RESUMEN
INTRODUCTION: ABP 938 is being developed as a biosimilar candidate to aflibercept reference product (RP), a biologic used for certain angiogenic eye disorders. This study was designed to provide a comparative analytical assessment of the structural and functional attributes of ABP 938 and aflibercept RP sourced from the United States (US) and the European Union (EU). METHODS: Structural and functional characterization studies were performed using state-of-the-art analytical techniques that were appropriate to assess relevant quality attributes and capable of detecting qualitative and quantitative differences in primary structure, higher-order structure and biophysical properties, product-related substances and impurities, general properties, and biological activities. RESULTS: ABP 938 had the same amino acid sequence and exhibited similar secondary and tertiary structures, and biological activity as aflibercept RP. There were minor differences in a small number of biochemical attributes which are not expected to impact clinical performance. In addition, aflibercept RP sourced from the US and EU were analytically similar. CONCLUSIONS: ABP 938 was structurally and functionally similar to aflibercept RP. Since aflibercept RP sourced from the US and EU were analytically similar, this allows for the development of a scientific bridge such that a single-source RP can be used in nonclinical and clinical studies.
Eylea® (aflibercept) is a biologic medication approved for the treatment of patients with certain eye diseases that can result in low vision or blindness. Biosimilars are biologic medications that are highly similar to an existing approved biologic medication, often called a reference product. Biosimilars have the potential to reduce medication costs despite having no clinically significant differences in quality, efficacy, and safety from their reference products. ABP 938 is currently being developed as a biosimilar to aflibercept reference product. We have conducted similarity studies to compare multiple batches of ABP 938 and aflibercept reference product sourced from both the United States and the European Union, using state-of-the-art analytical methods. The results demonstrated that ABP 938 had the same amino acid sequence and similar structural and biological activities as aflibercept reference product. Before biosimilars can be used as medicines, studies such as this one are required by the Food and Drug Administration and other regulatory authorities to ensure that biosimilars are as safe and effective as their reference products.
RESUMEN
The binding affinity of monoclonal antibodies (mAbs) for their intended therapeutic targets is often affected by chemical and post-translational modifications in the antigen binding (Fab) domains. A new two-dimensional analytical approach is described here utilizing native size exclusion chromatography (SEC) to separate populations of antibodies and bound antibody-antigen complexes for subsequent characterization of these modifications by reversed-phase (RP) liquid chromatography-mass spectrometry (LC-MS) at the intact antibody level. Previously, we utilized peptide mapping to measure modifications impacting binding. However, in this study, the large size of the modification (N-glycosylation) allowed assessing its impact from small amounts (â¼20 ug) of intact antibody, without the need for peptide mapping. Here, we apply the native SEC-based competitive binding assay to quickly and qualitatively investigate the effects of Fab glycosylation of four antispike protein mAbs that were developed for use in the treatment of COVID-19 disease. Three of the mAbs were observed to have consensus N-glycosylation sites (N-X-T/S) in the Fab domains, a relatively rare occurrence in therapeutic mAbs. The goal of the study was to characterize the levels of Fab glycosylation present, as well as determine the impact of glycosylation on binding to the spike protein receptor binding domain (RBD) and the ability of the mAbs to inhibit RBD-ACE2 interaction at the intact antibody level, with minimal sample treatment and preparation. The three mAbs with Fab N-glycans were found to have glycosylation profiles ranging from full occupancy at each Fab (in one mAb) to partially glycosylated with mixed populations of two, one, or no glycan moieties. Competitive SEC analysis of mAb-RBD revealed that the glycosylated antibody populations outcompete their nonglycosylated counterparts for the available RBD molecules. This competitive SEC binding analysis was applied to investigate the three-body interaction of a glycosylated mAb blocking the interaction between endogenous binding partners RBD-ACE2, finding that both glycosylated and nonglycosylated mAb populations bound to RBD with high enough affinity to block RBD-ACE2 binding.
Asunto(s)
COVID-19 , Humanos , Glicosilación , Cromatografía Liquida , Enzima Convertidora de Angiotensina 2/metabolismo , Espectrometría de Masas en Tándem , Anticuerpos Antivirales , Unión Proteica , Cromatografía en GelRESUMEN
Therapeutic antibody development requires discovery of an antibody molecule with desired specificities and drug-like properties. For toxicological studies, a therapeutic antibody must bind the ortholog antigen with a similar affinity to the human target to enable relevant dosing regimens, and antibodies falling short of this affinity design goal may not progress as therapeutic leads. Herein, we report the novel use of mammalian recombination signal sequence (RSS)-directed recombination for complementarity-determining region-targeted protein engineering combined with mammalian display to close the species affinity gap of human interleukin (IL)-13 antibody 731. This fully human antibody has not progressed as a therapeutic in part because of a 400-fold species affinity gap. Using this nonhypothesis-driven affinity maturation method, we generated multiple antibody variants with improved IL-13 affinity, including the highest affinity antibody reported to date (34 fM). Resolution of a cocrystal structure of the optimized antibody with the cynomolgus monkey (or nonhuman primate) IL-13 protein revealed that the RSS-derived mutations introduced multiple successive amino-acid substitutions resulting in a de novo formation of a π-π stacking-based protein-protein interaction between the affinity-matured antibody heavy chain and helix C on IL-13, as well as an introduction of an interface-distant residue, which enhanced the light chain-binding affinity to target. These mutations synergized binding of heavy and light chains to the target protein, resulting in a remarkably tight interaction, and providing a proof of concept for a new method of protein engineering, based on synergizing a mammalian display platform with novel RSS-mediated library generation.
Asunto(s)
Anticuerpos , Interleucina-13 , Señales de Clasificación de Proteína , Secuencia de Aminoácidos , Animales , Anticuerpos/genética , Anticuerpos/inmunología , Afinidad de Anticuerpos , Humanos , Interleucina-13/genética , Interleucina-13/inmunología , Macaca fascicularis , Mamíferos , Recombinación GenéticaRESUMEN
Triggering receptor expressed on myeloid cells 2 (TREM2) sustains microglia response to brain injury stimuli including apoptotic cells, myelin damage, and amyloid ß (Aß). Alzheimer's disease (AD) risk is associated with the TREM2R47H variant, which impairs ligand binding and consequently microglia responses to Aß pathology. Here, we show that TREM2 engagement by the mAb hT2AB as surrogate ligand activates microglia in 5XFAD transgenic mice that accumulate Aß and express either the common TREM2 variant (TREM2CV) or TREM2R47H scRNA-seq of microglia from TREM2CV-5XFAD mice treated once with control hIgG1 exposed four distinct trajectories of microglia activation leading to disease-associated (DAM), interferon-responsive (IFN-R), cycling (Cyc-M), and MHC-II expressing (MHC-II) microglia types. All of these were underrepresented in TREM2R47H-5XFAD mice, suggesting that TREM2 ligand engagement is required for microglia activation trajectories. Moreover, Cyc-M and IFN-R microglia were more abundant in female than male TREM2CV-5XFAD mice, likely due to greater Aß load in female 5XFAD mice. A single systemic injection of hT2AB replenished Cyc-M, IFN-R, and MHC-II pools in TREM2R47H-5XFAD mice. In TREM2CV-5XFAD mice, however, hT2AB brought the representation of male Cyc-M and IFN-R microglia closer to that of females, in which these trajectories had already reached maximum capacity. Moreover, hT2AB induced shifts in gene expression patterns in all microglial pools without affecting representation. Repeated treatment with a murinized hT2AB version over 10 d increased chemokines brain content in TREM2R47H-5XFAD mice, consistent with microglia expansion. Thus, the impact of hT2AB on microglia is shaped by the extent of TREM2 endogenous ligand engagement and basal microglia activation.
Asunto(s)
Enfermedad de Alzheimer/genética , Encéfalo/metabolismo , Glicoproteínas de Membrana/genética , Microglía/metabolismo , Receptores Inmunológicos/genética , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/metabolismo , Animales , Anticuerpos Monoclonales/farmacología , Anticuerpos Neutralizantes/farmacología , Encéfalo/efectos de los fármacos , Encéfalo/patología , Proliferación Celular , Quimiocinas/genética , Quimiocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica , Células HEK293 , Humanos , Cinética , Masculino , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Transgénicos , Microglía/clasificación , Microglía/efectos de los fármacos , Microglía/patología , Mutación , Unión Proteica , Receptores Inmunológicos/antagonistas & inhibidores , Receptores Inmunológicos/metabolismo , Factores SexualesRESUMEN
Transthyretin (TTR) is an abundant homotetrameric serum protein and was selected here for engineering higher-valency molecules because of its compact size, simple structure, and natural propensity to tetramerize. To demonstrate this utility, we fused TTR to the C terminus of conatumumab, an antibody that targets tumor necrosis factor-related apoptosis-inducing ligand receptor 2, as heavy chains to form antibody dimers and Fab heavy chains to form Fab tetramers. Moreover, we used constant heavy domain 3 heterodimerization substitutions to create TTR-mediated conatumumab tetramers. The conatumumab-TTR fusions displayed substantially enhanced potency in cell-based assays, as well as in murine tumor xenograft models. We conclude that antibody-TTR fusions may provide a powerful platform for multimerizing antibody and Fab fragments to enhance the capabilities of human therapeutics that benefit from target clustering and higher-order antigen-binding valency.
Asunto(s)
Anticuerpos Monoclonales , Antineoplásicos Inmunológicos , Fragmentos Fab de Inmunoglobulinas , Neoplasias Experimentales , Prealbúmina , Multimerización de Proteína , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/farmacocinética , Anticuerpos Monoclonales/farmacología , Antineoplásicos Inmunológicos/química , Antineoplásicos Inmunológicos/farmacocinética , Antineoplásicos Inmunológicos/farmacología , Línea Celular Tumoral , Humanos , Fragmentos Fab de Inmunoglobulinas/genética , Fragmentos Fab de Inmunoglobulinas/farmacología , Ratones , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Prealbúmina/genética , Prealbúmina/farmacocinética , Prealbúmina/farmacología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/farmacocinética , Proteínas Recombinantes de Fusión/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: The in vitro and in vivo pharmacologic assessment of ABP 980 similarity to its reference product is intended to compare the activity of ABP 980 and trastuzumab and support the overall conclusion of similarity based on a comprehensive analytical and functional evaluation. METHODS: This work complements the primary assessment of functional similarity with additional in vitro assays, binding studies, and non-clinical studies including human epidermal growth factor receptor-2 (HER2) kinetic binding, HER2 signaling, HER2 internalization, synergy with docetaxel chemotherapy, FcγR kinetic binding, primary natural killer and monocyte cell binding, antibody-dependent cellular phagocytosis activity, in vivo xenograft studies, and toxicokinetic parameters. RESULTS: The results contribute to the totality of evidence with respect to functional similarity and support that ABP 980 is similar to trastuzumab in all primary and secondary mechanisms of action. CONCLUSIONS: These results also support the scientific justification of extrapolation to all approved indications of trastuzumab given the established functional similarity of the two products and the same mechanisms of action across all conditions of use.
Asunto(s)
Antineoplásicos/química , Biosimilares Farmacéuticos/química , Trastuzumab/química , Animales , Unión Competitiva , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Femenino , Humanos , Cinética , Ratones Desnudos , Estructura Molecular , Neoplasias Experimentales , Unión Proteica , Receptor ErbB-2/química , Transducción de Señal , Neoplasias Gástricas/tratamiento farmacológico , Relación Estructura-ActividadRESUMEN
Glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) has been identified in multiple genome-wide association studies (GWAS) as a contributor to obesity, and GIPR knockout mice are protected against diet-induced obesity (DIO). On the basis of this genetic evidence, we developed anti-GIPR antagonistic antibodies as a potential therapeutic strategy for the treatment of obesity and observed that a mouse anti-murine GIPR antibody (muGIPR-Ab) protected against body weight gain, improved multiple metabolic parameters, and was associated with reduced food intake and resting respiratory exchange ratio (RER) in DIO mice. We replicated these results in obese nonhuman primates (NHPs) using an anti-human GIPR antibody (hGIPR-Ab) and found that weight loss was more pronounced than in mice. In addition, we observed enhanced weight loss in DIO mice and NHPs when anti-GIPR antibodies were codosed with glucagon-like peptide-1 receptor (GLP-1R) agonists. Mechanistic and crystallographic studies demonstrated that hGIPR-Ab displaced GIP and bound to GIPR using the same conserved hydrophobic residues as GIP. Further, using a conditional knockout mouse model, we excluded the role of GIPR in pancreatic ß-cells in the regulation of body weight and response to GIPR antagonism. In conclusion, these data provide preclinical validation of a therapeutic approach to treat obesity with anti-GIPR antibodies.
Asunto(s)
Receptor del Péptido 1 Similar al Glucagón/agonistas , Obesidad/tratamiento farmacológico , Receptores de la Hormona Gastrointestinal/antagonistas & inhibidores , Adipocitos/metabolismo , Animales , Anticuerpos/farmacología , Anticuerpos/uso terapéutico , Dieta , Quimioterapia Combinada , Conducta Alimentaria , Polipéptido Inhibidor Gástrico/metabolismo , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Péptidos Similares al Glucagón/análogos & derivados , Péptidos Similares al Glucagón/farmacología , Péptidos Similares al Glucagón/uso terapéutico , Humanos , Fragmentos Fc de Inmunoglobulinas/farmacología , Fragmentos Fc de Inmunoglobulinas/uso terapéutico , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Liraglutida/farmacología , Liraglutida/uso terapéutico , Ratones Obesos , Obesidad/patología , Primates , Receptores de la Hormona Gastrointestinal/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Proteínas Recombinantes de Fusión/uso terapéutico , Respiración , Aumento de Peso/efectos de los fármacos , Pérdida de Peso/efectos de los fármacosRESUMEN
Triggering receptor expressed on myeloid cells 2 (TREM2) is an immune receptor expressed on the surface of microglia, macrophages, dendritic cells, and osteoclasts. The R47H TREM2 variant is a significant risk factor for late-onset Alzheimer's disease (AD), and the molecular basis of R47H TREM2 loss of function is an emerging area of TREM2 biology. Here, we report three high-resolution structures of the extracellular ligand-binding domains (ECDs) of R47H TREM2, apo-WT, and phosphatidylserine (PS)-bound WT TREM2 at 1.8, 2.2, and 2.2 Å, respectively. The structures reveal that Arg47 plays a critical role in maintaining the structural features of the complementarity-determining region 2 (CDR2) loop and the putative positive ligand-interacting surface (PLIS), stabilizing conformations capable of ligand interaction. This is exemplified in the PS-bound structure, in which the CDR2 loop and PLIS drive critical interactions with PS via surfaces that are disrupted in the variant. Together with in vitro and in vivo characterization, our structural findings elucidate the molecular mechanism underlying loss of ligand binding, putative oligomerization, and functional activity of R47H TREM2. They also help unravel how decreased in vitro and in vivo stability of TREM2 contribute to loss of function in disease.
Asunto(s)
Enfermedad de Alzheimer/genética , Predisposición Genética a la Enfermedad , Glicoproteínas de Membrana/química , Proteínas Mutantes/química , Receptores Inmunológicos/química , Enfermedad de Alzheimer/patología , Cristalografía por Rayos X , Células Dendríticas/química , Células Dendríticas/patología , Variación Genética , Humanos , Ligandos , Macrófagos/química , Macrófagos/patología , Glicoproteínas de Membrana/genética , Microglía/química , Microglía/patología , Proteínas Mutantes/genética , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Osteoclastos/química , Osteoclastos/patología , Conformación Proteica , Dominios Proteicos/genética , Receptores Inmunológicos/genéticaRESUMEN
Triggering receptor expressed on myeloid cells 2 (TREM2) is an orphan immune receptor expressed on cells of myeloid lineage such as macrophages and microglia. The rare variant R47H TREM2 is associated with an increased risk for Alzheimer's disease, supporting the hypothesis that TREM2 loss of function may exacerbate disease progression. However, a complete knockout of the TREM2 gene in different genetic models of neurodegenerative diseases has been reported to result in both protective and deleterious effects on disease-related end points and myeloid cell function. Here, we describe a Trem2R47H transgenic mouse model and report that even in the absence of additional genetic perturbations, this variant clearly confers a loss of function on myeloid cells. The Trem2R47H variant-containing myeloid cells exhibited subtle defects in survival and migration and displayed an unexpected dysregulation of cytokine responses in a lipopolysaccharide challenge environment. These subtle phenotypic defects with a gradation in severity across genotypes were confirmed in whole-genome RNA-Seq analyses of WT, Trem2-/-, and Trem2R47H myeloid cells under challenge conditions. Of note, TREM2-activating antibodies that boost proximal signaling abrogated survival defects conferred by the variant and also modulated migration and cytokine responses in an antibody-, ligand-, and challenge-dependent manner. In some instances, these antibodies also boosted WT myeloid cell function. Our studies provide a first glimpse into the boost in myeloid cell function that can be achieved by pharmacological modulation of TREM2 activity that can potentially be ameliorative in neurodegenerative diseases such as Alzheimer's disease.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Anticuerpos Monoclonales/farmacología , Apoptosis , Movimiento Celular , Glicoproteínas de Membrana/fisiología , Mutación , Células Mieloides/patología , Receptores Inmunológicos/fisiología , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Animales , Biomarcadores/metabolismo , Encéfalo/inmunología , Encéfalo/metabolismo , Encéfalo/patología , Células Cultivadas , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microglía/inmunología , Microglía/metabolismo , Microglía/patología , Células Mieloides/inmunología , Células Mieloides/metabolismoRESUMEN
Agonism of cell surface receptors by monoclonal antibodies is dependent not only on its ability to bind the target, but also to deliver a biological signal through receptors to the cell. Immunoglobulin G2 antibodies (IgG2s) are made up of a mixture of distinct isoforms (IgG2-A, -B and A/B), which differ by the disulfide connectivity at the hinge region. When evaluating panels of agonistic antibodies against CD200 receptor (CD200R) or ßklotho receptor (ßklotho), we noticed striking activity differences of IgG1 or IgG2 antibodies with the same variable domains. For the CD200R antibody, the IgG2 antibody demonstrated higher activity than the IgG1 or IgG4 antibody. More significantly, for ßklotho, agonist antibodies with higher biological activity as either IgG2 or IgG1 were identified. In both cases, ion exchange chromatography was able to isolate the bioactivity to the IgG2-B isoform from the IgG2 parental mixture. The subclass-related increase in agonist activity was not correlated with antibody aggregation or binding affinity, but was driven by enhanced avidity for the CD200R antibody. These results add to the growing body of evidence that show that conformational differences in the antibody hinge region can have a dramatic impact on the antibody activity and must be considered when screening and engineering therapeutic antibody candidates. The results also demonstrate that the IgG1 (IgG2-A like) or the IgG2-B form may provide the most active form of agonist antibodies for different antibodies and targets.
Asunto(s)
Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Antígenos de Superficie/química , Antígenos de Superficie/inmunología , Proteínas de la Membrana/química , Proteínas de la Membrana/inmunología , Receptores de Superficie Celular/química , Receptores de Superficie Celular/inmunología , Animales , Células CHO , Cricetulus , Disulfuros/química , Disulfuros/inmunología , Mapeo Epitopo/métodos , Proteínas Klotho , Receptores de Orexina , Isoformas de Proteínas/química , Isoformas de Proteínas/inmunología , Relación Estructura-ActividadRESUMEN
Since the late 1990s, the use of transgenic animal platforms has transformed the discovery of fully human therapeutic monoclonal antibodies. The first approved therapy derived from a transgenic platform--the epidermal growth factor receptor antagonist panitumumab to treat advanced colorectal cancer--was developed using XenoMouse(®) technology. Since its approval in 2006, the science of discovering and developing therapeutic monoclonal antibodies derived from the XenoMouse(®) platform has advanced considerably. The emerging array of antibody therapeutics developed using transgenic technologies is expected to include antibodies and antibody fragments with novel mechanisms of action and extreme potencies. In addition to these impressive functional properties, these antibodies will be designed to have superior biophysical properties that enable highly efficient large-scale manufacturing methods. Achieving these new heights in antibody drug discovery will ultimately bring better medicines to patients. Here, we review best practices for the discovery and bio-optimization of monoclonal antibodies that fit functional design goals and meet high manufacturing standards.
Asunto(s)
Anticuerpos Monoclonales Humanizados/biosíntesis , Anticuerpos Monoclonales Humanizados/uso terapéutico , Biotecnología , Descubrimiento de Drogas , Ratones Transgénicos , Animales , Anticuerpos Monoclonales Humanizados/química , Anticuerpos Monoclonales Humanizados/genética , Formación de Anticuerpos , Ingeniería Genética , Humanos , Hibridomas/inmunología , Hibridomas/metabolismo , Isotipos de Inmunoglobulinas/biosíntesis , Isotipos de Inmunoglobulinas/química , Isotipos de Inmunoglobulinas/genética , RatonesRESUMEN
Epidermal growth factor receptor variant III (EGFRvIII) is a cancer-specific deletion mutant observed in approximately 25% to 50% of glioblastoma multiforme (GBM) patients. An antibody drug conjugate, AMG 595, composed of the maytansinoid DM1 attached to a highly selective anti-EGFRvIII antibody via a noncleavable linker, was developed to treat EGFRvIII-positive GBM patients. AMG 595 binds to the cell surface and internalizes into the endo-lysosomal pathway of EGFRvIII-expressing cells. Incubation of AMG 595 with U251 cells expressing EGFRvIII led to potent growth inhibition. AMG 595 treatment induced significant tumor mitotic arrest, as measured by phospho-histone H3, in GBM subcutaneous xenografts expressing EGFRvIII. A single intravenous injection of AMG 595 at 17 mg/kg (250 µg DM1/kg) generated complete tumor regression in the U251vIII subcutaneous xenograft model. AMG 595 mediated tumor regression in the D317 subcutaneous xenograft model that endogenously expresses EGFRvIII. Finally, AMG 595 treatment inhibited the growth of D317 xenografts orthotopically implanted into the brain as determined by magnetic resonance imaging. These results demonstrate that AMG 595 is a promising candidate to evaluate in EGFRvIII-expressing GBM patients.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Receptores ErbB/inmunología , Glioblastoma/tratamiento farmacológico , Inmunoconjugados/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/inmunología , Neoplasias Encefálicas/inmunología , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Receptores ErbB/genética , Receptores ErbB/metabolismo , Femenino , Glioblastoma/inmunología , Glioblastoma/metabolismo , Humanos , Inmunoconjugados/administración & dosificación , Inmunoconjugados/inmunología , Inmunohistoquímica , Inyecciones Intravenosas , Maitansina/análogos & derivados , Maitansina/inmunología , Maitansina/farmacología , Ratones Desnudos , Ratones SCID , Mutación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Resultado del Tratamiento , Carga Tumoral/efectos de los fármacos , Carga Tumoral/inmunologíaRESUMEN
Programmed cell-death 1 ligand 1 (PD-L1) is a member of the B7/CD28 family of proteins that control T-cell activation. Many tumors can upregulate expression of PD-L1, inhibiting antitumor T-cell responses and avoiding immune surveillance and elimination. We have identified and characterized MEDI4736, a human IgG1 monoclonal antibody that binds with high affinity and specificity to PD-L1 and is uniquely engineered to prevent antibody-dependent cell-mediated cytotoxicity. In vitro assays demonstrate that MEDI4736 is a potent antagonist of PD-L1 function, blocking interaction with PD-1 and CD80 to overcome inhibition of primary human T-cell activation. In vivo MEDI4736 significantly inhibits the growth of human tumors in a novel xenograft model containing coimplanted human T cells. This activity is entirely dependent on the presence of transplanted T cells, supporting the immunological mechanism of action for MEDI4736. To further determine the utility of PD-L1 blockade, an anti-mouse PD-L1 antibody was investigated in immunocompetent mice. Here, anti-mouse PD-L1 significantly improved survival of mice implanted with CT26 colorectal cancer cells. The antitumor activity of anti-PD-L1 was enhanced by combination with oxaliplatin, which resulted in increased release of HMGB1 within CT26 tumors. Taken together, our results demonstrate that inhibition of PD-L1 function can have potent antitumor activity when used as monotherapy or in combination in preclinical models, and suggest it may be a promising therapeutic approach for the treatment of cancer. MEDI4736 is currently in several clinical trials both alone and in combination with other agents, including anti-CTLA-4, anti-PD-1, and inhibitors of IDO, MEK, BRAF, and EGFR.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/farmacología , Antígeno B7-H1/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales/administración & dosificación , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Antígeno B7-1/metabolismo , Antígeno B7-H1/metabolismo , Unión Competitiva , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/patología , Femenino , Humanos , Activación de Linfocitos/efectos de los fármacos , Prueba de Cultivo Mixto de Linfocitos , Melanoma/inmunología , Melanoma/patología , Melanoma/prevención & control , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Compuestos Organoplatinos/administración & dosificación , Oxaliplatino , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/prevención & control , Receptor de Muerte Celular Programada 1/metabolismo , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Producing pure and well behaved bispecific antibodies (bsAbs) on a large scale for preclinical and clinical testing is a challenging task. Here, we describe a new strategy for making monovalent bispecific heterodimeric IgG antibodies in mammalian cells. We applied an electrostatic steering mechanism to engineer antibody light chain-heavy chain (LC-HC) interface residues in such a way that each LC strongly favors its cognate HC when two different HCs and two different LCs are co-expressed in the same cell to assemble a functional bispecific antibody. We produced heterodimeric IgGs from transiently and stably transfected mammalian cells. The engineered heterodimeric IgG molecules maintain the overall IgG structure with correct LC-HC pairings, bind to two different antigens with comparable affinity when compared with their parental antibodies, and retain the functionality of parental antibodies in biological assays. In addition, the bispecific heterodimeric IgG derived from anti-HER2 and anti-EGF receptor (EGFR) antibody was shown to induce a higher level of receptor internalization than the combination of two parental antibodies. Mouse xenograft BxPC-3, Panc-1, and Calu-3 human tumor models showed that the heterodimeric IgGs strongly inhibited tumor growth. The described approach can be used to generate tools from two pre-existent antibodies and explore the potential of bispecific antibodies. The asymmetrically engineered Fc variants for antibody-dependent cellular cytotoxicity enhancement could be embedded in monovalent bispecific heterodimeric IgG to make best-in-class therapeutic antibodies.
Asunto(s)
Inmunoglobulina G/química , Electricidad Estática , Aminoácidos/química , Animales , Citotoxicidad Celular Dependiente de Anticuerpos , Células CHO , Línea Celular , Cricetulus , Dimerización , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunoglobulina G/inmunología , Ratones , Ingeniería de Proteínas , Resonancia por Plasmón de SuperficieRESUMEN
Death receptor agonist therapies have exhibited limited clinical benefit to date. Investigations into why Apo2L/TRAIL and AMG 655 preclinical data were not predictive of clinical response revealed that coadministration of Apo2L/TRAIL with AMG 655 leads to increased antitumor activity in vitro and in vivo. The combination of Apo2L/TRAIL and AMG 655 results in enhanced signaling and can sensitize Apo2L/TRAIL-resistant cells. Structure determination of the Apo2L/TRAIL-DR5-AMG 655 ternary complex illustrates how higher order clustering of DR5 is achieved when both agents are combined. Enhanced agonism generated by combining Apo2L/TRAIL and AMG 655 provides insight into the limited efficacy observed in previous clinical trials and suggests testable hypotheses to reconsider death receptor agonism as a therapeutic strategy.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Animales , Anticuerpos Monoclonales/química , Antineoplásicos/química , Línea Celular Tumoral , Supervivencia Celular , Cristalografía por Rayos X , Resistencia a Antineoplásicos , Sinergismo Farmacológico , Humanos , Ratones , Modelos Moleculares , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/antagonistas & inhibidores , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/química , Transducción de Señal , Ligando Inductor de Apoptosis Relacionado con TNF/química , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Antibody-dependent cellular cytotoxicity (ADCC) is mediated through the engagement of the Fc segment of antibodies with Fcγ receptors (FcγRs) on immune cells upon binding of tumor or viral antigen. The co-crystal structure of FcγRIII in complex with Fc revealed that Fc binds to FcγRIII asymmetrically with two Fc chains contacting separate regions of the FcγRIII by utilizing different residues. To fully explore this asymmetrical nature of the Fc-FcγR interaction, we screened more than 9,000 individual clones in Fc heterodimer format in which different mutations were introduced at the same position of two Fc chains using a high throughput competition AlphaLISA® assay. To this end, we have identified a panel of novel Fc variants with significant binding improvement to FcγRIIIA (both Phe-158 and Val-158 allotypes), increased ADCC activity in vitro, and strong tumor growth inhibition in mice xenograft human tumor models. Compared with previously identified Fc variants in conventional IgG format, Fc heterodimers with asymmetrical mutations can achieve similar or superior potency in ADCC-mediated tumor cell killing and demonstrate improved stability in the CH2 domain. Fc heterodimers also allow more selectivity toward activating FcγRIIA than inhibitory FcγRIIB. Afucosylation of Fc variants further increases the affinity of Fc to FcγRIIIA, leading to much higher ADCC activity. The discovery of these Fc variants will potentially open up new opportunities of building the next generation of therapeutic antibodies with enhanced ADCC effector function for the treatment of cancers and infectious diseases.
Asunto(s)
Anticuerpos Antineoplásicos , Citotoxicidad Celular Dependiente de Anticuerpos , Fragmentos Fc de Inmunoglobulinas/inmunología , Inmunoglobulina G , Ingeniería de Proteínas , Receptores de IgG/inmunología , Animales , Anticuerpos Antineoplásicos/genética , Anticuerpos Antineoplásicos/inmunología , Anticuerpos Antineoplásicos/farmacología , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Citotoxicidad Celular Dependiente de Anticuerpos/genética , Línea Celular Tumoral , Humanos , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/farmacología , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Inmunoglobulina G/farmacología , Ratones , Ratones SCID , Mutación , Neoplasias , Receptores de IgG/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Iron maldistribution has been implicated in the etiology of many diseases including the anemia of inflammation (AI), atherosclerosis, diabetes, and neurodegenerative disorders. Iron metabolism is controlled by hepcidin, a 25-amino-acid peptide. Hepcidin is induced by inflammation and causes iron to be sequestered within cells of the reticuloendothelial system, suppressing erythropoiesis and blunting the activity of erythropoiesis stimulating agents (ESAs). For this reason, neutralization of hepcidin has been proposed as a therapeutic treatment of AI. The aim of the current work was to generate fully human anti-hepcidin antibodies (Abs) as a potential human therapeutic for the treatment of AI and other iron maldistribution disorders. An enzyme-linked immunosorbent assay was established using these Abs to identify patients likely to benefit from either ESAs or anti-hepcidin agents. Using human hepcidin knock-in mice, the mechanism of action of the Abs was shown to be due to an increase in available serum iron leading to enhanced red cell hemoglobinization. One of the Abs, 12B9m, was validated in a mouse model of AI and demonstrated to modulate serum iron in cynomolgus monkeys. The 12B9m Ab was deemed to be an appropriate candidate for use as a potential therapeutic to treat AI in patients with kidney disease or cancer.
Asunto(s)
Anemia Ferropénica/tratamiento farmacológico , Anticuerpos Neutralizantes/farmacología , Eritrocitos/efectos de los fármacos , Hemoglobinas/biosíntesis , Hierro/sangre , Anemia Ferropénica/sangre , Anemia Ferropénica/patología , Animales , Anticuerpos Neutralizantes/biosíntesis , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Ensayo de Inmunoadsorción Enzimática , Eritrocitos/metabolismo , Eritrocitos/patología , Eritropoyesis/efectos de los fármacos , Femenino , Hematínicos/farmacología , Humanos , Inflamación/prevención & control , Macaca fascicularis , Masculino , RatonesRESUMEN
Fibroblast growth factor 21 (FGF21) is a distinctive member of the FGF family with potent beneficial effects on lipid, body weight, and glucose metabolism and has attracted considerable interest as a potential therapeutic for treating diabetes and obesity. As an alternative to native FGF21, we have developed a monoclonal antibody, mimAb1, that binds to ßKlotho with high affinity and specifically activates signaling from the ßKlotho/FGFR1c (FGF receptor 1c) receptor complex. In obese cynomolgus monkeys, injection of mimAb1 led to FGF21-like metabolic effects, including decreases in body weight, plasma insulin, triglycerides, and glucose during tolerance testing. Mice with adipose-selective FGFR1 knockout were refractory to FGF21-induced improvements in glucose metabolism and body weight. These results in obese monkeys (with mimAb1) and in FGFR1 knockout mice (with FGF21) demonstrated the essential role of FGFR1c in FGF21 function and suggest fat as a critical target tissue for the cytokine and antibody. Because mimAb1 depends on ßKlotho to activate FGFR1c, it is not expected to induce side effects caused by activating FGFR1c alone. The unexpected finding of an antibody that can activate FGF21-like signaling through cell surface receptors provided preclinical validation for an innovative therapeutic approach to diabetes and obesity.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Diabetes Mellitus/tratamiento farmacológico , Factores de Crecimiento de Fibroblastos/inmunología , Glucuronidasa/metabolismo , Obesidad/tratamiento farmacológico , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/metabolismo , Tejido Adiposo/patología , Animales , Anticuerpos Monoclonales/farmacología , Peso Corporal/genética , Diabetes Mellitus/sangre , Epítopos/química , Glucosa/metabolismo , Glucuronidasa/inmunología , Humanos , Proteínas Klotho , Macaca fascicularis , Ratones , Obesidad/sangre , Obesidad/complicaciones , Fosfatos/sangre , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/agonistas , Transducción de Señal/efectos de los fármacos , Triglicéridos/sangreRESUMEN
The Notch signaling pathway has been implicated in cell fate determination and differentiation in many tissues. Accumulating evidence points toward a pivotal role in blood vessel formation, and the importance of the Delta-like ligand (Dll) 4-Notch1 ligand-receptor interaction has been shown in both physiological and tumor angiogenesis. Disruption of this interaction leads to a reduction in tumor growth as a result of an increase in nonfunctional vasculature leading to poor perfusion of the tumor. MEDI0639 is an investigational human therapeutic antibody that targets Dll4 to inhibit the interaction between Dll4 and Notch1. The antibody cross-reacts to cynomolgus monkey but not mouse species orthologues. In vitro MEDI0639 inhibits the binding of Notch1 to Dll4, interacting via a novel epitope that has not been previously described. Binding to this epitope translates into MEDI0639 reversing Notch1-mediated suppression of human umbilical vein endothelial cell growth in vitro. MEDI0639 administration resulted in stimulation of tubule formation in a three-dimensional (3D) endothelial cell outgrowth assay, a phenotype driven by disruption of the Dll4-Notch signaling axis. In contrast, in a two-dimensional endothelial cell-fibroblast coculture model, MEDI0639 is a potent inhibitor of tubule formation. In vivo, MEDI0639 shows activity in a human endothelial cell angiogenesis assay promoting human vessel formation and reducing the number of vessels with smooth muscle actin-positive mural cells coverage. Collectively, the data show that MEDI0639 is a potent modulator of Dll4-Notch signaling pathway.