RESUMEN
Downstream purification of therapeutic antibodies requires candidates to be stable under various stress conditions, such as low pH. Current approaches to assess the conformational or colloidal stability of biologics may require significant amounts of material, and the testing may occur over an extended period of time. Furthermore, typical methodologies for early stability testing often do not directly address low pH stability, but focus more on conditions suitable for long-term drug product storage. Here we report a high-throughput method that measures protonation-induced unfolding of ligand binding sites for stability evaluation by surface plasmon resonance or PULSE SPR. This method, which requires only several micrograms of sample, is highly sensitive to the structural integrity of ligand binding sites and correlates well with thermal and chemical conformational stability. Combined with ligands targeting different regions of antibodies, this method allows a comprehensive assessment of antibody domain stability. By applying PULSE SPR, we found that antibodies with different complementarity-determining regions (CDRs), frameworks, formats, and interchain disulfide bonds showed different stabilities under acidic conditions. Additionally, biologics that aggregated in solution also had poor low pH stability. Taken together, PULSE SPR is a high-throughput and low sample consumption method that can be adopted to evaluate antibody domain stability and aggregation at low pH, which are two important aspects of therapeutic biologics.
Asunto(s)
Anticuerpos/química , Resonancia por Plasmón de Superficie , Regiones Determinantes de Complementariedad , Humanos , Concentración de Iones de Hidrógeno , Cinética , Ligandos , TemperaturaRESUMEN
Treatment of stage IV metastatic breast cancer patients is limited to palliative options and represents an unmet clinical need. Here, we demonstrate that pharmacological inhibition of miRNA-10b - a master regulator of metastatic cell viability - leads to elimination of distant metastases in a mouse model of metastatic breast cancer. This was achieved using the miRNA-10b inhibitory nanodrug, MN-anti-miR10b, which consists of magnetic nanoparticles, conjugated to LNA-based miR-10b antagomirs. Intravenous injection of MN-anti-miR10b into mice bearing lung, bone, and brain metastases from breast cancer resulted in selective accumulation of the nanodrug in metastatic tumor cells. Weekly treatments of mice with MN-anti-miR-10b and low-dose doxorubicin resulted in complete regression of pre-existing distant metastases in 65% of the animals and a significant reduction in cancer mortality. These observations were supported by dramatic reduction in proliferation and increase in apoptosis in metastatic sites. On a molecular level, we observed a significant increase in the expression of HOXD10, which is a known target of miRNA-10b. These results represent first steps into the uncharted territory of therapy targeted to the metastatic niche.
Asunto(s)
Neoplasias de la Mama/patología , Neoplasias de la Mama/terapia , Modelos Biológicos , Terapia Molecular Dirigida , Animales , Apoptosis/genética , Neoplasias de la Mama/diagnóstico por imagen , Neoplasias de la Mama/etiología , Línea Celular Tumoral , Proliferación Celular , Modelos Animales de Enfermedad , Doxorrubicina/administración & dosificación , Sistemas de Liberación de Medicamentos , Femenino , Humanos , Ratones , MicroARNs/administración & dosificación , MicroARNs/genética , Nanomedicina , Nanopartículas/química , Metástasis de la Neoplasia , Estadificación de Neoplasias , Imagen Óptica , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: The development of tools for the analysis of microRNA (miRNA) function in tumors can advance our diagnostic and prognostic capabilities. Here, we describe the development of technology for the profiling of miRNA expression in the tumors of live animals. PROCEDURES: The approach is based on miRNA nanosensors consisting of sensor oligonucleotides conjugated to magnetic nanoparticles for systemic delivery. Feasibility was demonstrated for the detection of miR-10b, implicated in epithelial to mesenchymal transition and the development of metastasis. The miR-10b nanosensor was tested in vivo in two mouse models of cancer. In the first model, mice were implanted subcutaneously with MDA-MB-231-luc-D3H2LN tumors, in which miR-10b was inhibited. In the second model, mice were implanted bilaterally with metastatic MDA-MB-231 and nonmetastatic MCF-7 cells. The nanosensors were injected intravenously, and fluorescence intensity in the tumors was monitored over time. RESULTS: We showed that the described nanosensors are capable of discriminating between tumors based on their expression of miR-10b. Radiant efficiency was higher in the miR-10b-active tumors than in the miR-10b-inhibited tumors and in the MDA-MB-231 tumors relative to the MCF-7 tumors. CONCLUSIONS: The described technology provides an important tool that could be used to answer questions about microRNA function in cancer.
Asunto(s)
Técnicas Biosensibles/métodos , MicroARNs/metabolismo , Nanopartículas/química , Neoplasias/genética , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Fluorescencia , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones Desnudos , MicroARNs/genética , Oligonucleótidos/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The ability to detect miRNA expression in live cells would leave these cells available for further manipulation or culture. Here, we describe the design of a miRNA sensor oligonucleotide whose sequence mimics the target mRNA. The sensor has a fluorescent label on one end of the oligo and a quencher on the other. When inside the cell, the sensor is recognized by its cognate miRNA-RISC and gets cleaved, setting the fluorophore free from its quencher. This results in fluorescence "turn on." Since cleavage by the RISC complex is an enzymatic process, the described approach has a very high level of sensitivity (nM). The rate of nonspecific cleavage of the sensor is very slow permitting the collection of meaningful signal over a long period of time.
Asunto(s)
MicroARNs/genética , MicroARNs/metabolismo , Imagen Molecular/métodos , Complejo Silenciador Inducido por ARN/metabolismo , Línea Celular Tumoral , Expresión Génica , Humanos , Microscopía Fluorescente , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/metabolismo , TransfecciónRESUMEN
The therapeutic promise of microRNA (miRNA) in cancer has yet to be realized. In this study, we identified and therapeutically exploited a new role for miR-10b at the metastatic site, which links its overexpression to tumor cell viability and proliferation. In the protocol developed, we combined a miR-10b-inhibitory nanodrug with low-dose anthracycline to achieve complete durable regressions of metastatic disease in a murine model of metastatic breast cancer. Mechanistic investigations suggested a potent antiproliferative, proapoptotic effect of the nanodrug in the metastatic cells, potentiated by a cell-cycle arrest produced by administration of the low-dose anthracycline. miR-10b was overexpressed specifically in cells with high metastatic potential, suggesting a role for this miRNA as a metastasis-specific therapeutic target. Taken together, our results implied the existence of pathways that regulate the viability and proliferation of tumor cells only after they have acquired the ability to grow at distant metastatic sites. As illustrated by miR-10b targeting, such metastasis-dependent apoptotic pathways would offer attractive targets for further therapeutic exploration.
Asunto(s)
Antibióticos Antineoplásicos/administración & dosificación , Neoplasias de la Mama/genética , Doxorrubicina/administración & dosificación , MicroARNs/genética , Nanopartículas , Animales , Apoptosis/genética , Neoplasias de la Mama/patología , Neoplasias de la Mama/terapia , Ciclo Celular/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular , Modelos Animales de Enfermedad , Femenino , Técnicas de Inactivación de Genes , Humanos , Ratones , Metástasis de la Neoplasia , Fenotipo , Carga Tumoral/efectos de los fármacos , Carga Tumoral/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Manipulation of brown adipose tissue (BAT) and browning of white adipose tissue (WAT) can be promising new approaches to counter metabolic disorder diseases in humans. Imaging probes that could consistently monitor BAT mass and browning of WAT are highly desirable. In the course of our imaging probe screening, we found that BAT could be imaged with curcumin analogues in mice. However, the poor BAT selectivity over WAT and short emissions of the lead probes promoted further lead optimization. Limited uptake mechanism studies suggested that CD36/FAT (fatty acid transporter) probably contributed to the facilitated uptake of the probes. By increasing the stereo-hindrance of the lead compound, we designed CRANAD-29 to extend the emission and increase the facilitated uptake, thus increasing its BAT selectivity. Our data demonstrated that CRANAD-29 had significantly improved selectivity for BAT over WAT, and could be used for imaging BAT mass change in a streptozotocin-induced diabetic mouse model, as well as for monitoring BAT activation under cold exposure. In addition, CRANAD-29 could be used for monitoring the browning of subcutaneous WAT (sWAT) induced by ß3-adrenoceptor agonist CL-316, 243.
Asunto(s)
Tejido Adiposo Pardo/fisiología , Curcumina/análogos & derivados , Curcumina/farmacocinética , Colorantes Fluorescentes/farmacocinética , Células 3T3-L1 , Tejido Adiposo Blanco/fisiología , Animales , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Microscopía Fluorescente , Coloración y Etiquetado , Imagen de Cuerpo EnteroRESUMEN
We describe a technology for the profiling of miRNA expression in intact cells. The technology is based on sensor oligonucleotides that are cleavable, completely complementary to a target miRNA, and dual-labeled with a fluorescent dye and a quencher. Upon entering the cell, the sensor oligonucleotide binds its specific miRNA target through complementary base-pairing. This triggers assembly of the endogenous RNA Induced Silencing Complex (RISC) around the miRNA-sensor duplex and cleavage of the sensor oligonucleotide, resulting in separation between the dye and quencher, and a fluorescence turn-on. In the presented feasibility studies, we focus on a specific miRNA (miR-10b) implicated in breast cancer metastasis. Using a human breast adenocarcinoma cell line, we illustrate the application of this technology for miRNA detection with nanomolar sensitivity in both a cell-free system and intact cells.
Asunto(s)
Técnicas Biosensibles , MicroARNs/análisis , Oligonucleótidos/metabolismo , Emparejamiento Base , Línea Celular Tumoral , Sistema Libre de Células , Citometría de Flujo , Colorantes Fluorescentes/química , Humanos , MicroARNs/metabolismo , Microscopía Fluorescente , Oligonucleótidos/química , Ribonucleasas/metabolismoRESUMEN
The establishment and validation of specific markers on the surfaces of pancreatic beta-cells would have a significant impact on the development of agents that specifically target these cells for imaging and/or image-guided therapy in diabetes patient samples. We have recently described unique, cholesterol-stabilized sphingomyelin (SM) patches on the surfaces of beta-cells using the IC2 antibody. To further investigate the utility of SM patches as a unique beta-cell biomarker, we embarked on the current study to correlate the expression of this antigen with the insulin secretory capacity of beta-cells in tissue samples from patients and animals with type 1 and type 2 diabetes and compared this with samples from normal subjects. We found that the locations of SM patches were consistent with the insulin status of islets in all tissues studied. Using immunohistochemistry and staining with an IC2 antibody, we demonstrated a direct correlation between the reduced expression of SM patches and insulin production in diabetic individuals, indicating that the former could potentially serve as a functional biomarker of beta-cells. We believe that our results have significant implications for the further development of ligands with SM specificity for the non-invasive functional assessment of beta-cells and/or for targeted therapeutic delivery in diabetic patients.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Esfingomielinas/metabolismo , Animales , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Tipo 1/patología , Diabetes Mellitus Tipo 2/patología , Femenino , Humanos , Secreción de Insulina , Células Secretoras de Insulina/patología , Ratones , Ratones Endogámicos NOD , Ratones Transgénicos , Ratas , Células Tumorales CultivadasRESUMEN
Increasing evidence links the complement system with complications of human diabetes. The complement regulatory protein CD59, an inhibitor of formation of membrane attack complex (MAC), is inhibited by hyperglycemia-induced glycation fostering increased deposition of MAC, a major effector of complement-mediated tissue damage. CD59, an ubiquitous GPI-anchored membrane protein, is shed from cell membranes by phospholipases generating a soluble form present in blood and urine. We established an enzyme-linked immunosorbent assay (ELISA) to measure serum/plasma glycated human CD59 (hCD59) (GCD59) and evaluated its potential as a diabetes biomarker. We used a synthetic peptide strategy to generate (a) a mouse monoclonal antibody to capture hCD59, (b) a rabbit monoclonal antibody to detect GCD59, and (c) a GCD59 surrogate for assay standardization. ELISA conditions were optimized for precision, reproducibility, and clinical sensitivity. The clinical utility of the assay was initially evaluated in 24 subjects with or without diabetes and further validated in a study that included 100 subjects with and 90 subjects without a diagnosis of diabetes. GCD59 (a) was significantly higher in individuals with than in individual without diabetes, (b) was independently associated with HbA1c, and (c) identified individuals with diabetes with high specificity and sensitivity. We report the development and standardization of a novel, sensitive, and specific ELISA for measuring GCD59 in blood. The assay distinguished individuals with diabetes from those without, and showed strong correlation between GCD59 and HbA1c. Because GCD59 likely contributes to the pathogenesis of diabetes complications, measurement of blood levels of GCD59 may be useful in the diagnosis and management of diabetes.
Asunto(s)
Antígenos CD59/sangre , Diabetes Mellitus Tipo 2/sangre , Adolescente , Adulto , Animales , Anticuerpos Monoclonales de Origen Murino/química , Anticuerpos Monoclonales de Origen Murino/inmunología , Biomarcadores/sangre , Biomarcadores/química , Antígenos CD59/química , Antígenos CD59/inmunología , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Hemoglobina Glucada/inmunología , Hemoglobina Glucada/metabolismo , Glicosilación , Humanos , Masculino , Ratones , Persona de Mediana Edad , Ratas , Sensibilidad y EspecificidadRESUMEN
To devise successful imaging and therapeutic strategies, the identification of ß-cell surface markers is one of the challenges in diabetes research that has to be resolved. We previously showed that IC2, a rat monoclonal IgM antibody, can be used for ex vivo determination of ß-cell mass by imaging. Further progress toward the development of an antibody-based imaging agent was hampered by the lack of knowledge regarding the nature and composition of the IC2 antigen. Here, we show a series of systematic experiments involving classical lipid extraction and chromatography techniques combined with immunochemistry, which led to the identification of sphingomyelin as the target antigen assembled in the form of patches on the ß-cell surface. Our findings were verified by modulating SM by enzymatic cleavage, downregulation, upregulation, and perturbation of membrane SM and observation of corresponding changes in IC2 binding. Cholesterol participates in stabilization of these patches, as its removal results in loss of IC2 binding. We believe that these findings have implications for identifying future ligands for the proposed antigen for imaging purposes as well as for potential therapy, as sphingomyelin has been shown to play a role in the apoptotic cascade in pancreatic ß cells.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos/inmunología , Antígenos/aislamiento & purificación , Células Secretoras de Insulina/inmunología , Esfingomielinas/inmunología , Esfingomielinas/aislamiento & purificación , Animales , Anticuerpos Monoclonales/química , Antígenos/química , Biomarcadores/metabolismo , Línea Celular , Cromatografía/métodos , Inhibidores Enzimáticos/farmacología , Fumonisinas/farmacología , Humanos , Células Secretoras de Insulina/química , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/efectos de los fármacos , Lípidos/química , Ratones , Ratones Endogámicos C57BL , Unión Proteica , Ratas , Esfingomielinas/química , Toxinas Biológicas/química , beta-Ciclodextrinas/farmacologíaRESUMEN
The incidence of life threatening mycoses caused by opportunistic fungi has increased dramatically in recent years with Candida and Aspergillus being the most commonly encountered species. Candida albicans ranks among the four most common causes of bloodstream infections and is responsible for vulvovaginal candidiasis in the majority of women in their reproductive years. Limited spectrum of antifungal activity of currently available antifungals and emergence of resistance has become a serious problem. Therefore, in search of an alternative form of treatment of candidiasis, in the present study a monoclonal antibody (MAb-G5) of IgA isotype was identified from the hybridoma produced by the fusion of lymphocytes of C. albicans immunized mouse with Sp2/O cells. The MAb-G5 exhibited in vitro candidacidal activity and was also found to be useful for treatment and prophylactic use under experimental conditions in vivo.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Candida albicans/inmunología , Candidiasis Vulvovaginal/terapia , Proteínas Fúngicas/inmunología , Inmunización Pasiva/métodos , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/aislamiento & purificación , Western Blotting , Candidiasis Vulvovaginal/inmunología , Supervivencia Celular/inmunología , Pared Celular/inmunología , Pared Celular/microbiología , Ensayo de Inmunoadsorción Enzimática , Epítopos/inmunología , Femenino , Citometría de Flujo , Formazáns/química , Inmunoglobulina A/farmacología , Inmunoglobulina A/uso terapéutico , Ratones , Ratones Endogámicos BALB C , Sales de Tetrazolio/químicaRESUMEN
It is becoming clear that antimicrobial peptides are important components of the innate defences of all species of life. They kill very rapidly, do not easily select resistant mutants and are synergistic with potentially toxic conventional therapeutic agents against microbes. This paper describes an attempt to expand a lead hexapeptide motif synthesized through combinatorial approach. A cationic peptide H-Arg-Trp-Trp-Arg-D-Trp-D-Phe-Ile-D-Phe-His-NH2 was found to be active with a therapeutic index of >17. I was proposed that the combination of peptide with known antifungal agents may identify synergistic combinations that would ideally reduce the dosage of conventional antifungals as well as their associated toxicity. Nine different pathogenic strains and species of Candida and two of Cryptococcus neoformans were employed in chequerboard method and in time kill assays to evaluate the synergistic effect of the lead peptide in combination with amphotericin B, 5-flucytosine, ketoconazole and fluconazole. We found synergistic interaction between the peptide and all four drugs against Cryptococcus isolates whilst both synergistic and additive combinations occurred when Candida isolates were used.