RESUMEN
BACKGROUND: Existing therapeutic strategies are challenged by long times to achieve effect and often require frequent administration. Peanut allergic individuals would benefit from a therapeutic that provides rapid protection against accidental exposure within days of administration while carrying little risk of adverse reactions. OBJECTIVE: Guided by the repertoire of human IgE monoclonal antibodies (mAbs) from allergic individuals, we sought to develop a treatment approach leveraging the known protective effects of allergen-specific IgG4 antibodies. METHODS: We applied our single-cell RNA sequencing SEQ SIFTER™ platform to whole blood samples from peanut allergic individuals to discover IgE mAbs. These were then class-switched by replacing the IgE constant region with IgG4 while retaining the allergen-specific variable regions. In vitro mast cell activation tests (MATs), basophil activation tests (BATs), enzyme-linked immunosorbent assays (ELISAs), and an in vivo peanut allergy mouse model were used to evaluate the specificity, affinity, and activity of these recombinant IgG4 mAbs. RESULTS: We determined that human peanut-specific IgE mAbs predominantly target immunodominant epitopes on Ara h 2 and Ara h 6 and that recombinant IgG4 mAbs effectively blocked these epitopes. IGNX001, a mixture of two such high-affinity IgG4 mAbs, provided robust protection against peanut-mediated mast cell activation in vitro as well as against anaphylaxis upon intragastric peanut challenge in a peanut allergy mouse model. CONCLUSION: We developed a peanut-specific IgG4 antibody therapeutic with convincing preclinical efficacy starting from a large repertoire of human monoclonal IgE antibodies from demographically and geographically diverse individuals. These results warrant further clinical investigation of IGNX001 and underscore the opportunity for the application of this therapeutic development strategy in other food and environmental allergies.
RESUMEN
BACKGROUND: Despite their central role in peanut allergy, human monoclonal IgE antibodies have eluded characterization. OBJECTIVE: We sought to define the sequences, affinities, clonality, and functional properties of human monoclonal IgE antibodies in peanut allergy. METHODS: We applied our single-cell RNA sequencing-based SEQ SIFTER discovery platform to samples from allergic individuals who varied by age, sex, ethnicity, and geographic location in order to understand commonalities in the human IgE response to peanut allergens. Select antibodies were then recombinantly expressed and characterized for their allergen and epitope specificity, affinity, and functional properties. RESULTS: We found striking convergent evolution of IgE monoclonal antibodies (mAbs) from several clonal families comprising both memory B cells and plasmablasts. These antibodies bound with subnanomolar affinity to the immunodominant peanut allergen Ara h 2, specifically a linear, repetitive motif. Further characterization of these mAbs revealed their ability to single-handedly cause affinity-dependent degranulation of human mast cells and systemic anaphylaxis on peanut allergen challenge in humanized mice. Finally, we demonstrated that these mAbs, reengineered as IgGs, inhibit significant, but variable, amounts of Ara h 2- and peanut-mediated degranulation of mast cells sensitized with allergic plasma. CONCLUSIONS: Convergent evolution of IgE mAbs in peanut allergy is a common phenomenon that can reveal immunodominant epitopes on major allergenic proteins. Understanding the functional properties of these molecules is key to developing therapeutics, such as competitive IgG inhibitors, that are able to stoichiometrically outcompete endogenous IgE for allergen and thereby prevent allergic cascade in cases of accidental allergen exposure.
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Hipersensibilidad al Cacahuete , Humanos , Animales , Ratones , Epítopos Inmunodominantes , Antígenos de Plantas , Glicoproteínas , Inmunoglobulina E , Epítopos , Anticuerpos Monoclonales , Alérgenos , Arachis , Albuminas 2S de PlantasRESUMEN
Nanoparticles provide a unique opportunity to explore the benefits of selective distribution and release of cancer therapeutics at sites of disease through varying particle sizes and compositions that exploit the enhanced permeability of tumor-associated blood vessels. Though delivery of larger as opposed to smaller and/or actively transported molecules to the brain is prima facie a challenging endeavor, we wondered whether nanoparticles could improve the therapeutic index of existing drugs for use in treating brain tumors via these vascular effects. We therefore selected a family of nanoparticles composed of cabazitaxel-carboxymethyl cellulose amphiphilic polymers to investigate the potential for delivering a brain-penetrant taxane to intracranial brain tumors in mice. Among a small set of nanoparticle formulations, we found evidence for nanoparticle accumulation in the brain, and one such formulation demonstrated activity in an orthotopic model of glioma, suggesting that such nanoparticles could be useful for the treatment of glioma and brain metastases of other tumor types.
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Antineoplásicos/administración & dosificación , Neoplasias Encefálicas/tratamiento farmacológico , Carboximetilcelulosa de Sodio/química , Nanopartículas/química , Polietilenglicoles/química , Taxoides/administración & dosificación , Animales , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapéutico , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Sistemas de Liberación de Medicamentos , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Nanopartículas/ultraestructura , Taxoides/química , Taxoides/farmacocinética , Taxoides/uso terapéuticoRESUMEN
Omalizumab (Xolair®) is a recombinant humanized monoclonal antibody that selectively binds to human immunoglobulin E (IgE). Omalizumab is used to treat IgE-mediated diseases such as chronic idiopathic urticaria (CIU) and moderate to severe allergic asthma. In pre-marketing clinical trials in patients with asthma, anaphylaxis was reported in 3 of 3,507 (0.1%) patients. In post-marketing spontaneous reports, the frequency of anaphylaxis attributed to omalizumab use was estimated to be at least 0.2% of patients based on an estimated exposure of about 57,300 patients from June 2003 through December 2006. To better understand the risk of anaphylaxis in patients with allergic asthma receiving omalizumab, a post-marketing pharmacosurveillance study was initiated in 2009. As part of this study, an assay was developed to detect antibodies of IgE isotype to omalizumab. Serum samples from patients in the study were evaluated using this assay. Our results indicated that there was no observable correlation between either anaphylaxis or skin test reactivity and the presence of antibodies of IgE isotype to omalizumab. Here, we discuss the development of this assay as well as the results of the immunogenicity assessment.
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Anafilaxia/epidemiología , Antialérgicos/inmunología , Anticuerpos/análisis , Inmunoglobulina E/análisis , Omalizumab/inmunología , Adulto , Anafilaxia/etiología , Antialérgicos/efectos adversos , Anticuerpos/genética , Asma/tratamiento farmacológico , Humanos , Inmunoglobulina E/genética , Omalizumab/efectos adversos , Vigilancia de Productos Comercializados , Pruebas CutáneasRESUMEN
The increased proteolytic activity of membrane-bound and secreted proteases on the surface of cancer cells and in the transformed stroma is a common characteristic of aggressive metastatic prostate cancer. We describe here the development of an active site-specific probe for detecting a secreted peritumoral protease expressed by cancer cells and the surrounding tumor microenvironment. Using a human fragment antigen-binding phage display library, we identified a human antibody termed U33 that selectively inhibited the active form of the protease urokinase plasminogen activator (uPA, PLAU). In the full-length immunoglobulin form, U33 IgG labeled with near-infrared fluorophores or radionuclides allowed us to noninvasively detect active uPA in prostate cancer xenograft models using optical and single-photon emission computed tomography imaging modalities. U33 IgG labeled with (111)In had a remarkable tumor uptake of 43.2% injected dose per gram (%ID/g) 72 hours after tail vein injection of the radiolabeled probe in subcutaneous xenografts. In addition, U33 was able to image active uPA in small soft-tissue and osseous metastatic lesions using a cardiac dissemination prostate cancer model that recapitulated metastatic human cancer. The favorable imaging properties were the direct result of U33 IgG internalization through an uPA receptor-mediated mechanism in which U33 mimicked the function of the endogenous inhibitor of uPA to gain entry into the cancer cell. Overall, our imaging probe targets a prostate cancer-associated protease, through a unique mechanism, allowing for the noninvasive preclinical imaging of prostate cancer lesions.
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Neoplasias de la Próstata/enzimología , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Animales , Línea Celular Tumoral , Técnica del Anticuerpo Fluorescente , Expresión Génica , Humanos , Radioisótopos de Indio , Masculino , Ratones Desnudos , Trasplante de Neoplasias , Especificidad de Órganos , Neoplasias de la Próstata/diagnóstico por imagen , Radiofármacos , Tomografía Computarizada de Emisión de Fotón Único , Tomografía Óptica , Activador de Plasminógeno de Tipo Uroquinasa/genéticaRESUMEN
Probodies are proteolytically activated antibodies engineered to remain inert until activated locally in diseased tissue. In principle, any therapeutic antibody can be converted into Probody™ form. In this perspective, we highlight the emerging therapeutic potential of the Probody approach in the form of conventional IgG-based Probodies as well as in the form of 'empowered Probody' formats such as Probody-drug conjugates.
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Anticuerpos Biespecíficos/uso terapéutico , Inmunoconjugados/uso terapéutico , Terapia Molecular Dirigida/métodos , Profármacos/uso terapéutico , Animales , Antígenos/inmunología , Humanos , Inmunoconjugados/química , Inmunoglobulina G/metabolismo , Inmunoglobulina G/uso terapéutico , Profármacos/metabolismo , Ingeniería de ProteínasRESUMEN
Target-mediated toxicity constitutes a major limitation for the development of therapeutic antibodies. To redirect the activity of antibodies recognizing widely distributed targets to the site of disease, we have applied a prodrug strategy to create an epidermal growth factor receptor (EGFR)-directed Probody therapeutic-an antibody that remains masked against antigen binding until activated locally by proteases commonly active in the tumor microenvironment. In vitro, the masked Probody showed diminished antigen binding and cell-based activities, but when activated by appropriate proteases, it regained full activity compared to the parental anti-EGFR antibody cetuximab. In vivo, the Probody was largely inert in the systemic circulation of mice, but was activated within tumor tissue and showed antitumor efficacy that was similar to that of cetuximab. The Probody demonstrated markedly improved safety and increased half-life in nonhuman primates, enabling it to be dosed safely at much higher levels than cetuximab. In addition, we found that both Probody-responsive xenograft tumors and primary tumor samples from patients were capable of activating the Probody ex vivo. Probodies may therefore improve the safety profile of therapeutic antibodies without compromising efficacy of the parental antibody and may enable the wider use of empowered antibody formats such as antibody-drug conjugates and bispecifics.
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Anticuerpos Antineoplásicos/uso terapéutico , Receptores ErbB/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Profármacos/uso terapéutico , Animales , Anticuerpos Monoclonales Humanizados/farmacología , Anticuerpos Monoclonales Humanizados/uso terapéutico , Anticuerpos Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Cetuximab , Humanos , Inmunohistoquímica , Macaca fascicularis , Ratones , Ratones Desnudos , Profármacos/toxicidad , Piel/efectos de los fármacos , Piel/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: To investigate the underlying cause for the observed differences in self-associating and viscoelastic behavior between two monoclonal antibodies, MAb1, and MAb2. METHODS: Several mutants were designed by swapping charged residues in MAb1 with those present in MAb2 at their respective positions and vice versa. Rheological analysis was done at low and high shear rates. Dynamic light scattering quantified intermolecular interactions in dilute solutions; sedimentation equilibrium analysis determined the corrected weight average molecular weight (M (wc)) to assess the self-associating behavior in high concentration. The molecular charge was estimated from electrophoretic mobility measurements. RESULTS: Replacing the charged residues in the CDR of MAb1 resulted in a lower M (wc) and solution viscosity. The corresponding changes in either just the variable light (VL) or variable heavy (VH) chain showed only a partial decrease in viscosity, whereas changes in both VL and VH chains resulted in a dramatic reduction in viscosity. The converse case where the VL and VH chains of MAb2 were made to look like MAb1 did not self-associate or show increased viscosity. CONCLUSIONS: Exposed charged residues in the CDR of MAb1 are critical in determining the self-associating and highly viscous behavior observed at high concentrations.
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Anticuerpos Monoclonales/química , Secuencia de Aminoácidos , Anticuerpos Monoclonales/genética , Dicroismo Circular , Glicosilación , Humanos , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Mutación , Sustancias ViscoelásticasRESUMEN
Clinical response to the anti-CD20 antibody rituximab has been demonstrated to correlate with the polymorphism in the FcγRIIIa receptor where patients homozygous for the higher affinity V158 allotype showed a better response rate. This finding suggests that engineering of anti-CD20 for increased FcγRIIIa affinity could result in improved clinical outcome. To identify variants with increased affinity to FcγRIIIa, we developed quantitative assays using soluble receptors as well as engineered cell lines expressing FcγRI or FcγRIIIa on the cell surface. We assayed a set of anti-CD20 IgG(1) variants that had identical Fab regions, but alterations in the Fc regions, in both the soluble receptor-based and cell-based FcγRIIIa binding assays. We obtained similar relative binding affinity increases and assay precisions. The increase in affinity for FcγRIIIa correlated with the increase in activity in the antibody-dependent cellular cytotoxicity assay. These variants had unaltered FcγRI binding. In addition to Fcγ receptors, IgG also binds to FcRn, the receptor responsible for the long circulating half-life of IgG. The mutations in the anti-CD20 variants were previously found not to affect FcRn binding in the soluble receptor-based assays; consequently, we used anti-Her2 variants with different binding affinities to FcRn to study FcRn binding assays. We generated a cell line expressing FcRn on the cell surface to measure IgG binding and obtained similar ranking of these anti-Her2 variants in the cell-based and the soluble receptor-based FcRn binding assays. In conclusion, both the soluble receptor-based and cell-based binding assays can be used to identify IgG(1) variants with increased affinity to FcγRIIIa and unaltered affinity to FcγRI and FcRn.
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Antígenos de Histocompatibilidad Clase I/metabolismo , Inmunoglobulina G/genética , Inmunoglobulina G/metabolismo , Receptores Fc/metabolismo , Receptores de IgG/metabolismo , Sustitución de Aminoácidos , Animales , Anticuerpos Monoclonales de Origen Murino/genética , Anticuerpos Monoclonales de Origen Murino/metabolismo , Afinidad de Anticuerpos , Citotoxicidad Celular Dependiente de Anticuerpos , Antígenos CD20/inmunología , Linfocitos B/inmunología , Células CHO , Membrana Celular/inmunología , Cricetinae , Cricetulus , Ensayo de Inmunoadsorción Enzimática , Variación Genética , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Técnicas In Vitro , Células Asesinas Naturales/inmunología , Cinética , Ratones , Ratones Transgénicos , Unión Proteica , Ingeniería de Proteínas , Receptor ErbB-2/antagonistas & inhibidores , Receptor ErbB-2/inmunología , Receptores Fc/genética , Receptores de IgG/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rituximab , SolubilidadRESUMEN
Bevacizumab [Avastin; anti-vascular endothelial growth factor (VEGF) antibody] is an antiangiogenic IgG approved for treating patients with certain types of colon, breast, and lung cancer. In these indications, bevacizumab is administered every 2 to 3 weeks, prompting us to study ways to reduce the frequency of administration. Increasing affinity to neonatal Fc receptor (FcRn) may extend the pharmacokinetic half-life of an antibody, but the quantitative effect of FcRn affinity on clearance has not been clearly elucidated. To gain further insight into this relationship, we engineered a series of anti-VEGF antibody variants with minimal amino acid substitutions and showed a range of half-life improvements in primates. These results suggest that, if proven clinically safe and effective, a modified version of bevacizumab could potentially provide clinical benefit to patients on long-term anti-VEGF therapy through less-frequent dosing and improved compliance with drug therapy. Moreover, despite having half-life similar to that of wild-type in mice due to the species-specific FcRn binding effects, the variant T307Q/N434A exhibited superior in vivo potency in slowing the growth of certain human tumor lines in mouse xenograft models. These results further suggest that FcRn variants may achieve increased potency through unidentified mechanisms in addition to increased systemic exposure.
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Inhibidores de la Angiogénesis/farmacocinética , Anticuerpos Monoclonales/farmacocinética , Anticuerpos/química , Factor A de Crecimiento Endotelial Vascular/inmunología , Animales , Anticuerpos Monoclonales Humanizados , Bevacizumab , Línea Celular Tumoral , Femenino , Humanos , Inmunoglobulina G/química , Macaca fascicularis , Ratones , Trasplante de Neoplasias , Ingeniería de Proteínas/métodos , Receptores Fc/química , Resonancia por Plasmón de Superficie , Factor A de Crecimiento Endotelial Vascular/farmacocinéticaRESUMEN
The neonatal Fc receptor (FcRn) plays a critical role in maintaining homeostasis of IgG antibodies. Recent studies have shown that the FcRn-IgG interaction can be modulated to alter the pharmacokinetics of the antibody. This has been achieved by altering amino acid residues in the FcRn-binding domain of the antibody, resulting in a change in the pH-dependent binding affinity of the antibody to FcRn. The purpose of this study was to examine the impact of the pH-dependent FcRn binding affinity on the pharmacokinetics of the antibody with changes in the Asn434 residue. Two anti-tumor necrosis factor-alpha monoclonal antibody (mAb) FcRn variants (N434A and N434H) were engineered, and pharmacokinetic studies of the two FcRn variants together with the wild type (WT) were conducted in mice and cynomolgus monkeys. N434A, which had binding properties to murine FcRn similar to those of the WT, had the same pharmacokinetic profile as the WT in mice. N434H, with the highest binding affinity to murine FcRn at pH 7.4, had a faster clearance (16.1 ml/day/kg) and a lower bioavailability (61.3%) compared with the WT (5.07 ml/day/kg, 73.2%) and N434A (5.90 ml/day/kg, 72.4%) in mice. N434A and N434H, which had higher binding affinity at pH 6.0 to monkey FcRn with comparable affinity at pH 7.4, had significantly higher areas under the serum concentration-time curve from time 0 to day 7 than the WT (749 +/- 71.9 and 819 +/- 81.5 versus 592 +/- 56.8 microg/ml . day) in monkeys. Thus, increasing the binding affinity of mAbs to FcRn at pH 6.0 while keeping a low binding affinity at pH 7.4 improves the pharmacokinetics of these molecules.
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Anticuerpos Bloqueadores/metabolismo , Anticuerpos Monoclonales/farmacocinética , Receptores Fc/metabolismo , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/inmunología , Animales , Animales Recién Nacidos , Anticuerpos Bloqueadores/administración & dosificación , Anticuerpos Monoclonales/administración & dosificación , Disponibilidad Biológica , Humanos , Concentración de Iones de Hidrógeno , Inyecciones Intravenosas , Inyecciones Subcutáneas , Macaca fascicularis , Ratones , Ratones SCID , Especificidad de la EspecieRESUMEN
The pH-dependent binding of Igs to the neonatal FcR (FcRn) plays a critical role in the in vivo homeostasis of IgGs. Modulating the interaction between Fc and FcRn through protein engineering is one method for improving the pharmacokinetics of therapeutic Abs. Recent studies disputed the direct relationship between increasing FcRn affinity and improved pharmacokinetic properties. In this work, we studied the pharmacokinetics of two human IgG1 Fc variants in cynomolgus monkey to further clarify the affinity-pharmacokinetic relationship. First, we report a number of novel Fc point mutations and combination variants, including some with primate-specific FcRn-binding improvements. By studying these variants along with some previously described variants across a wide range of affinities, we discovered a direct correlation of pH 6 affinity improvements with neutral pH improvements, suggesting that all of the tested variants exhibit similar pH dependency in FcRn binding. We then evaluated the pharmacokinetics of variants N434A and N434W, which, respectively, gave approximately 4- and 80-fold improvements in pH 6-binding affinity to both human and nonhuman primate FcRn. Surprisingly, clearance of N434W was similar to that of wild type. N434W is the first variant studied in primates that exhibits significant binding to FcRn at pH 7.4, and its clearance substantiates the principle that too much affinity improvement, i.e., beyond that of N434W, does not yield improved pharmacokinetics. In contrast, N434A exhibited a approximately 2-fold decrease in clearance in cynomolgus monkey, supporting the notion that modest increases in pH 6 FcRn affinity can result in improved pharmacokinetics in primates.
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Afinidad de Anticuerpos/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Inmunoglobulina G/inmunología , Inmunoglobulina G/farmacología , Macaca fascicularis/inmunología , Receptores Fc/inmunología , Secuencia de Aminoácidos , Animales , Humanos , Concentración de Iones de Hidrógeno , Inmunoglobulina G/genética , Inmunoglobulina G/metabolismo , Ratones , Modelos Moleculares , Mutación/genética , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologíaRESUMEN
A single-dose cynomolgus monkey pharmacokinetic study was performed comparing two monoclonal anti-TNF antibodies (mAbs), GNExTNFvF and Humira. Normal pharmacokinetic profiles were observed over the first week of the study, followed by a rapid drop in serum mAb levels after day 8. In order to determine whether an anti-therapeutic antibody (ATA) response led to the abnormal clearance of antibody in this study, ATA assays were developed using two electrochemiluminescent technologies, BioVeris and Meso Scale Discovery (MSD). Characterization of the assays demonstrated that the two platforms gave similar sensitivities and tolerance to the presence of therapeutic antibody. Analysis of the cynomolgus monkey serum samples revealed that all animals developed significant ATA titers with log titer values of 2-4, with the BioVeris and MSD technologies giving very similar results. Immunodepletion studies confirmed the CDR-specificity of the ATA response for the GNExTNFvF-dosed cynos, although the Humira-dosed cynos showed both CDR-specific and human IgG1 framework-specific ATAs. To further characterize the ATA response, neutralizing antibody (NAb) assays were developed using two different approaches, flow cytometry and MSD. Flow cytometry and MSD cell-binding assays used Jurkat cells transfected with noncleavable TNF (huTNF(NC)). Neutralizing activity was assessed by the ability of ATA-positive serum samples to block the binding of biotinylated anti-TNF to huTNF(NC) Jurkat cells, showing that all but one animal developed neutralizing antibodies. Although both technologies displayed similar trends, the MSD approach showed greater differentiation between samples and could detect a broader range of neutralizing activities.
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Anticuerpos Antiidiotipos/sangre , Anticuerpos Monoclonales/farmacocinética , Técnicas Electroquímicas/métodos , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Adalimumab , Animales , Anticuerpos Monoclonales/sangre , Anticuerpos Monoclonales Humanizados , Técnicas Electroquímicas/instrumentación , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Humanos , Células Jurkat , Mediciones Luminiscentes/instrumentación , Mediciones Luminiscentes/métodos , Macaca fascicularis , Masculino , Pruebas de Neutralización , Sensibilidad y EspecificidadRESUMEN
HAE1, a high-affinity anti-IgE monoclonal antibody, is discussed here as a case study in the use of quantitative pharmacology in the development of a second-generation molecule. In vitro, preclinical, and clinical data from the first-generation molecule, omalizumab, were heavily leveraged in the HAE1 program. A preliminary mechanism-based pharmacokinetic/pharmacodynamic (PK/PD) model for HAE1 was developed using an existing model for omalizumab, together with in vitro binding data for HAE1 and omalizumab. When phase I data were available, the model was refined by simultaneously modeling PK/PD data from omalizumab studies with the available HAE1 phase I data. The HAE1 clinical program was based on knowledge of the quantitative relationship between a pharmacodynamic biomarker, suppression of free IgE, and clinical response (e.g., lower exacerbation rates) obtained in pivotal studies with omalizumab. A clinical trial simulation platform was developed to predict free IgE levels and clinical responses following attainment of a target free IgE level (=10 IU/ml). The simulation platform enabled selection of four doses for the phase II dose-ranging trial by two independent methods: dose-response non-linear fitting and linear mixed modeling. Agreement between the two methods provided confidence in the doses selected. Modeling and simulation played a large role in supporting acceleration of the HAE1 program by enabling data-driven decision-making, often based on confirmation of projections and/or learning from incoming new data.
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Anticuerpos Monoclonales , Afinidad de Anticuerpos/inmunología , Diseño de Fármacos , Inmunoglobulina E/inmunología , Anticuerpos Antiidiotipos , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales Humanizados , Asma/tratamiento farmacológico , Línea Celular , Humanos , Omalizumab , Receptores de IgE/inmunologíaRESUMEN
Antibody-drug conjugates enhance the antitumor effects of antibodies and reduce adverse systemic effects of potent cytotoxic drugs. However, conventional drug conjugation strategies yield heterogenous conjugates with relatively narrow therapeutic index (maximum tolerated dose/curative dose). Using leads from our previously described phage display-based method to predict suitable conjugation sites, we engineered cysteine substitutions at positions on light and heavy chains that provide reactive thiol groups and do not perturb immunoglobulin folding and assembly, or alter antigen binding. When conjugated to monomethyl auristatin E, an antibody against the ovarian cancer antigen MUC16 is as efficacious as a conventional conjugate in mouse xenograft models. Moreover, it is tolerated at higher doses in rats and cynomolgus monkeys than the same conjugate prepared by conventional approaches. The favorable in vivo properties of the near-homogenous composition of this conjugate suggest that our strategy offers a general approach to retaining the antitumor efficacy of antibody-drug conjugates, while minimizing their systemic toxicity.
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Anticuerpos Antineoplásicos/farmacología , Antineoplásicos/farmacología , Citotoxinas/farmacología , Inmunotoxinas/farmacocinética , Neoplasias Ováricas/inmunología , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/farmacología , Anticuerpos Antineoplásicos/genética , Especificidad de Anticuerpos , Sitios de Unión , Antígeno Ca-125/inmunología , Línea Celular Tumoral , Química Farmacéutica/métodos , Cisteína/genética , Femenino , Humanos , Macaca fascicularis , Proteínas de la Membrana/inmunología , Ratones , Mutagénesis Sitio-Dirigida , Oligopéptidos/farmacología , Neoplasias Ováricas/tratamiento farmacológico , Ratas , Ratas Sprague-Dawley , Compuestos de Sulfhidrilo/farmacologíaRESUMEN
Preclinical pharmacokinetic (PK) assays are important to help evaluate the safety and efficacy of a potential biotherapeutic before clinical studies. The assay typically requires a biotherapeutic-specific reagent to minimize matrix effects especially when the host species are non-human primates such as cynomolgus monkeys and the biotherapeutic is a humanized monoclonal antibody (MAb). Recombinant humanized mAb 2H7 (rhuMAb2H7) binds to the extracellular domain of CD20 that is expressed on B cells and results in B cell depletion. It is currently being evaluated for its therapeutic potential in rheumatoid arthritis (RA) in clinical studies. During the early development of rhuMAb2H7, a cynomolgus monkey PK assay was needed to help assess the pharmacokinetic parameters of rhuMAb2H7 in a pilot cynomolgus monkey study. However, development of a cynomolgus monkey PK assay was challenging due to lack of rhuMAb2H7-specific reagents. Here we describe an alternative method for detection of rhuMAb2H7 in cynomolgus monkey serum using polyclonal antibodies against human IgGs. This assay quantifies rhuMAb2H7 in 10% cynomolgus monkey serum with high sensitivity, accuracy, and precision. This assay successfully supported the rhuMAb2H7 development, and has the potential to be used to quantify other humanized MAb biotherapeutics in serum from a variety of non-human species.
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Anticuerpos Monoclonales/sangre , Anticuerpos , Antígenos CD20/inmunología , Antirreumáticos/sangre , Ensayo de Inmunoadsorción Enzimática , Inmunoglobulina G/inmunología , Juego de Reactivos para Diagnóstico , Animales , Anticuerpos Monoclonales/farmacocinética , Anticuerpos Monoclonales Humanizados , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Antirreumáticos/farmacocinética , Bovinos , Humanos , Macaca fascicularis , Proteínas Recombinantes/sangre , Reproducibilidad de los Resultados , OvinosRESUMEN
PURPOSE: Bevacizumab is a humanized anti-human VEGF-A monoclonal antibody (mAb) approved by the United States Food and Drug Administration for cancer therapy and used off label to treat neovascular age-related macular degeneration. Earlier studies characterized bevacizumab as species specific and lacking the ability to neutralize murine (m) VEGF-A. However, a recent study reported that bevacizumab is a potent inhibitor of hemangiogenesis and lymphangiogenesis in murine models. The authors sought to reassess the interaction between bevacizumab and mVEGF-A. METHODS: The authors performed Western blot analysis, plasmon resonance by BIAcore, and endothelial cell proliferation assays to characterize the interaction between bevacizumab and mVEGF-A. They also tested whether bevacizumab had any effects in two in vivo murine models, laser-induced choroidal neovascularization (CNV) and melanoma growth. RESULTS: Western blot detected a very weak interaction, but BIAcore detected no measurable interaction between mVEGF and bevacizumab. Bevacizumab failed to inhibit mVEGF-stimulated endothelial cell proliferation. In addition, bevacizumab was indistinguishable from the control antibody in the CNV and tumor models, whereas a cross-reactive anti-VEGF-A mAb had dramatic inhibitory effects. CONCLUSIONS: Bevacizumab has an extremely weak interaction with mVEGF-A, which fails to result in immunoneutralization as assessed by several bioassays.
Asunto(s)
Inhibidores de la Angiogénesis/metabolismo , Anticuerpos Monoclonales/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Inhibidores de la Angiogénesis/farmacología , Animales , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales Humanizados , Bevacizumab , Western Blotting , Bovinos , Proliferación Celular/efectos de los fármacos , Neovascularización Coroidal/patología , Interacciones Farmacológicas , Electroforesis en Gel de Poliacrilamida , Endotelio Vascular/efectos de los fármacos , Femenino , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Neoplasias Cutáneas/patología , Resonancia por Plasmón de Superficie , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
Cysteines with reactive thiol groups are attractive tools for site-specific labeling of proteins. Engineering a reactive cysteine residue into proteins with multiple disulfide bonds is often a challenging task as it may interfere with structural and functional properties of the protein. Here we developed a phage display-based biochemical assay, PHESELECTOR (Phage ELISA for Selection of Reactive Thiols) to rapidly screen reactive thiol groups on antibody fragments without interfering with their antigen binding, using trastuzumab-Fab (hu4D5Fab) as a model system. The solvent accessibility values for all the amino acid residues in the hu4D5Fab were calculated using available crystal structure information. Serine, alanine and valine residues with highest solvent accessibility values were selected and tested to compare structure-based design with the PHESELECTOR biochemical method. Cysteine substitutions at partially solvent-accessible alanine or valine residues exhibited better thiol reactivity values than substitutions at serine residues. The poor correlation between fractional solvent accessibility and thiol reactivity of the engineered hu4D5Fab variants indicated the value of PHESELECTOR biochemical assay to identify reactive thiol groups on the antibody-Fab surface. Mass spectrometric analysis of biotinylated ThioFab (Fab with engineered cysteine) variants confirmed that conjugation occurred only at the engineered cysteine thiols of either light or heavy chains. ThioFabs with engineered cysteine residues in the constant domains (CL and CH(1)) should allow universal application for site-specific conjugation of antibody-Fabs.
Asunto(s)
Anticuerpos Monoclonales/química , Cisteína/química , Fragmentos Fab de Inmunoglobulinas/química , Anticuerpos Monoclonales/inmunología , Reacciones Antígeno-Anticuerpo , Sitios de Unión/genética , Sitios de Unión/inmunología , Biotina/química , Línea Celular Tumoral , Cromatografía Liquida/métodos , Cisteína/genética , Cisteína/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Citometría de Flujo/métodos , Regulación de la Expresión Génica/genética , Ingeniería Genética , Variación Genética , Humanos , Fragmentos Fab de Inmunoglobulinas/genética , Fragmentos Fab de Inmunoglobulinas/inmunología , Maleimidas/química , Espectrometría de Masas/métodos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Plásmidos/genética , Sensibilidad y Especificidad , Coloración y Etiquetado/métodos , Propiedades de SuperficieRESUMEN
Neovascular age-related macular degeneration (AMD) is the leading cause of blindness in older adults in the Western world. Ranibizumab (Lucentis), a humanized antibody fragment directed against vascular endothelial growth factor (VEGF-A), was recently approved by the US Food and Drug Administration (FDA) for the treatment of neovascular AMD. The objective of this study was to characterize the binding affinity and pharmacological activity of ranibizumab for 3 biologically active forms of VEGF-A: VEGF165, VEGF121, and VEGF110. The apparent equilibrium binding affinity of ranibizumab for VEGF-A molecules was determined by Biacore analysis; the biological activity of VEGF-A was demonstrated in a human umbilical vein endothelial cell (HUVEC) proliferation-inhibition assay. Inhibition of VEGF-A-induced vascular permeability by ranibizumab was assessed in vivo using hairless guinea pigs and a modified Miles assay. Ranibizumab was capable of binding to recombinant human VEGF165, VEGF121, and VEGF110 (KD < or = 192 pM), inhibiting VEGF-A-induced HUVEC proliferation in a concentration-dependent manner. Ranibizumab also exerted potent dose-dependent inhibition (IC(50) of 0.4-1.2 nM) of the vascular permeability-enhancing activity of VEGF165, VEGF121, and VEGF110 in the Miles assay. In conclusion, these results show that ranibizumab is capable of binding to and specifically inhibiting the activities of 3 biologically active forms of VEGF-A. As VEGF-A plays a pivotal role in the pathogenesis of neovascular AMD, ranibizumab activity, as demonstrated in this study, supports its clinical utility in the treatment of this disease.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Anticuerpos Monoclonales/farmacología , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Inhibidores de la Angiogénesis/metabolismo , Animales , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales Humanizados , Permeabilidad Capilar/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Cobayas , Humanos , Ranibizumab , Proteínas Recombinantes/metabolismo , Piel/irrigación sanguínea , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
VEGF-A is important in tumor angiogenesis, and a humanized anti-VEGF-A monoclonal antibody (bevacizumab) has been approved by the FDA as a treatment for metastatic colorectal and nonsquamous, non-small-cell lung cancer in combination with chemotherapy. However, contributions of both tumor- and stromal-cell derived VEGF-A to vascularization of human tumors grown in immunodeficient mice hindered direct comparison between the pharmacological effects of anti-VEGF antibodies with different abilities to block host VEGF. Therefore, by gene replacement technology, we engineered mice to express a humanized form of VEGF-A (hum-X VEGF) that is recognized by many anti-VEGF antibodies and has biochemical and biological properties comparable with WT mouse and human VEGF-A. The hum-X VEGF mouse model was then used to compare the activity and safety of a panel of VEGF Mabs with different affinities for VEGF-A. Although in vitro studies clearly showed a correlation between binding affinity and potency at blocking endothelial cell proliferation stimulated by VEGF, in vivo experiments failed to document any consistent correlation between antibody affinity and the ability to inhibit tumor growth and angiogenesis in most animal models. However, higher-affinity antibodies were more likely to result in glomerulosclerosis during long-term treatment.