar Primaria Familiar/fisiopatología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Osteoprotegerina/genética , Unión Proteica , Arteria Pulmonar/citología , Arteria Pulmonar/metabolismo , Arteria Pulmonar/fisiopatología , Ratas , Ratas Wistar , Remodelación Vascular/efectos de los fármacosRESUMEN
If immunized with an antigen of interest, transgenic mice with large portions of unrearranged human immunoglobulin loci can produce fully human antigen-specific antibodies; several such antibodies are in clinical use. However, technical limitations inherent to conventional transgenic technology and sequence divergence between the human and mouse immunoglobulin constant regions limit the utility of these mice. Here, using repetitive cycles of genome engineering in embryonic stem cells, we have inserted the entire human immunoglobulin variable-gene repertoire (2.7 Mb) into the mouse genome, leaving the mouse constant regions intact. These transgenic mice are viable and fertile, with an immune system resembling that of wild-type mice. Antigen immunization results in production of high-affinity antibodies with long human-like complementarity-determining region 3 (CDR3H), broad epitope coverage and strong signatures of somatic hypermutation. These mice provide a robust system for the discovery of therapeutic human monoclonal antibodies; as a surrogate readout of the human antibody response, they may also aid vaccine design efforts.
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Anticuerpos Monoclonales/genética , Ingeniería Genética/métodos , Región Variable de Inmunoglobulina/genética , Transgenes/genética , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Linfocitos B/inmunología , Linfocitos B/fisiología , Cromosomas Artificiales Bacterianos/genética , Femenino , Humanos , Masculino , Ratones , Ratones Transgénicos , Datos de Secuencia MolecularRESUMEN
Age-related macular degeneration (AMD) is a leading cause of legal blindness in the Western world. There are effective treatments for the vascular complications of neo-vascular AMD, but no effective therapies are available for the dry/atrophic form of the disease. A previously described transgenic CFH-gene deficient mouse model, (cfh-/-), shows hallmarks of early AMD. The ocular phenotype has been further analysed to demonstrate amyloid beta (Aß) rich basement membrane deposits associated with activated complement C3. Cfh-/- mice were treated systemically in both prophylactic and therapeutic regimes with an anti-Aß monoclonal antibody (mAb), 6F6, to determine the effect on the cfh-/- retinal phenotype. Prophylactic treatment with 6F6 demonstrated a dose dependent reduction in the accumulation of both Aß and activated C3 deposition. A similar reduction in the retinal endpoints could be seen after therapeutic treatment. Serum Aß levels after systemic administration of 6F6 show accumulation of Aß in the periphery suggestive of a peripheral sink mechanism. In summary, anti-Aß mAb treatment can partially prevent or reverse ocular phenotypes of the cfh-/- mouse. The data support this therapeutic approach in humans potentially modulating two key elements in the pathogenesis of AMD - Aß and activated, complement C3.