RESUMEN
Protein glycation is a common, normally innocuous, post-translational modification in therapeutic monoclonal antibodies. However, when glycation occurs on complementarity-determining regions (CDRs) of a therapeutic monoclonal antibody, its biological activities (e.g., potency) may be impacted. Here, we present a comprehensive approach to understanding the mechanism of protein glycation using a bispecific antibody. Cation exchange chromatography and liquid chromatography-mass spectrometry were used to characterize glycation at a lysine residue within a heavy chain (HC) CDR (HC-CDR3-Lys98) of a bispecific antibody. Thermodynamic analysis revealed that this reaction is reversible and can occur under physiological conditions with an apparent affinity of 8-10 mM for a glucose binding to HC-CDR3-Lys98. Results from kinetic analysis demonstrated that this reaction follows Arrhenius behavior in the temperature range of 5°C-45°C and can be well predicted in vitro and in a non-human primate. In addition, this glycation reaction was found to be driven by an unusually low pKa on the ε-amino group of HC-CDR3-Lys98. Van't Hoff analysis and homology modeling suggested that this reaction is enthalpically driven, with this lysine residue surrounded by a microenvironment with low polarity. This study provides, to our knowledge, new insights toward a mechanistic understanding of protein glycation and strategies to mitigate the impact of protein glycation during pharmaceutical development.
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Regiones Determinantes de Complementariedad , Lisina , Animales , Anticuerpos Monoclonales/química , Cromatografía Liquida , Regiones Determinantes de Complementariedad/química , Regiones Determinantes de Complementariedad/metabolismo , Glicosilación , Cinética , Lisina/metabolismoRESUMEN
BACKGROUND: Dupilumab, a fully human monoclonal antibody that binds IL-4Rα and inhibits signaling of both IL-4 and IL-13, has shown efficacy across multiple diseases with underlying type 2 signatures and is approved for treatment of asthma, atopic dermatitis, and chronic sinusitis with nasal polyposis. We sought to provide a comprehensive analysis of the redundant and distinct roles of IL-4 and IL-13 in type 2 inflammation and report dupilumab mechanisms of action. METHODS: Using primary cell assays and a mouse model of house dust mite-induced asthma, we compared IL-4 vs IL-13 vs IL-4Rα blockers. RESULTS: Intranasal administration of either IL-4 or IL-13 confers an asthma-like phenotype in mice by inducing immune cell lung infiltration, including eosinophils, increasing cytokine/chemokine expression and mucus production, thus demonstrating redundant functions of these cytokines. We further teased out their respective contributions using human in vitro culture systems. Then, in a mouse asthma model by comparing in head-to-head studies, either IL-4 or IL-13 inhibition to dual IL-4/IL-13 inhibition, we demonstrate that blockade of both IL-4 and IL-13 is required to broadly block type 2 inflammation, which translates to protection from allergen-induced lung function impairment. Notably, only dual IL-4/IL-13 blockade prevented eosinophil infiltration into lung tissue without affecting circulating eosinophils, demonstrating that tissue, but not circulating eosinophils, contributes to disease pathology. CONCLUSIONS: Overall, these data support IL-4 and IL-13 as key drivers of type 2 inflammation and help provide insight into the therapeutic mechanism of dupilumab, a dual IL-4/IL-13 blocker, in multiple type 2 diseases.
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Interleucina-13 , Animales , Anticuerpos Monoclonales Humanizados , Inflamación , Interleucina-4 , RatonesRESUMEN
Background: For most classes of drugs, rapid development of therapeutics to treat emerging infections is challenged by the timelines needed to identify compounds with the desired efficacy, safety, and pharmacokinetic profiles. Fully human monoclonal antibodies (mAbs) provide an attractive method to overcome many of these hurdles to rapidly produce therapeutics for emerging diseases. Methods: In this study, we deployed a platform to generate, test, and develop fully human antibodies to Zaire ebolavirus. We obtained specific anti-Ebola virus (EBOV) antibodies by immunizing VelocImmune mice that use human immunoglobulin variable regions in their humoral responses. Results: Of the antibody clones isolated, 3 were selected as best at neutralizing EBOV and triggering FcγRIIIa. Binding studies and negative-stain electron microscopy revealed that the 3 selected antibodies bind to non-overlapping epitopes, including a potentially new protective epitope not targeted by other antibody-based treatments. When combined, a single dose of a cocktail of the 3 antibodies protected nonhuman primates (NHPs) from EBOV disease even after disease symptoms were apparent. Conclusions: This antibody cocktail provides complementary mechanisms of actions, incorporates novel specificities, and demonstrates high-level postexposure protection from lethal EBOV disease in NHPs. It is now undergoing testing in normal healthy volunteers in preparation for potential use in future Ebola epidemics.
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Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Neutralizantes/uso terapéutico , Anticuerpos Antivirales/uso terapéutico , Fiebre Hemorrágica Ebola/tratamiento farmacológico , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Glicoproteínas/inmunología , Cobayas , Células HEK293 , Humanos , Macaca mulatta , Masculino , RatonesRESUMEN
Anti-vascular endothelial growth factor (VEGF) therapies have improved clinical outcomes for patients with cancers and retinal vascular diseases. Three anti-VEGF agents, pegaptanib, ranibizumab, and aflibercept, are approved for ophthalmic indications, while bevacizumab is approved to treat colorectal, lung, and renal cancers, but is also used off-label to treat ocular vascular diseases. The efficacy of bevacizumab relative to ranibizumab in treating neovascular age-related macular degeneration has been assessed in several trials. However, questions persist regarding its safety, as bevacizumab can form large complexes with dimeric VEGF165, resulting in multimerization of the Fc domain and platelet activation. Here, we compare binding stoichiometry, Fcγ receptor affinity, platelet activation, and binding to epithelial and endothelial cells in vitro for bevacizumab and aflibercept, in the absence or presence of VEGF. In contrast to bevacizumab, aflibercept forms a homogenous 1:1 complex with each VEGF dimer. Unlike multimeric bevacizumab:VEGF complexes, the monomeric aflibercept:VEGF complex does not exhibit increased affinity for low-affinity Fcγ receptors, does not activate platelets, nor does it bind to the surface of epithelial or endothelial cells to a greater degree than unbound aflibercept or control Fc. The latter finding reflects the fact that aflibercept binds VEGF in a unique manner, distinct from antibodies not only blocking the amino acids necessary for VEGFR1/R2 binding but also occluding the heparin-binding site on VEGF165.
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Bevacizumab/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/metabolismo , Inhibidores de la Angiogénesis/efectos adversos , Inhibidores de la Angiogénesis/metabolismo , Inhibidores de la Angiogénesis/uso terapéutico , Animales , Complejo Antígeno-Anticuerpo/química , Complejo Antígeno-Anticuerpo/metabolismo , Bevacizumab/efectos adversos , Bevacizumab/uso terapéutico , Línea Celular , Células Endoteliales de la Vena Umbilical Humana , Humanos , Técnicas In Vitro , Degeneración Macular/inmunología , Degeneración Macular/metabolismo , Degeneración Macular/terapia , Ratones , Ratones Transgénicos , Activación Plaquetaria , Unión Proteica , Multimerización de Proteína , Receptores de IgG/genética , Receptores de IgG/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular/uso terapéutico , Proteínas Recombinantes de Fusión/efectos adversos , Proteínas Recombinantes de Fusión/uso terapéutico , Trombocitopenia/etiología , Trombosis/etiología , Factor A de Crecimiento Endotelial Vascular/inmunologíaRESUMEN
A cell-based assay was developed to detect neutralizing anti-drug antibodies (NAbs) against odronextamab, a CD20xCD3 bispecific monoclonal antibody (mAb) under investigation for treatment of CD20+ B cell malignancies. In this assay, odronextamab bridges between two cell types, CD20-expressing HEK293 cells and CD3-expressing Jurkat T cells that generate a luciferase signal upon CD3 clustering. Patient samples containing NAbs directed to either arm of the bispecific drug block the odronextamab bridge formation between the cell lines thus preventing the generation of the luciferase signal. We determined that other anti-CD20 therapeutics also block bridge formation, resulting in false-positive results. In patient samples from odronextamab clinical trials, approximately 30% of baseline samples had a strong false-positive NAb signal that correlated with the presence of prior rituximab (anti-CD20) therapy. We determined that rituximab interference can be minimized by the addition of anti-rituximab antibodies in the NAb assay. Understanding and mitigating the impact of prior biologic exposure is increasingly important for implementing a successful bioanalytical strategy to support clinical drug development, especially in the immuno-oncology field. Odronextamab neutralizing antibody assay, interference, and mitigation. A Design of the odronextamab neutralizing antibody (NAb) assay where anti-CD20xCD3 drug bridges between CD20-expressing HEK293 cells and Jurkat T cells expressing an NFAT response element and luciferase reporter. True NAb prevents odronextamab from bridging between target and effector cells, thus preventing the expression of luciferase. B Interference with odronextamab from other anti-CD20 therapeutic antibodies (e.g., rituximab) from prior disease treatment generates a false-positive NAb result. Assay interference can be mitigated with an anti-idiotypic antibody against the interfering therapy.
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Anticuerpos Biespecíficos , Antineoplásicos , Anticuerpos Biespecíficos/farmacología , Anticuerpos Biespecíficos/uso terapéutico , Anticuerpos Monoclonales , Anticuerpos Neutralizantes , Antígenos CD20 , Células HEK293 , Humanos , RituximabRESUMEN
Although many patients with diffuse large B cell lymphoma (DLBCL) may achieve a complete response to frontline chemoimmunotherapy, patients with relapsed/refractory disease typically have poor outcomes. Odronextamab, a CD20xCD3 bispecific antibody that provides "signal 1" through the activation of the T cell receptor/CD3 complex, has exhibited early, promising activity for patients with highly refractory DLBCL in phase 1 trials. However, not all patients achieve complete responses, and many relapse, thus representing a high unmet medical need. Here, we investigated whether adding a costimulatory "signal 2" by engaging CD28 receptors on T cells could augment odronextamab activity. We demonstrate that REGN5837, a bispecific antibody that cross-links CD22-expressing tumor cells with CD28-expressing T cells, enhances odronextamab by potentiating T cell activation and cytolytic function. In preclinical DLBCL studies using human immune system-reconstituted animals, REGN5837 promotes the antitumor activity of odronextamab and induces intratumoral expansion of reprogrammable T cells while skewing away from a dysfunctional state. Although REGN5837 monotherapy shows limited activity and no toxicity in primate studies, it augments T cell activation when dosed in combination with odronextamab. In addition, analysis of non-Hodgkin lymphoma clinical samples reveals an increase in CD28+CD8+ T cells after odronextamab treatment, demonstrating the presence of a population that could potentially be targeted by REGN5837. Collectively, our data demonstrate that REGN5837 can markedly enhance the antitumor activity of odronextamab in preclinical NHL models, and the combination of these two bispecific antibodies may provide a chemotherapy-free approach for the treatment of DLBCL.
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Anticuerpos Biespecíficos , Antineoplásicos , Linfoma de Células B Grandes Difuso , Linfoma no Hodgkin , Animales , Humanos , Anticuerpos Biespecíficos/farmacología , Anticuerpos Biespecíficos/uso terapéutico , Antígenos CD28 , Linfocitos T CD8-positivos , Antígenos CD19 , Recurrencia Local de Neoplasia/tratamiento farmacológico , Linfoma no Hodgkin/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Antineoplásicos/farmacología , Lectina 2 Similar a Ig de Unión al Ácido Siálico/uso terapéuticoRESUMEN
Assessment of immune-cell subsets within the tumor immune microenvironment is a powerful approach to better understand cancer immunotherapy responses. However, the use of biopsies to assess the tumor immune microenvironment poses challenges, including the potential for sampling error, restricted sampling over time, and inaccessibility of some tissues/organs, as well as the fact that single biopsy analyses do not reflect discordance across multiple intrapatient tumor lesions. Immuno-positron emission tomography (PET) presents a promising translational imaging approach to address the limitations and assess changes in the tumor microenvironment. We have developed 89Zr-DFO-REGN5054, a fully human CD8A-specific antibody conjugate, to assess CD8+ tumor-infiltrating lymphocytes (TIL) pre- and posttherapy. We used multiple assays, including in vitro T-cell activation, proliferation, and cytokine production, and in vivo viral clearance and CD8 receptor occupancy, to demonstrate that REGN5054 has minimal impact on T-cell activity. Preclinical immuno-PET studies demonstrated that 89Zr-DFO-REGN5054 specifically detected CD8+ T cells in lymphoid tissues of CD8-genetically humanized immunocompetent mice (VelociT mice) and discerned therapy-induced changes in CD8+ TILs in two models of response to a CD20xCD3 T-cell activating bispecific antibody (REGN1979, odronextamab). Toxicology studies in cynomolgus monkeys showed no overt toxicity, and immuno-PET imaging in cynomolgus monkeys demonstrated dose-dependent clearance and specific targeting to lymphoid tissues. This work supports the clinical investigation of 89Zr-DFO-REGN5054 to monitor T-cell responses in patients undergoing cancer immunotherapy.
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Anticuerpos Biespecíficos , Neoplasias , Animales , Linfocitos T CD8-positivos , Citocinas/uso terapéutico , Humanos , Linfocitos Infiltrantes de Tumor , Macaca fascicularis , Ratones , Tomografía de Emisión de Positrones/métodos , Radioisótopos , Microambiente Tumoral , CirconioRESUMEN
We evaluated interleukin-6 (IL-6) receptor-α subunit (IL-6Rα) signaling inhibition with sarilumab and tocilizumab, the association between IL-6Rα receptor occupancy (RO) and C-reactive protein (CRP), and the potential clinical relevance of any differences. For this, we measured IL-6Rα binding and signaling inhibition with sarilumab and tocilizumab in vitro, simulated soluble IL-6Rα RO over time for approved sarilumab subcutaneous (SC) and tocilizumab intravenous (IV) and SC doses, and assessed associations between calculated RO and CRP reduction, 28-joint Disease Activity Score based on CRP, and 20%/50%/70% improvement in American College of Rheumatology responses from clinical data. Sarilumab binds IL-6Rα in vitro with 15- to 22-fold higher affinity than tocilizumab, and inhibits IL-6-mediated classical and trans signaling via membrane-bound and soluble IL-6Rα. Sarilumab 200 and 150 mg SC every 2 weeks achieved >90% RO after first and second doses, respectively, maintained throughout the treatment period. At steady-state trough, RO was greater with sarilumab 200 mg (98%) and 150 mg SC every 2 weeks (94%), and tocilizumab 162 mg SC weekly (>99%) and 8 mg/kg IV every 4 weeks (99%), vs tocilizumab 162 mg SC every 2 weeks (84%) and 4 mg/kg IV every 4 weeks (60%). Higher RO was associated with greater CRP reduction and 28-joint Disease Activity Score based on CRP reduction, and more sarilumab patients achieving 20%/50%/70% improvement in American College of Rheumatology responses. The greatest reduction in CRP levels was observed with sarilumab (both doses) and tocilizumab 8 mg/kg IV every 4 weeks (reductions proportionally smaller with 4 mg/kg IV every 4 weeks). Higher IL-6Rα binding affinity translated into higher RO with sarilumab vs tocilizumab 4 mg/kg every 4 weeks or 162 mg every 2 weeks; tocilizumab required the higher dose or increased frequency to maintain the same degree of RO and CRP reduction. Higher RO was associated with clinical parameter improvements.
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Anticuerpos Monoclonales Humanizados/farmacología , Antirreumáticos/farmacología , Proteína C-Reactiva/efectos de los fármacos , Receptores de Interleucina-6/antagonistas & inhibidores , Anticuerpos Monoclonales Humanizados/farmacocinética , Antirreumáticos/farmacocinética , Humanos , Modelos Biológicos , Unión ProteicaRESUMEN
PURPOSE: Recent clinical data demonstrate that tumors harboring MET genetic alterations (exon 14 skip mutations and/or gene amplification) respond to small-molecule tyrosine kinase inhibitors, validating MET as a therapeutic target. Although antibody-mediated blockade of the MET pathway has not been successful in the clinic, the failures are likely the result of inadequate patient selection strategies as well as suboptimal antibody design. Thus, our goal was to generate a novel MET blocking antibody with enhanced efficacy. EXPERIMENTAL DESIGN: Here, we describe the activity of a biparatopic MET×MET antibody that recognizes two distinct epitopes in the MET Sema domain. We use a combination of in vitro assays and tumor models to characterize the effect of our antibody on MET signaling, MET intracellular trafficking, and the growth of MET-dependent cells/tumors. RESULTS: In MET-driven tumor models, our biparatopic antibody exhibits significantly better activity than either of the parental antibodies or the mixture of the two parental antibodies and outperforms several clinical-stage MET antibodies. Mechanistically, the biparatopic antibody inhibits MET recycling, thereby promoting lysosomal trafficking and degradation of MET. In contrast to the parental antibodies, the biparatopic antibody fails to activate MET-dependent biological responses, consistent with the observation that it recycles inefficiently and induces very transient downstream signaling. CONCLUSIONS: Our results provide strong support for the notion that biparatopic antibodies are a promising therapeutic modality, potentially having greater efficacy than that predicted from the properties of the parental antibodies.
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Anticuerpos Monoclonales/farmacología , Epítopos/inmunología , Amplificación de Genes , Neoplasias/terapia , Proteínas Proto-Oncogénicas c-met/metabolismo , Animales , Línea Celular Tumoral , Epítopos/genética , Humanos , Ratones , Ratones SCID , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/patología , Transporte de Proteínas , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-met/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
T cell activation is initiated upon binding of the T cell receptor (TCR)/CD3 complex to peptide-major histocompatibility complexes ("signal 1"); activation is enhanced by engagement of a second "costimulatory" receptor, such as the CD28 receptor on T cells binding to its cognate ligand(s) on the target cell ("signal 2"). CD3-based bispecific antibodies act by replacing conventional signal 1, linking T cells to tumor cells by binding a tumor-specific antigen (TSA) with one arm of the bispecific and bridging to TCR/CD3 with the other. Although some of these so-called TSAxCD3 bispecifics have demonstrated promising antitumor efficacy in patients with cancer, their activity remains to be optimized. Here, we introduce a class of bispecific antibodies that mimic signal 2 by bridging TSA to the costimulatory CD28 receptor on T cells. We term these TSAxCD28 bispecifics and describe two such bispecific antibodies: one specific for ovarian and the other for prostate cancer antigens. Unlike CD28 superagonists, which broadly activate T cells and resulted in profound toxicity in early clinical trials, these TSAxCD28 bispecifics show limited activity and no toxicity when used alone in genetically humanized immunocompetent mouse models or in primates. However, when combined with TSAxCD3 bispecifics, they enhance the artificial synapse between a T cell and its target cell, potentiate T cell activation, and markedly improve antitumor activity of CD3 bispecifics in a variety of xenogeneic and syngeneic tumor models. Combining this class of CD28-costimulatory bispecific antibodies with the emerging class of TSAxCD3 bispecifics may provide well-tolerated, off-the-shelf antibody therapies with robust antitumor efficacy.
Asunto(s)
Anticuerpos Biespecíficos/inmunología , Antígenos CD28/inmunología , Complejo CD3/inmunología , Neoplasias/inmunología , Animales , Antígenos de Neoplasias/inmunología , Línea Celular Tumoral , Proliferación Celular , Citocinas/metabolismo , Citotoxicidad Inmunológica , Femenino , Células HEK293 , Humanos , Sinapsis Inmunológicas/metabolismo , Activación de Linfocitos/inmunología , Macaca fascicularis , Ratones , Neoplasias/patología , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
In the tumor microenvironment, multiple inhibitory checkpoint receptors can suppress T-cell function, thereby enabling tumor immune evasion. Blockade of one of these checkpoint receptors, PD-1, with therapeutic antibodies has produced positive clinical responses in various cancers; however, the efficacy of this approach can be further improved. Simultaneously targeting multiple inhibitory checkpoint receptors has emerged as a promising therapeutic strategy. Here, we report the development and characterization of REGN3767, a fully human IgG4 antibody targeting LAG-3, another inhibitory receptor on T cells. REGN3767 binds human and monkey LAG-3 with high affinity and specificity and blocks the interaction of LAG-3 with its ligand, MHC class II. In an engineered T-cell/antigen-presenting cell bioassay, REGN3767 alone, or in combination with cemiplimab (REGN2810, human anti-PD-1 antibody), blocked inhibitory signaling to T cells mediated by hLAG-3/MHCII in the presence of PD-1/PD-L1. To test the in vivo activity of REGN3767 alone or in combination with cemiplimab, we generated human PD-1xLAG-3 knockin mice, in which the extracellular domains of mouse Pdcd1 and Lag3 were replaced with their human counterparts. In these humanized mice, treatment with cemiplimab and REGN3767 showed increased efficacy in a mouse tumor model and enhanced the secretion of proinflammatory cytokines by tumor-specific T cells. The favorable pharmacokinetics and toxicology of REGN3767 in nonhuman primates, together with enhancement of antitumor efficacy of anti-PD-1 antibody in preclinical tumor models, support its clinical development.
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Anticuerpos Monoclonales Humanizados/administración & dosificación , Antígenos CD/química , Antígenos CD/genética , Antineoplásicos Inmunológicos/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Neoplasias/tratamiento farmacológico , Receptor de Muerte Celular Programada 1/genética , Animales , Anticuerpos Monoclonales Humanizados/farmacología , Antígenos CD/metabolismo , Antineoplásicos Inmunológicos/química , Antineoplásicos Inmunológicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Técnicas de Sustitución del Gen , Haplorrinos , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Ratones , Neoplasias/genética , Unión Proteica/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Microambiente Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , Proteína del Gen 3 de Activación de LinfocitosRESUMEN
Harnessing complement-mediated cytotoxicity by therapeutic antibodies has been limited because of dependency on size and density of antigen, structural constraints resulting from orientation of antibody binding, and blockade of complement activation by inhibitors expressed on target cells. We developed a modular bispecific antibody platform that directs the complement-initiating protein C1q to target cells, increases local complement deposition and induces cytotoxicity against target antigens with a wide-range of expression. The broad utility of this approach to eliminate both prokaryotic and eukaryotic cells was demonstrated by pairing a unique C1q-recruiting arm with multiple targeting arms specific for Staphylococcus aureus, Pseudomonas aeruginosa, B-cells and T-cells, indicating applicability for diverse indications ranging from infectious diseases to cancer. Generation of C1q humanized mice allowed for demonstration of the efficacy of this approach to clear disease-inducing cells in vivo. In summary, we present a novel, broadly applicable, and versatile therapeutic modality for targeted cell depletion.
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Anticuerpos Biespecíficos/inmunología , Proteínas del Sistema Complemento/inmunología , Citotoxicidad Inmunológica , Animales , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Activación de Complemento , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Transgénicos , Unión Proteica , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/metabolismo , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/inmunologíaRESUMEN
Advanced ovarian cancer is frequently treated with combination chemotherapy, but high recurrence rates show the need for therapies that can produce durable responses and extend overall survival. Bispecific antibodies that interact with tumor antigens on cancer cells and activating receptors on immune cells offer an innovative immunotherapy approach. Here, we describe a human bispecific antibody (REGN4018) that binds both Mucin 16 (MUC16), a glycoprotein that is highly expressed on ovarian cancer cells, and CD3, thus bridging MUC16-expressing cells with CD3+ T cells. REGN4018 induced T cell activation and killing of MUC16-expressing tumor cells in vitro. Binding and cytotoxicity of REGN4018 in vitro were minimally affected by high concentrations of CA-125, the shed form of MUC16, which is present in patients. In preclinical studies with human ovarian cancer cells and human T cells in immunodeficient mice, REGN4018 potently inhibited growth of intraperitoneal ovarian tumors. Moreover, in a genetically engineered immunocompetent mouse expressing human CD3 and human MUC16 [humanized target (HuT) mice], REGN4018 inhibited growth of murine tumors expressing human MUC16, and combination with an anti-PD-1 antibody enhanced this efficacy. Immuno-PET imaging demonstrated localization of REGN4018 in MUC16-expressing tumors and in T cell-rich organs such as the spleen and lymph nodes. Toxicology studies in cynomolgus monkeys showed minimal and transient increases in serum cytokines and C-reactive protein after REGN4018 administration, with no overt toxicity. Collectively, these data demonstrate potent antitumor activity and good tolerability of REGN4018, supporting clinical evaluation of REGN4018 in patients with MUC16-expressing advanced ovarian cancer.
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Anticuerpos Biespecíficos/inmunología , Anticuerpos Biespecíficos/uso terapéutico , Antígeno Ca-125/inmunología , Antígeno Ca-125/metabolismo , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/inmunología , Linfocitos T/metabolismo , Animales , Antígenos CD13/inmunología , Antígenos CD13/metabolismo , Femenino , Citometría de Flujo , Humanos , Inmunoglobulina G/inmunología , Inmunoglobulina G/metabolismo , Células Jurkat , Macaca fascicularis , Ratones , Neoplasias Ováricas/metabolismo , Linfocitos T/inmunologíaRESUMEN
Previously, it was shown that cationic alpha-peptides derived from the human immunodeficiency virus TAT protein transduction domain blocked herpes simplex virus type 1 (HSV-1) entry. We now show that cationic oligomers of beta-amino acids ("beta-peptides") inhibit HSV-1 infection. Among three cationic beta-peptides tested, the most effective inhibition was observed for the one with a strong propensity to adopt a helical conformation in which cationic and hydrophobic residues are segregated from one another ("globally amphiphilic helix"). The antiviral effect was not cell type specific. Inhibition of virus infection by the beta-peptides occurred at the postattachment penetration step, with a 50% effective concentration of 3 muM for the most-effective beta-peptide. The beta-peptides did not inactivate virions in solution, nor did they induce resistance to infection when cells were pretreated with the beta-peptides. The beta-peptides showed little if any toxicity toward Vero cells. These results raise the possibility that cationic beta-peptides may be useful antiviral agents for HSV-1 and demonstrate the potential of beta-peptides as novel antiviral drugs.
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Antivirales/farmacología , Herpesvirus Humano 1/efectos de los fármacos , Péptidos/farmacología , Animales , Antivirales/síntesis química , Antivirales/química , Cationes/química , Células Cultivadas , Chlorocebus aethiops , Córnea/citología , Córnea/virología , Fibroblastos/virología , Herpesvirus Humano 1/patogenicidad , Humanos , Pruebas de Sensibilidad Microbiana/métodos , Péptidos/síntesis química , Péptidos/química , Conformación Proteica , Células VeroRESUMEN
Oxidative stress is a deleterious force that must be combated relentlessly by aerobic organisms and is known to underlie many human diseases including atherosclerosis, Parkinson's disease, and Alzheimer's disease. Information available about the oxidative stress response has come primarily from studies using reactive oxygen species (ROS) with ill-defined locations within the cell. Thus, existing models do not account for possible differences between stress originating within particular regions of the cell. Here, oxidative stress is studied at the subcellular level using ROS-generating compounds localizing within two different organelles: the nucleus and the mitochondrion. Differences in cytotoxicity, gene expression, and survival pathway activation are detected as a function of the subcellular origin of oxidative stress, indicating that independent mechanisms are used to cope with oxidative stress arising in different cellular compartments. These comparative studies, enabled by the development of organelle-specific oxidants, examine the cellular responses to site-specific oxidative stress with heightened precision.
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Orgánulos , Estrés Oxidativo , Péptidos/metabolismo , Línea Celular , Citometría de Flujo , Humanos , Reacción en Cadena de la Polimerasa , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The Prolactin Receptor (PRLR) is a type 1 cytokine receptor that is expressed in a subset of breast cancers and may contribute to its pathogenesis. It is relatively overexpressed in approximately 25% of human breast tumors while expressed at low levels in some normal human tissues including the mammary gland. We developed an anti-PRLR antibody-drug conjugate (ADC), to target PRLR-positive breast cancer. REGN2878-DM1 is comprised of a fully human high-affinity function-blocking anti-PRLR IgG1 antibody (REGN2878) conjugated via a noncleavable SMCC linker to the cytotoxic maytansine derivative DM1. Both unconjugated REGN2878 and conjugated REGN2878-DM1 block PRL-mediated activation in vitro and are rapidly internalized into lysosomes. REGN2878-DM1 induces potent cell-cycle arrest and cytotoxicity in PRLR-expressing tumor cell lines. In vivo, REGN2878-DM1 demonstrated significant antigen-specific antitumor activity against breast cancer xenograft models. In addition, REGN2878-DM1 showed additive activity when combined with the antiestrogen agent fulvestrant. These results illustrate promising antitumor activity against PRLR-positive breast cancer xenografts and support the evaluation of anti-PRLR ADCs as potential therapeutic agents in breast cancer. Mol Cancer Ther; 16(7); 1299-311. ©2017 AACR.
Asunto(s)
Anticuerpos Monoclonales Humanizados/administración & dosificación , Neoplasias de la Mama/tratamiento farmacológico , Inmunoconjugados/administración & dosificación , Receptores de Prolactina/inmunología , Animales , Anticuerpos Antiidiotipos/administración & dosificación , Anticuerpos Antiidiotipos/inmunología , Anticuerpos Monoclonales Humanizados/inmunología , Neoplasias de la Mama/genética , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , Inmunoconjugados/inmunología , Ratones , Receptores de Prolactina/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The Programmed Death-1 (PD-1) receptor delivers inhibitory checkpoint signals to activated T cells upon binding to its ligands PD-L1 and PD-L2 expressed on antigen-presenting cells and cancer cells, resulting in suppression of T-cell effector function and tumor immune evasion. Clinical antibodies blocking the interaction between PD-1 and PD-L1 restore the cytotoxic function of tumor antigen-specific T cells, yielding durable objective responses in multiple cancers. This report describes the preclinical characterization of REGN2810, a fully human hinge-stabilized IgG4(S228P) high-affinity anti-PD-1 antibody that potently blocks PD-1 interactions with PD-L1 and PD-L2. REGN2810 was characterized in a series of binding, blocking, and functional cell-based assays, and preclinical in vivo studies in mice and monkeys. In cell-based assays, REGN2810 reverses PD-1-dependent attenuation of T-cell receptor signaling in engineered T cells and enhances responses of human primary T cells. To test the in vivo activity of REGN2810, which does not cross-react with murine PD-1, knock-in mice were generated to express a hybrid protein containing the extracellular domain of human PD-1, and transmembrane and intracellular domains of mouse PD-1. In these mice, REGN2810 binds the humanized PD-1 receptor and inhibits growth of MC38 murine tumors. As REGN2810 binds to cynomolgus monkey PD-1 with high affinity, pharmacokinetic and toxicologic assessment of REGN2810 was performed in cynomolgus monkeys. High doses of REGN2810 were well tolerated, without adverse immune-related effects. These preclinical studies validate REGN2810 as a potent and promising candidate for cancer immunotherapy. Mol Cancer Ther; 16(5); 861-70. ©2017 AACR.
Asunto(s)
Anticuerpos Monoclonales Humanizados/administración & dosificación , Proliferación Celular/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Receptor de Muerte Celular Programada 1/genética , Animales , Anticuerpos Monoclonales Humanizados/inmunología , Línea Celular Tumoral , Técnicas de Sustitución del Gen , Humanos , Inmunoterapia , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Ratones , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/patología , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/inmunología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunologíaRESUMEN
Bacteriorhodopsin (bR) is solubilized efficiently as a monomer by a novel surfactant, a tripod amphiphile (TPA), which permits the formation of purple hexagonal bR crystals under several conditions. The crystals, although small, diffract to 2.5 A resolution using synchrotron radiation. TPA may be useful for the solubilization, purification, and crystallization of other membrane proteins.
Asunto(s)
Bacteriorodopsinas/química , Halobacterium salinarum/química , Tensoactivos/química , Bacteriorodopsinas/aislamiento & purificación , Cristalización , Cristalografía por Rayos X , Membrana Púrpura/químicaRESUMEN
BACKGROUND: Loss of skeletal muscle mass and function in humans is associated with significant morbidity and mortality. The role of myostatin as a key negative regulator of skeletal muscle mass and function has supported the concept that inactivation of myostatin could be a useful approach for treating muscle wasting diseases. METHODS: We generated a myostatin monoclonal blocking antibody (REGN1033) and characterized its effects in vitro using surface plasmon resonance biacore and cell-based Smad2/3 signaling assays. REGN1033 was tested in mice for the ability to induce skeletal muscle hypertrophy and prevent atrophy induced by immobilization, hindlimb suspension, or dexamethasone. The effect of REGN1033 on exercise training was tested in aged mice. Messenger RNA sequencing, immunohistochemistry, and ex vivo force measurements were performed on skeletal muscle samples from REGN1033-treated mice. RESULTS: The human monoclonal antibody REGN1033 is a specific and potent myostatin antagonist. Chronic treatment of mice with REGN1033 increased muscle fiber size, muscle mass, and force production. REGN1033 prevented the loss of muscle mass induced by immobilization, glucocorticoid treatment, or hindlimb unweighting and increased the gain of muscle mass during recovery from pre-existing atrophy. In aged mice, REGN1033 increased muscle mass and strength and improved physical performance during treadmill exercise. CONCLUSIONS: We show that specific myostatin antagonism with the human antibody REGN1033 enhanced muscle mass and function in young and aged mice and had beneficial effects in models of skeletal muscle atrophy.
RESUMEN
EGFR blocking antibodies are approved for the treatment of colorectal cancer and head and neck squamous cell carcinoma (HNSCC). Although ERBB3 signaling has been proposed to limit the effectiveness of EGFR inhibitors, the underlying molecular mechanisms are not fully understood. To gain insight into these mechanisms, we generated potent blocking antibodies against ERBB3 (REGN1400) and EGFR (REGN955). We show that EGFR and ERBB3 are coactivated in multiple HNSCC cell lines and that combined blockade of EGFR and ERBB3 inhibits growth of these cell lines more effectively than blockade of either receptor alone. Blockade of EGFR with REGN955 strongly inhibited activation of ERK in HNSCC cell lines, whereas blockade of ERBB3 with REGN1400 strongly inhibited activation of Akt; only the combination of the 2 antibodies blocked both of these essential downstream pathways. We used a HER2 blocking antibody to show that ERBB3 phosphorylation in HNSCC and colorectal cancer cells is strictly dependent on association with HER2, but not EGFR, and that neuregulin 1 activates ERBB3/HER2 signaling to reverse the effect of EGFR blockade on colorectal cancer cell growth. Finally, although REGN1400 and REGN955 as single agents slowed the growth of HNSCC and colorectal cancer xenografts, the combination of REGN1400 plus REGN955 caused significant tumor regression. Our results indicate that activation of the Akt survival pathway by ERBB3/HER2 limits the effectiveness of EGFR inhibition, suggesting that REGN1400, which is currently in a phase I clinical trial, could provide benefit when combined with EGFR blocking antibodies.