RESUMEN
BACKGROUND: Tick transmission of Borrelia spirochetes to humans results in significant morbidity from Lyme disease worldwide. Serum concentrations of antibodies against outer surface protein A (OspA) were shown to correlate with protection from infection with Borrelia burgdorferi, the primary cause of Lyme disease in the United States. METHODS: Mice transgenic for human immunoglobulin genes were immunized with OspA from B. burgdorferi to generate human monoclonal antibodies (HuMabs) against OspA. HuMabs were generated and tested in in vitro borreliacidal assays and animal protection assays. RESULTS: Nearly 100 unique OspA-specific HuMabs were generated, and 4 HuMabs (221-7, 857-2, 319-44, and 212-55) were selected as lead candidates on the basis of borreliacidal activity. HuMabs 319-44, 857-2, and 212-55 were borreliacidal against 1 or 2 Borrelia genospecies, whereas 221-7 was borreliacidal (half maximal inhibitory concentration, < 1 nM) against B. burgdorferi, Borrelia afzelii, and Borrelia garinii, the 3 main genospecies endemic in the United States, Europe, and Asia. All 4 HuMabs completely protected mice from infection at 10 mg/kg in a murine model of tick-mediated transmission of B. burgdorferi CONCLUSIONS: Our study indicates that OspA-specific HuMabs can prevent the transmission of Borrelia and that administration of these antibodies could be employed as preexposure prophylaxis for Lyme disease.
Asunto(s)
Anticuerpos Antibacterianos/administración & dosificación , Anticuerpos Monoclonales/administración & dosificación , Proteínas de la Membrana Bacteriana Externa/antagonistas & inhibidores , Vacunas Bacterianas/antagonistas & inhibidores , Transmisión de Enfermedad Infecciosa/prevención & control , Factores Inmunológicos/administración & dosificación , Lipoproteínas/antagonistas & inhibidores , Enfermedad de Lyme/prevención & control , Profilaxis Pre-Exposición/métodos , Animales , Anticuerpos Antibacterianos/aislamiento & purificación , Anticuerpos Monoclonales/aislamiento & purificación , Antígenos de Superficie , Modelos Animales de Enfermedad , Inmunización Pasiva/métodos , Factores Inmunológicos/aislamiento & purificación , Enfermedad de Lyme/transmisión , Ratones Endogámicos C3H , Ratones Transgénicos , Mordeduras de Garrapatas/complicaciones , Resultado del TratamientoRESUMEN
Angiogenic biomarkers, including soluble fms-like tyrosine kinase 1 (sFlt1), are thought to be predictors of preeclampsia onset; however, improvement is needed before a widespread diagnostic test can be utilized. Here we describe the development and use of diagnostic monoclonal antibodies specific to the two main splice variants of sFlt1, sFlt1-1 and sFlt1-14. These antibodies were selected for their sensitivity and specificity to their respective sFlt1 isoform in a capture ELISA format. Data from this pilot study suggest that sFlt1-1 may be more predictive of preeclampsia than total sFlt1. It may be possible to improve current diagnostic platforms if more specific antibodies are utilized.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Preeclampsia/diagnóstico , Proteínas Gestacionales/sangre , Receptor 1 de Factores de Crecimiento Endotelial Vascular/sangre , Receptor 1 de Factores de Crecimiento Endotelial Vascular/inmunología , Empalme Alternativo/inmunología , Líquido Amniótico/inmunología , Líquido Amniótico/metabolismo , Animales , Células CHO , Cricetulus , Femenino , Humanos , Ratones , Proyectos Piloto , Preeclampsia/sangre , Preeclampsia/inmunología , Embarazo , Proteínas Gestacionales/genética , Proteínas Gestacionales/inmunología , Isoformas de Proteínas/sangre , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Sensibilidad y Especificidad , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genéticaRESUMEN
New immune checkpoints are emerging in a bid to improve response rates to immunotherapeutic drugs. The adenosine A2A receptor (A2AR) has been proposed as a target for immunotherapeutic development due to its participation in immunosuppression of the tumor microenvironment. Blockade of A2AR could restore tumor immunity and, consequently, improve patient outcomes. Here, we describe the discovery of a potent, selective, and tumor-suppressing antibody antagonist of human A2AR (hA2AR) by phage display. We constructed and screened four single-chain variable fragment (scFv) libraries-two synthetic and two immunized-against hA2AR and antagonist-stabilized hA2AR. After biopanning and ELISA screening, scFv hits were reformatted to human IgG and triaged in a series of cellular binding and functional assays to identify a lead candidate. Lead candidate TB206-001 displayed nanomolar binding of hA2AR-overexpressing HEK293 cells; cross-reactivity with mouse and cynomolgus A2AR but not human A1, A2B, or A3 receptors; functional antagonism of hA2AR in hA2AR-overexpressing HEK293 cells and peripheral blood mononuclear cells (PBMCs); and tumor-suppressing activity in colon tumor-bearing HuCD34-NCG mice. Given its therapeutic properties, TB206-001 is a good candidate for incorporation into next-generation bispecific immunotherapeutics.
Asunto(s)
Antagonistas del Receptor de Adenosina A2 , Receptor de Adenosina A2A , Humanos , Animales , Receptor de Adenosina A2A/metabolismo , Receptor de Adenosina A2A/inmunología , Células HEK293 , Ratones , Antagonistas del Receptor de Adenosina A2/farmacología , Antagonistas del Receptor de Adenosina A2/uso terapéutico , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/farmacología , Macaca fascicularis , Biblioteca de PéptidosRESUMEN
Cardiac hypertrophy, whether pathological or physiological, induces a variety of additional morphological and physiological changes in the heart, including altered contractility and hemodynamics. Events exacerbating these changes are documented during later stages of hypertrophy (usually termed pathological hypertrophy). Few studies document the morphological and physiological changes during early physiological hypertrophy. We define acute cardiac remodeling events in response to transverse aortic constriction (TAC), including temporal changes in hypertrophy, collagen deposition, capillary density, and the cell populations responsible for these changes. Cardiac hypertrophy induced by TAC in mice was detected 2 days after surgery (as measured by heart weight, myocyte width, and wall thickness) and peaked by day 7. Picrosirius staining revealed increased collagen deposition 7 days after TAC; immunostaining and flow cytometry indicated a concurrent increase in fibroblasts. The findings correlated with angiogenesis in TAC hearts; a decrease in capillary density was observed at day 2, with recovery to sham-surgery levels by day 7. Increased pericyte levels, which were observed 2 days after TAC, may mediate this angiogenic transition. Gene expression suggests a coordinated response in growth, extracellular matrix, and angiogenic factors to mediate the observed morphological changes. Our data demonstrate that morphological changes in response to cardiovascular injury occur rapidly, and the present findings allow correlation of specific events that facilitate these changes.
Asunto(s)
Cardiomegalia/patología , Miocardio/patología , Animales , Aorta Torácica/metabolismo , Aorta Torácica/patología , Aorta Torácica/fisiopatología , Aorta Torácica/cirugía , Cardiomegalia/fisiopatología , Proliferación Celular , Colágeno/metabolismo , Constricción Patológica , Modelos Animales de Enfermedad , Matriz Extracelular/metabolismo , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica , Masculino , Ratones , Ratones Endogámicos C57BL , Miocardio/ultraestructura , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Coloración y Etiquetado , Remodelación VentricularRESUMEN
OBJECTIVE: Although significant research has detailed angiogenesis during development and cancer, little is known about cardiac angiogenesis, yet it is critical for survival following pathological insult. The transcription factor c-Myc is a target of anticancer therapies because of its mitogenic and proangiogenic induction. In the current study, we investigate its role in cardiac angiogenesis in a cell-dependent and gene-specific context. METHODS AND RESULTS: Angiogenesis assays using c-Myc-deficient cardiac endothelial cells and fibroblasts demonstrate that c-Myc is essential to vessel formation, and fibroblast-mediated vessel formation is dependent on c-Myc expression in fibroblasts. Gene analyses revealed that c-Myc-mediated gene expression is unique in cardiac angiogenesis and varies in a cell-dependent manner. In vitro 3-dimensional cultures demonstrated c-Myc's role in the expression of secreted angiogenic factors, while also providing evidence for c-Myc-mediated cell-cell interactions. Additional in vivo vascular analyses support c-Myc's critical role in capillary formation and vessel patterning during development and also in response to a pathological stimulus where its expression in myocytes is required for angiogenic remodeling. CONCLUSIONS: These data demonstrate that proper c-Myc expression in cardiac fibroblasts and myocytes is essential to cardiac angiogenesis. These results have the potential for novel therapeutic applications involving the angiogenic response during cardiac remodeling.
Asunto(s)
Vasos Coronarios/citología , Neovascularización Fisiológica/genética , Proteínas Proto-Oncogénicas c-myc/biosíntesis , ARN/genética , Transducción de Señal , Animales , Comunicación Celular , Células Cultivadas , Vasos Coronarios/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas c-myc/genéticaRESUMEN
In vivo VHH discovery approaches have been limited by the lack of methodologies for camelid B cell interrogation. Here, we report a novel application of the Beacon® optofluidic platform to the discovery of heavy-chain-only antibodies by screening alpaca B cells. Methods for alpaca B cell enrichment, culture, IgG2/3 detection, and sequencing were developed and used to discover target-specific VHH from an alpaca immunized with prostate-specific membrane antigen (PSMA) or a second target. PSMA-specific hits were expressed as VHH-Fc and characterized using label-free techniques. Anti-PSMA IgG2/3 titer plateaued on day 153, when on-Beacon IgG2/3 secretion and target binding rates peaked. Of 13 recombinantly expressed VHH-Fc, all but one bound with nanomolar affinity, and five were successfully humanized. Repertoire sequencing uncovered additional variants within the clonal lineages of the validated hits. The establishment of this workflow extends the powerful Beacon technology to enable rapid VHH discovery directly from natural camelid immune repertoires.
RESUMEN
CD276/B7-H3 represents a promising target for cancer therapy based on widespread overexpression in both cancer cells and tumor-associated stroma. In previous preclinical studies, CD276 antibody-drug conjugates (ADCs) exploiting a talirine-type pyrrolobenzodiazepine (PBD) payload showed potent activity against various solid tumors but with a narrow therapeutic index and dosing regimen higher than that tolerated in clinical trials using other antibody-talirine conjugates. Here, we describe the development of a modified talirine PBD-based fully human CD276 ADC, called m276-SL-PBD, that is cross-species (human/mouse) reactive and can eradicate large 500-1,000-mm3 triple-negative breast cancer xenografts at doses 10- to 40-fold lower than the maximum tolerated dose. By combining CD276 targeting with judicious genetic and chemical ADC engineering, improved ADC purification, and payload sensitivity screening, these studies demonstrate that the therapeutic index of ADCs can be substantially increased, providing an advanced ADC development platform for potent and selective targeting of multiple solid tumor types.
Asunto(s)
Inmunoconjugados , Neoplasias , Humanos , Ratones , Animales , Inmunoconjugados/farmacología , Línea Celular Tumoral , Ensayos Antitumor por Modelo de Xenoinjerto , Anticuerpos Monoclonales Humanizados , Factores de Transcripción , Neoplasias/tratamiento farmacológico , Antígenos B7RESUMEN
The permanent cellular constituents of the heart include cardiac fibroblasts, myocytes, endothelial cells, and vascular smooth muscle cells. Previous studies have demonstrated that there are undulating changes in cardiac cell populations during embryonic development, through neonatal development and into the adult. Transient cell populations include lymphocytes, mast cells, and macrophages, which can interact with these permanent cell types to affect cardiac function. It has also been observed that there are marked differences in the makeup of the cardiac cell populations depending on the species, which may be important when examining myocardial remodeling. Current dogma states that the fibroblast makes up the largest cell population of the heart; however, this appears to vary for different species, especially mice. Cardiac fibroblasts play a critical role in maintaining normal cardiac function, as well as in cardiac remodeling during pathological conditions such as myocardial infarct and hypertension. These cells have numerous functions, including synthesis and deposition of extracellular matrix, cell-cell communication with myocytes, cell-cell signaling with other fibroblasts, as well as with endothelial cells. These contacts affect the electrophysiological properties, secretion of growth factors and cytokines, as well as potentiating blood vessel formation. Although a plethora of information is known about several of these processes, relatively little is understood about fibroblasts and their role in angiogenesis during development or cardiac remodeling. In this review, we provide insight into the various properties of cardiac fibroblasts that helps illustrate their importance in maintaining proper cardiac function, as well as their critical role in the remodeling heart.
Asunto(s)
Fibroblastos/metabolismo , Cardiopatías/metabolismo , Miocardio/metabolismo , Transducción de Señal , Animales , Comunicación Celular , Matriz Extracelular/metabolismo , Fibroblastos/patología , Cardiopatías/patología , Cardiopatías/fisiopatología , Humanos , Potenciales de la Membrana , Miocardio/patología , Neovascularización Fisiológica , Remodelación VentricularRESUMEN
BACKGROUND: Rapid deployment of technologies capable of high-throughput and high-resolution screening is imperative for timely response to viral outbreaks. Risk mitigation in the form of leveraging multiple advanced technologies further increases the likelihood of identifying efficacious treatments in aggressive timelines. METHODS: In this study, we describe two parallel, yet distinct, in vivo approaches for accelerated discovery of antibodies targeting the severe acute respiratory syndrome coronavirus-2 spike protein. Working with human transgenic Alloy-GK mice, we detail a single B-cell discovery workflow to directly interrogate antibodies secreted from plasma cells for binding specificity and ACE2 receptor blocking activity. Additionally, we describe a concurrent accelerated hybridoma-based workflow utilizing a DiversimAb™ mouse model for increased diversity. RESULTS: The panel of antibodies isolated from both workflows revealed binding to distinct epitopes with both blocking and non-blocking profiles. Sequence analysis of the resulting lead candidates uncovered additional diversity with the opportunity for straightforward engineering and affinity maturation. CONCLUSIONS: By combining in vivo models with advanced integration of screening and selection platforms, lead antibody candidates can be sequenced and fully characterized within one to three months.
RESUMEN
Disrupting transmission of Borrelia burgdorferi sensu lato complex (B. burgdorferi) from infected ticks to humans is one strategy to prevent the significant morbidity from Lyme disease. We have previously shown that an anti-OspA human mAb, 2217, prevents transmission of B. burgdorferi from infected ticks in animal models. Maintenance of a protective plasma concentration of a human mAb for tick season presents a significant challenge for a preexposure prophylaxis strategy. Here, we describe the optimization of mAb 2217 by amino acid substitutions (2217LS: M428L and N434S) in the Fc domain. The LS mutation led to a 2-fold increase in half-life in cynomolgus monkeys. In a rhesus macaque model, 2217LS protected animals from tick transmission of spirochetes at a dose of 3 mg/kg. Crystallographic analysis of Fab in complex with OspA revealed that 2217 bound an epitope that was highly conserved among the B. burgdorferi, B. garinii, and B. afzelii species. Unlike most vaccines that may require boosters to achieve protection, our work supports the development of 2217LS as an effective preexposure prophylaxis in Lyme-endemic regions, with a single dose at the beginning of tick season offering immediate protection that remains for the duration of exposure risk.
Asunto(s)
Anticuerpos Antibacterianos , Anticuerpos Monoclonales/farmacología , Borrelia burgdorferi , Enfermedad de Lyme , Sustitución de Aminoácidos , Animales , Anticuerpos Antibacterianos/genética , Anticuerpos Antibacterianos/inmunología , Anticuerpos Antibacterianos/farmacología , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Antígenos de Superficie/genética , Antígenos de Superficie/inmunología , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/inmunología , Vacunas Bacterianas/genética , Vacunas Bacterianas/inmunología , Borrelia burgdorferi/genética , Borrelia burgdorferi/inmunología , Modelos Animales de Enfermedad , Humanos , Lipoproteínas/genética , Lipoproteínas/inmunología , Enfermedad de Lyme/tratamiento farmacológico , Enfermedad de Lyme/genética , Enfermedad de Lyme/inmunología , Enfermedad de Lyme/transmisión , Macaca fascicularis , Macaca mulatta , Masculino , Ratones , Ratones Transgénicos , Mutación Missense , Garrapatas/inmunología , Garrapatas/microbiologíaRESUMEN
The formation and the patterning of the coronary vasculature are critical to the development and pathology of the heart. Alterations in cytokine signaling and biomechanical load can alter the vascular distribution of the vessels within the heart. Changes in the physical patterning of the vasculature can have significant impacts on the relationships of the pressure-flow network and distribution of critical growth and survival factors to the tissue. Interleukin-6 (IL-6) is a pleiotropic cytokine that regulates several biological processes, including vasculogenesis. Using both immunohistological and cardioangiographic analyses, we tested the hypothesis that IL-6-loss will result in decreased vessel density, along with changes in vascular distribution. Moreover, given the impact of vascular patterning on pressure-flow and distribution mechanics, we utilized non-Euclidean geometrical fractal analysis to quantify the changes in patterning resulting from IL-6-loss. Our analyses revealed that IL-6-loss results in a decreased capillary density and increase in intercapillary distances, but does not alter vessel size or diameter. We also observed that the IL-6-/- coronary vasculature had a marked increase in fractal dimension (D value), indicating that IL-6-loss alters vascular patterning. Characterization of IL-6-loss on coronary vasculature may lend insight into the role of IL-6 in the formation and patterning of the vascular bed.
Asunto(s)
Vasos Coronarios/crecimiento & desarrollo , Interleucina-6/fisiología , Neovascularización Fisiológica , Angiografía , Animales , Inmunohistoquímica , Interleucina-6/deficiencia , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Antibodies targeting the Ebola virus surface glycoprotein (EBOV GP) are implicated in protection against lethal disease, but the characteristics of the human antibody response to EBOV GP remain poorly understood. We isolated and characterized 349 GP-specific monoclonal antibodies (mAbs) from the peripheral B cells of a convalescent donor who survived the 2014 EBOV Zaire outbreak. Remarkably, 77% of the mAbs neutralize live EBOV, and several mAbs exhibit unprecedented potency. Structures of selected mAbs in complex with GP reveal a site of vulnerability located in the GP stalk region proximal to the viral membrane. Neutralizing antibodies targeting this site show potent therapeutic efficacy against lethal EBOV challenge in mice. The results provide a framework for the design of new EBOV vaccine candidates and immunotherapies.
Asunto(s)
Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Neutralizantes/aislamiento & purificación , Anticuerpos Antivirales/aislamiento & purificación , Ebolavirus/inmunología , Fiebre Hemorrágica Ebola/inmunología , Proteínas del Envoltorio Viral/inmunología , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/uso terapéutico , Anticuerpos Antivirales/química , Anticuerpos Antivirales/uso terapéutico , Formación de Anticuerpos , Complejo Antígeno-Anticuerpo/química , República Democrática del Congo/epidemiología , Brotes de Enfermedades , Vacunas contra el Virus del Ébola/inmunología , Vacunas contra el Virus del Ébola/uso terapéutico , Fiebre Hemorrágica Ebola/epidemiología , Fiebre Hemorrágica Ebola/terapia , Humanos , Inmunización Pasiva , Ratones , Sobrevivientes , Donantes de Tejidos , Proteínas del Envoltorio Viral/química , Virión/inmunologíaRESUMEN
Immunoglobulin G (IgG) has an unusually long serum half-life in comparison to proteins of a similar size. It is well-known that this phenomenon is due to IgG's ability to bind the neonatal Fc receptor (FcRn) in a pH-dependent manner. FcRn binding properties can vary among IgGs, resulting in altered in vivo half-lives, and therefore it would be beneficial to accurately predict the FcRn binding properties of therapeutic IgG monoclonal antibodies (mAbs). Here we describe the development of an in vitro model capable of predicting the in vivo half-life of human IgG. Using a high-throughput biolayer interferometry (BLI) platform, the human FcRn association rate at acidic pH and subsequent dissociation rate at physiological pH was determined for 5 human IgG1 mAbs. Comparing the combined FcRn association and dissociation rates to the Phase 1 clinical study half-lives of the mAbs resulted in a strong correlation. The correlation was also verified in vivo using mice transgenic for human FcRn. The model was used to characterize various factors that may influence FcRn-mAb binding, including mAb variable region sequence differences and constant region glycosylation patterns. Results indicated that the complementarity-determining regions of the heavy chain significantly influence the mAb's FcRn binding properties, while the absence of glycosylation does not alter mAb-FcRn binding. Development of this high-throughput FcRn binding model could potentially predict the half-life of therapeutic IgGs and aid in selection of lead candidates while also serving as a screening tool for the development of mAbs with desired pharmacokinetic properties.