RESUMEN
Myofibroblasts, or activated fibroblasts, play a critical role in the process of renal fibrosis. Targeting myofibroblasts to inhibit their activation or induce specific cell death has been considered to be an effective strategy to attenuate renal fibrosis. However, specific biomarkers for myofibroblasts are needed to ensure the efficacy of these strategies. Here, we verified that CD248 was mainly expressed in myofibroblasts in patients with chronic kidney disease, which was inversely correlated with renal function. The same result was also confirmed in renal fibrotic mice induced by unilateral ureteral obstruction and aristolochic acid nephropathy. By using an antibody-drug conjugate (ADC) named IgG78-DM1, in which maytansinoid (DM1) was linked to a fully human antibody IgG78 through an uncleavable SMCC linker, we demonstrated that it could effectively bind with and kill CD248+ fibroblasts in vitro and alleviate renal fibrosis in mice models. Besides, we confirmed that IgG78-DM1 had qualified biosafety in vivo. Our results confirmed that CD248 can be used as a specific marker for myofibroblasts, and specific killing of CD248+ myofibroblasts by IgG78-DM1 has excellent anti-fibrotic effect in renal fibrotic mice. Our study expanded the application of ADC and provided a novel strategy for the treatment of renal fibrosis.
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Antígenos CD/metabolismo , Antígenos de Neoplasias/metabolismo , Sistemas de Liberación de Medicamentos , Inmunoconjugados/farmacología , Maitansina/farmacología , Miofibroblastos/metabolismo , Insuficiencia Renal Crónica , Animales , Fibrosis , Masculino , Ratones , Insuficiencia Renal Crónica/tratamiento farmacológico , Insuficiencia Renal Crónica/metabolismoRESUMEN
BACKGROUND: Chronic liver injury induces pathological repair, resulting in fibrosis, during which hepatic stellate cells (HSCs) are activated and transform into myofibroblasts. CD248 is mainly expressed on myofibroblasts and was considered as a promising target to treat fibrosis. The primary aim of this study was to generate a CD248 specific antibody-drug conjugate (ADC) and evaluate its therapeutic efficacy for liver fibrosis and its safety in vivo. METHODS: CD248 expression was examined in patients with liver cirrhosis and in mice with CCl4-induced liver fibrosis. The ADC IgG78-DM1, which targets CD248, was prepared and its bioactivity on activated primary HSCs was studied. The anti-fibrotic effects of IgG78-DM1 on liver fibrosis were evaluated in CCl4-induced mice. The reproductive safety and biosafety of IgG78-DM1 were also evaluated in vivo. RESULTS: CD248 expression was upregulated in patients with liver cirrhosis and in CCl4-induced mice, and was mainly expressed on alpha smooth muscle actin (α-SMA)+ myofibroblasts. IgG78-DM1 was successfully generated, which could effectively bind with and kill CD248+ activated HSCs in vitro and inhibit liver fibrosis in vivo. In addition, IgG78-DM1 was demonstrated to have qualified biosafety and reproductive safety in vivo. CONCLUSIONS: Our study demonstrated that CD248 could be an ideal target for myofibroblasts in liver fibrosis, and CD248-targeting IgG78-DM1 had excellent anti-fibrotic effects in mice with liver fibrosis. Our study provided a novel strategy to treat liver fibrosis and expanded the application of ADCs beyond tumors.
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Inmunoconjugados , Miofibroblastos , Animales , Antígenos CD/metabolismo , Antígenos de Neoplasias/efectos adversos , Antígenos de Neoplasias/metabolismo , Fibrosis , Células Estrelladas Hepáticas/metabolismo , Humanos , Inmunoconjugados/efectos adversos , Inmunoconjugados/metabolismo , Hígado/metabolismo , Cirrosis Hepática/tratamiento farmacológico , Cirrosis Hepática/metabolismo , Ratones , Miofibroblastos/metabolismoRESUMEN
Tumor endothelial marker 1 (TEM1) has been identified as a novel surface marker upregulated on the blood vessels and stroma in many solid tumors. We previously isolated a novel single-chain variable fragment (scFv) 78 against TEM1 from a yeast display scFv library. Here we evaluated the potential applications of scFv78 as a tool for tumor molecular imaging, immunotoxin-based therapy and nanotherapy. Epitope mapping, three-dimensional (3D) structure docking and affinity measurements indicated that scFv78 could bind to both human and murine TEM1, with equivalent affinity, at a well-conserved conformational epitope. The rapid internalization of scFv78 and scFv78-labeled nanoparticles was triggered after specific TEM1 binding. The scFv78-saporin immunoconjugate also exerted dose-dependent cytotoxicity with high specificity to TEM1-positive cells in vitro. Finally, specific and sensitive tumor localization of scFv78 was confirmed with optical imaging in a mouse tumor model that has highly endogenous mTEM1 expression in the vasculature. Our data indicate that scFv78, the first fully human anti-TEM1 recombinant antibody, recognizes both human and mouse TEM1 and has unique and favorable features that are advantageous for the development of imaging probes or antibody-toxin conjugates for a large spectrum of human TEM1-positive solid tumors.
RESUMEN
Tumor endothelial marker 1 (TEM1) has been identified as a novel surface marker upregulated on the blood vessels and stroma in many solid tumors. We previously isolated a novel single-chain variable fragment (scFv) 78 against TEM1 from a yeast display scFv library. Here, we evaluated the potential applications of scFv78 as a tool for tumor molecular imaging, immunotoxin-based therapy and nanotherapy. Epitope mapping, three-dimensional structure docking and affinity measurements indicated that scFv78 could bind to both human and murine TEM1, with equivalent affinity, at a well-conserved conformational epitope. The rapid internalization of scFv78 and scFv78-labeled nanoparticles was triggered after specific TEM1 binding. The scFv78-saporin immunoconjugate also exerted dose-dependent cytotoxicity with high specificity to TEM1-positive cells in vitro. Finally, specific and sensitive tumor localization of scFv78 was confirmed with optical imaging in a tumor mouse model that has highly endogenous mTEM1 expression in the vasculature. Our data indicated that scFv78, the first fully human anti-TEM1 recombinant antibody, recognizes both human and mouse TEM1 and has unique and favorable features that are advantageous for the development of imaging probes or antibody-toxin conjugates for a large spectrum of human TEM1-positive solid tumors.
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Antígenos CD/inmunología , Antígenos de Neoplasias/inmunología , Fragmentos de Inmunoglobulinas/inmunología , Inmunotoxinas/inmunología , Nanopartículas/administración & dosificación , Proteínas de Neoplasias/inmunología , Neoplasias/inmunología , Neoplasias/terapia , Secuencia de Aminoácidos , Animales , Antígenos CD/biosíntesis , Epítopos/inmunología , Humanos , Inmunoterapia/métodos , Inmunotoxinas/farmacocinética , Ratones , Ratones Desnudos , Simulación del Acoplamiento Molecular , Nanopartículas/metabolismo , Proteínas de Neoplasias/biosíntesisRESUMEN
Phage display technology (PDT), a combinatorial screening approach, provides a molecular diversity tool for creating libraries of peptides/proteins and discovery of new recombinant therapeutics. Expression of proteins such as monoclonal antibodies (mAbs) on the surface of filamentous phage can permit the selection of high affinity and specificity therapeutic mAbs against virtually any target antigen. Using a number of diverse selection platforms (e.g. solid phase, solution phase, whole cell and in vivo biopannings), phage antibody libraries (PALs) from the start point provides great potential for the isolation of functional mAb fragments with diagnostic and/or therapeutic purposes. Given the pivotal role of PDT in the discovery of novel therapeutic/diagnostic mAbs, in the current review, we provide an overview on PALs and discuss their impact in the advancement of engineered mAbs.
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Anticuerpos Monoclonales , Inmunoterapia , Biblioteca de Péptidos , Animales , Humanos , RatonesRESUMEN
Over the past decade, yeast display technology has emerged as a powerful tool for the isolation of high-affinity immunoglobulin fragments with potential utility as clinical diagnostic and therapeutic reagents. Despite significant refinement of the various methodologies underpinning library construction and selections, certain aspects remain challenging and process limiting. We have sought to significantly improve the robustness of the single-chain Fv (scFv) library construction step by overcoming the technical inefficiencies frequently encountered during the PCR-mediated assembly of scFvs from the discrete heavy and light V-domain repertoires. Using a novel primer set designed to provide maximum amplification coverage of the known germ-line V-domain repertoire, we have exploited the potential of the in vivo homologous gap-repair apparatus of Saccharomyces cerevisiae to assemble intact scFvs directly from co-transformed PBMC-derived VH, VL, and linearized vector component fragments. We have successfully applied this three-fragment assembly strategy to construct a large (>10(9)) scFv yeast display library from the ascites immune repertoire of ovarian cancer patients and validated the approach by applying FACS-based sorting to readily isolate scFvs that recognize various tumor marker antigens (TMAs). It is expected that this simplified construction method may find general utility, both for de novo scFv library construction and for subsequent combinatorial affinity maturation manipulations that require more than two fragments.
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Antígenos de Neoplasias/análisis , Técnicas de Visualización de Superficie Celular/métodos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/metabolismo , Ascitis , Linfocitos B/inmunología , Femenino , Humanos , Región Variable de Inmunoglobulina , Neoplasias Ováricas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Recombinación GenéticaRESUMEN
RATIONALE: Phosphorylation of ß(2)-adrenergic receptor (ß(2)AR) by a family of serine/threonine kinases known as G protein-coupled receptor kinase (GRK) and protein kinase A (PKA) is a critical determinant of cardiac function. Upregulation of G protein-coupled receptor kinase 2 (GRK2) is a well-established causal factor of heart failure, but the underlying mechanism is poorly understood. OBJECTIVE: We sought to determine the relative contribution of PKA- and GRK-mediated phosphorylation of ß(2)AR to the receptor coupling to G(i) signaling that attenuates cardiac reserve and contributes to the pathogenesis of heart failure in response to pressure overload. METHODS AND RESULTS: Overexpression of GRK2 led to a G(i)-dependent decrease of contractile response to ßAR stimulation in cultured mouse cardiomyocytes and in vivo. Importantly, cardiac-specific transgenic overexpression of a mutant ß(2)AR lacking PKA phosphorylation sites (PKA-TG) but not the wild-type ß(2)AR (WT-TG) or a mutant ß(2)AR lacking GRK sites (GRK-TG) led to exaggerated cardiac response to pressure overload, as manifested by markedly exacerbated cardiac maladaptive remodeling and failure and early mortality. Furthermore, inhibition of G(i) signaling with pertussis toxin restores cardiac function in heart failure associated with increased ß(2)AR to G(i) coupling induced by removing PKA phosphorylation of the receptor and in GRK2 transgenic mice, indicating that enhanced phosphorylation of ß(2)AR by GRK and resultant increase in G(i)-biased ß(2)AR signaling play an important role in the development of heart failure. CONCLUSIONS: Our data show that enhanced ß(2)AR phosphorylation by GRK, in addition to PKA, leads the receptor to G(i)-biased signaling, which, in turn, contributes to the pathogenesis of heart failure, marking G(i)-biased ß(2)AR signaling as a primary event linking upregulation of GRK to cardiac maladaptive remodeling, failure and cardiodepression.
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Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Insuficiencia Cardíaca/enzimología , Miocitos Cardíacos/enzimología , Receptores Adrenérgicos beta 2/metabolismo , Transducción de Señal , Agonistas Adrenérgicos beta/farmacología , Animales , Cardiomegalia/enzimología , Cardiomegalia/genética , Cardiomegalia/fisiopatología , Células Cultivadas , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Quinasa 2 del Receptor Acoplado a Proteína-G/genética , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/antagonistas & inhibidores , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/fisiopatología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación , Contracción Miocárdica , Miocitos Cardíacos/efectos de los fármacos , Toxina del Pertussis/farmacología , Fosforilación , Receptores Adrenérgicos beta 2/genética , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Transfección , Regulación hacia Arriba , Función Ventricular Izquierda , Presión Ventricular , Remodelación VentricularRESUMEN
In spite of popularity of TNF-α antagonist in the treatment of rheumatoid arthritis (RA), their modes of action are not fully understood. In the present study, we further explore the effects of gene transfer route of a TNF-α antagonist on arthritis. Recombinant adeno-associated virus 2 (rAAV2) encoding rat TNF receptor-immunoglobulin Fc (ratTNFR:Fc) fusion gene was injected intraarticularly in rats with collagen-induced arthritis (CIA). As revealed by examination of the clinical, radiographical, and histological aspects, local gene transfer of rAAV2/ratTNFR:Fc ameliorated the arthritis symptoms and inhibited the development of CIA. Compared with the vector control group, expressions of TNF-α, IL-1, and IFN-γ were down-regulated, and IL-10 release was up-regulated in the rAAV2/ratTNFR:Fc-treated group. Furthermore, administration of rAAV2/ratTNFR:Fc ameliorated the enlargement of spleen and significantly reduced spleen cell proliferation. Low level of nitric oxide (NO) in spleen was observed in CIA rats following the delivery of rAAV2/ratTNFR:Fc when compared to the vector control group. This study provides the evidence that intraarticular delivery of rAAV2/ratTNFR:Fc suppress the progression of arthritis by restoring the balance between pro-inflammatory and anti-inflammatory cytokines and inhibiting spleen cell proliferation. Our findings also implicate that the down-regulation of NO release on arthritis is involved in the anti-inflammatory mechanisms of TNF-α antagonist.
Asunto(s)
Inmunidad Adaptativa/fisiología , Artritis Experimental/prevención & control , Artritis Experimental/fisiopatología , Terapia Genética , Inmunoglobulina G/genética , Inmunoglobulina G/uso terapéutico , Inflamación/fisiopatología , Receptores del Factor de Necrosis Tumoral/genética , Receptores del Factor de Necrosis Tumoral/uso terapéutico , Animales , Artritis Experimental/metabolismo , Proliferación Celular , Citocinas/metabolismo , Dependovirus/genética , Modelos Animales de Enfermedad , Etanercept , Femenino , Técnicas de Transferencia de Gen , Inmunoglobulina G/administración & dosificación , Inyecciones Intraarticulares , Óxido Nítrico/metabolismo , Ratas , Ratas Wistar , Receptores del Factor de Necrosis Tumoral/administración & dosificación , Bazo/metabolismo , Bazo/patología , Resultado del TratamientoRESUMEN
Fibroblasts and macrophages play key roles in the development of hepatocellular carcinoma (HCC). However, cross-talk between these two kinds of cells has not been well studied. Endosialin (CD248/TEM1) is a transmembrane glycoprotein that is expressed in certain cancer cells, tumor stromal cells, and pericytes. In this study, we found that endosialin is mainly expressed in cancer-associated fibroblasts (CAF) in HCC and its expression inversely correlates with patient prognosis. Endosialin interacted with CD68 to recruit macrophages and regulated expression of GAS6 in CAFs to mediate M2 polarization of macrophages. The fully human antibody IgG78 bound glycosylated endosialin and induced its internalization in CAFs, thus weakening the cross-talk between CAFs and macrophages. In subcutaneous and orthotopic xenograft models of HCC in nude mice, treatment with IgG78 significantly inhibited tumor growth. These results indicate that endosialin-positive CAFs promote HCC progression and highlight IgG78 as a promising therapeutic candidate for HCC treatment. SIGNIFICANCE: These findings highlight CAF-expressed endosialin as a primary regulator of macrophage recruitment and polarization and demonstrate endosialin inhibition as a potential treatment strategy for HCC. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/18/3892/F1.large.jpg.
Asunto(s)
Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Antígenos de Neoplasias/metabolismo , Fibroblastos Asociados al Cáncer/metabolismo , Carcinoma Hepatocelular/metabolismo , Comunicación Celular , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Neoplasias Hepáticas/metabolismo , Macrófagos Asociados a Tumores/metabolismo , Animales , Especificidad de Anticuerpos , Antígenos CD/efectos de los fármacos , Antígenos CD/inmunología , Antígenos de Neoplasias/efectos de los fármacos , Antígenos de Neoplasias/inmunología , Fibroblastos Asociados al Cáncer/fisiología , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/terapia , Línea Celular Tumoral , Movimiento Celular , Polaridad Celular , Progresión de la Enfermedad , Glicosilación , Humanos , Inmunoglobulina G/uso terapéutico , Factores Inmunológicos/uso terapéutico , Hígado/metabolismo , Hígado/patología , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/terapia , Ratones , Ratones Desnudos , Macrófagos Asociados a Tumores/fisiologíaRESUMEN
AIMS: A combination of biomarker and instrument technology diagnosis methods, especially antigen-targeted imaging methods, is required to increase the accuracy of the diagnosis of cancer. Currently, the targeting efficiency is limited by the conjugation methods used for the conjugation of antibodies and imaging materials. Here, a simple strategy for the conjugation of a probe and a single-chain fragment antibody (scFv) that does not change the characteristics of the antibody was shown. MAIN METHODS: An ScFv was conjugated with superparamagnetic iron oxide (SPIO) or indocyanine green (ICG) via a linker by utilizing the reaction between cysteine and maleimide. The characterization of the probe was performed by flow cytometry, confocal imaging, optical imaging and magnetic resonance imaging (MRI). KEY FINDINGS: After conjugation, the scFv retained high affinity, antigen specificity, and strong internalization ability. The application of the conjugated probe was also confirmed by optical imaging and MRI. SIGNIFICANCE: The proposed strategy provides a simple method for the production of high efficiency antigen-targeted imaging probes for tumor diagnosis.
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Anticuerpos Monoclonales/química , Imagen por Resonancia Magnética/métodos , Anticuerpos de Cadena Única/química , Anticuerpos Monoclonales/metabolismo , Línea Celular Tumoral , Medios de Contraste , Compuestos Férricos/química , Citometría de Flujo , Humanos , Verde de Indocianina/química , Nanopartículas de Magnetita , Anticuerpos de Cadena Única/metabolismoRESUMEN
INTRODUCTION: Prostate-specific membrane antigen (PSMA) was originally found to be specifically expressed in normal prostate, and its expression was upregulated in almost all stages of prostate cancer. In recent years, PSMA was also found to be expressed in tumor-associated vasculature in many nonprostatic solid tumors. However, the expression pattern of PSMA in hepatocellular carcinoma (HCC) is not well studied. METHODS: In this study, we examined PSMA expression in 103 HCC tissues using immunohistochemical staining and analyzed the association between PSMA expression and other clinicopathological features and prognosis. RESULTS: Among the 103 cases, 27 cases (26%) showed PSMA expression in more than 50% of tumor-associated vasculature, 49 cases (48%) showed PSMA expression in less than 50% of vasculature, and 27 cases (26%) did not have detectable PSMA expression. Vascular PSMA expression was associated with several clinicopathological features, such as tumor stage, tumor differentiation, lymph node metastasis, and Ki-67 index. Furthermore, high vascular PSMA expression was also associated with poor prognosis in patients with HCC. Univariate and multivariate analyses showed that high vascular PSMA expression can be used as an independent prognostic marker for HCC. DISCUSSION: Our study provides the evidence that PSMA is specifically expressed in tumor-associated vasculature of HCC, and vascular PSMA expression may be used as a novel prognostic marker and a vascular therapeutic target for HCC.
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Antígenos de Superficie/metabolismo , Biomarcadores de Tumor/metabolismo , Carcinoma Hepatocelular/mortalidad , Glutamato Carboxipeptidasa II/metabolismo , Neoplasias Hepáticas/mortalidad , Neovascularización Patológica/patología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Antígenos de Superficie/análisis , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/antagonistas & inhibidores , Carcinoma Hepatocelular/irrigación sanguínea , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/terapia , Femenino , Estudios de Seguimiento , Glutamato Carboxipeptidasa II/análisis , Glutamato Carboxipeptidasa II/antagonistas & inhibidores , Hepatectomía , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Hígado/irrigación sanguínea , Hígado/patología , Hígado/cirugía , Neoplasias Hepáticas/irrigación sanguínea , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/terapia , Metástasis Linfática/patología , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Neovascularización Patológica/mortalidad , Neovascularización Patológica/terapia , Pronóstico , Factores de Tiempo , Adulto JovenRESUMEN
Prostate cancer is the most commonly diagnosed malignancy in men and the second leading cause of cancer-related death. It is of vital importance to develop new strategies for prostate cancer therapy. PSMA (prostate-specific membrane antigen) is specifically expressed in prostate cancer and the neovasculature of certain cancer types, thus is considered to be an ideal target for cancer therapy. In our previous study, we have obtained a PSMA-specific single-chain variable fragment (scFv), named gy1, from a large yeast display naïve human scFv library. In this study, we reconstructed the PSMA scFv into a fully human antibody (named PSMAb) and evaluated its characterization both in vitro and in vivo We showed that PSMAb can specifically bind with and internalize into PSMA+ cells. The binding affinity of PSMAb is measured to be at nanomolar level, and PSMAb has very good thermostability. In vivo study showed that near IR dye-labeled PSMAb can specifically localize at PSMA+ tumors, and the application of PSMAb in vivo significantly inhibited the growth of PSMA+ tumors, but not PSMA- tumors. At the studied doses, no obvious toxicity was observed when applied in vivo, as shown by the relative normal liver and kidney function and normal structure of important organs, shown by hematoxylin and eosin staining. In addition, PSMAb may inhibit tumor growth through antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity mechanisms. Our results indicated that the novel fully human antibody, PSMAb, deserve further study for PSMA-targeted diagnosis and therapy for prostate cancer and other cancer types with vascular PSMA expression.
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Glutamato Carboxipeptidasa II/genética , Glicoproteínas de Membrana/genética , Neoplasias de la Próstata/genética , Animales , Línea Celular Tumoral , Humanos , Masculino , Ratones , Ratones Desnudos , Neoplasias de la Próstata/patologíaRESUMEN
Background and Aims: Prostate specific membrane antigen (PSMA) is specifically expressed on prostate epithelial cells and markedly overexpressed in almost all prostate cancers. TRIM24 is also up-regulated from localized prostate cancer to metastatic castration-resistant prostate cancer (CRPC). Because of the high relevance of TRIM24 for cancer development and the universal expression of PSMA in CPRC, we investigated the efficacy of human monoclonal PSMA antibody (PSMAb)-based platform for the targeted TRIM24 siRNA delivery and its therapeutic efficacy in CRPC in vivo and in vitro. Methods: The therapeutic complexes were constructed by conjugating PSMAb and sulfo-SMCC-protamine, and encapsulating TRIM24 siRNA. Flow cytometry, immunofluorescence, and fluorescence imaging were performed to detect the receptor-binding, internalization, and targeted delivery of PSMAb-sulfo-SMCC-protamine (PSP)-FAM-siRNA complex (PSPS) in vitro and in vivo. CCK-8, plate-colony formation, apoptosis, cell cycle, and Transwell assays were performed to evaluate the therapeutic potential of the PSP-TRIM24 siRNA complex in vitro, whereas the in vivo therapeutic efficacy was monitored by small animal imaging, radiography, and micro CT. Results: We confirmed that PSP could efficiently protect siRNA from enzymatic digestion, enable targeted delivery of siRNA, and internalize and release siRNA into PSMA-positive (PSMA+) prostate cancer cells in vitro and in vivo. Silencing TRIM24 expression by the PSP-TRIM24 siRNA complex could dramatically suppress proliferation, colony-formation, and invasion of PSMA+ CRPC cells in vitro, and inhibit tumor growth of PSMA+ CRPC xenografts and bone loss in PSMA+ CRPC bone metastasis model without obvious toxicity at therapeutic doses in vivo. Conclusion: PSMAb mediated TRIM24 siRNA delivery platform could significantly inhibit cell proliferation, colony-formation, and invasion in PSMA+ CRPC in vitro and suppressed tumor growth and bone loss in PSMA+ CRPC xenograft and bone metastasis model.
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Anticuerpos Monoclonales/administración & dosificación , Antígenos de Superficie/inmunología , Proteínas Portadoras/antagonistas & inhibidores , Glutamato Carboxipeptidasa II/inmunología , Terapia Molecular Dirigida/métodos , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , ARN Interferente Pequeño/administración & dosificación , Animales , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Humanos , Masculino , Ratones Desnudos , Modelos Teóricos , Usos Terapéuticos , Ensayo de Tumor de Célula Madre , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Xihuang pill (XHP), a traditional Chinese herbal formula, has long been used as an effective agent against multiple tumors. The aim of this study is to evaluate the effects of XHP on the growth inhibition and apoptosis in glioblastoma U-87 MG cells. Gas chromatography-mass spectrometry (GC-MS) was performed for constituent analysis of XHP. Cell viability, cell cycle arrest, generation of reactive oxygen species (ROS), and apoptosis were measured by CCK-8 assay, PI/RNase staining, DCFH-DA assay, TUNEL assay, Annexin V-FITC/PI double staining, and JC-1 assay, respectively. The role of XHP in the regulation of Akt/mTOR/FOXO1 interaction was clarified by using Western Blotting (WB), immunofluorescence (IF), pharmacological inhibitor or antioxidant, and siRNA silencing. The results suggested that XHP could inhibit U-87 MG cells growth and arrest cells in S-phase cell cycle significantly and that the generation of ROS, collapse of mitochondrial membrane potential, enhancement of Bax/Bcl-xL ratio, and reduction of the precursor forms of caspase-9 and caspase-3 caused by XHP prompted that a ROS-mediated mitochondria-dependent apoptosis was possibly involved. Furthermore, XHP affected the Akt/mTOR/FOXO1 pathway via inhibiting the phosphorylation of Akt, mTOR, and FOXO1 and increasing both prototype and nuclear translocation of FOXO1. Inhibition of Akt, mTOR, and FOXO1 by specific inhibitors or siRNA could interpose the apoptotic induction. In conclusion, we demonstrate for the first time that XHP may regulate glioblastoma U-87 MG cell apoptosis via ROS-mediated Akt/mTOR/FOXO1 pathway.
RESUMEN
A single chain Fv fragment (scFv) is a fusion of the variable regions of heavy (VH ) and light (VL ) chains of immunoglobulins. They are important elements of chimeric antigen receptors for cancer therapy. We sought to produce a panel of 16 extracellular protein domains of tumor markers for use in scFv yeast library screenings. A series of vectors comprising various combinations of expression elements was made, but expression was unpredictable and more than half of the protein domains could not be produced using any of the constructs. Here we describe a novel fusion expression system based on mouse TEM7 (tumor endothelial marker 7), which could facilitate protein expression. With this approach we could produce all but one of the tumor marker domains that could not otherwise be expressed. In addition, we demonstrated that the tumor associated antigen hFZD10 produced as a fusion protein with mTEM7 could be used to enrich scFv antibodies from a yeast display library. Collectively our study demonstrates the potential of specific fusion proteins based on mTEM7 in enabling mammalian cell production of tumor targeting protein domains for therapeutic development.
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Receptores Frizzled/biosíntesis , Neoplasias/tratamiento farmacológico , Receptores de Superficie Celular/biosíntesis , Anticuerpos de Cadena Única/metabolismo , Animales , Células Cultivadas , Células HEK293 , Humanos , Ratones , Neoplasias/metabolismo , Dominios Proteicos , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/aislamiento & purificaciónRESUMEN
To develop reagents for early diagnosis and therapeutic drugs against SARS-associated coronavirus (SARS-CoV), a large (3 x 10(9)) immunized human antibody library was constructed from peripheral blood mononuclear cells from six SARS convalescent patients. A single chain variable fragment antibody (N18) with high affinity against N protein of SARS-CoV was isolated. Sequence analysis revealed that the VL gene was composed of VL3h (V lambda subgroup) and JL2 regions and the VH gene was composed of VH1-69 (VH1 subgroup), D2-15, D3-22 and JH6 regions. Soluble N18 antibody was expressed in Escherichia coli HB2151, purified by Ni-NTA affinity chromatography and verified by SDS-PAGE and Western blot. The potential application for early diagnosis was evaluated using N protein capture ELISA in which N18 antibody demonstrated high sensitive activity in detecting N protein of SARS-CoV. Finally, the potential usefulness of the N18 antibody in prophylaxis, vaccine design and therapy of SARS is discussed.
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Anticuerpos Antivirales/inmunología , Especificidad de Anticuerpos , Fragmentos de Inmunoglobulinas/inmunología , Proteínas de la Nucleocápside/inmunología , Biblioteca de Péptidos , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/inmunología , Secuencia de Aminoácidos , Secuencia de Bases , Proteínas de la Nucleocápside de Coronavirus , Humanos , Fragmentos de Inmunoglobulinas/química , Fragmentos de Inmunoglobulinas/genética , Datos de Secuencia Molecular , Análisis de Secuencia de ADNRESUMEN
The development of patient-tailored anti-tumor therapies requires large-scale production of antibodies for the purpose of screening specific tumor antibodies. Thus, the generation of a single chain fragment of variable region (scFv) library with a large repertoire is required for the selection of specific antibodies with high affinity. Presently, the generation of large scFv libraries is impeded by the low efficiency of cloning PCR fragments into phage display vectors, which is due to the low efficiency of the restriction digestion step required in the process. The aim of this study was to increase the efficiency of this critical step. We found methods that are formally believed to facilitate the digestion efficiency, such as adding longer tails to the primers or prolonging the incubation time, were inefficient. We then investigated the feasibility of using T/A cloning to improve the efficiency of cloning and found that when PCR fragments were first cloned into T-vector, and then subsequently subcloned into a phagemid, the cloning efficiency was dramatically increased. Our findings show that by utilizing this method the construction of a large scFv library can be easily accomplished.
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Clonación Molecular/métodos , Región Variable de Inmunoglobulina/química , Biblioteca de Péptidos , Secuencia de Bases , Enzimas de Restricción del ADN/metabolismo , Eficiencia , Estudios de Factibilidad , Humanos , Fragmentos de Inmunoglobulinas/química , Datos de Secuencia MolecularRESUMEN
Recombinant adeno-associated virus (rAAV) 2 vector gene therapy offers promise for the healing of Rheumatoid arthritis. To support the clinical development of the candidate gene therapeutic product in China, a comprehensive preclinical safety assessment of rAAV2 encoding human TNF receptor-immunoglobulin Fc fusion gene (rAAV2/human TNFR:Fc), were conducted in 3 species of experimental animals. No abnormal findings were observed in mice following single intravenous administration with test article. Compared with the control group, no differences in mean body weight, food consumption in rats and monkeys following the repeated intraarticular administration with rAAV2/human TNFR:Fc. There were also no significant adverse effects due to treatment noted by clinical chemistry, hematology and pathology assessments. After intraarticular administration with rAAV2/human TNFR:Fc, the vector DNA initially distributed to spleen, lymph nodes, and joint synovium. The vector DNA cleared rapidly as it could be detected mainly at the site of injection by 91 d post-administration (182 d for monkey). Taken together, localized delivery of rAAV2/human TNFR:Fc showed no significant toxicity in mice, rats, and monkeys, which support the planned clinical evaluation of this product.
Asunto(s)
Artritis Reumatoide/terapia , Dependovirus/genética , Terapia Genética/efectos adversos , Vectores Genéticos/efectos adversos , Fragmentos Fc de Inmunoglobulinas/efectos adversos , Factores Inmunológicos/efectos adversos , Proteínas Recombinantes de Fusión/efectos adversos , Animales , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/epidemiología , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/patología , Femenino , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/metabolismo , Factores Inmunológicos/genética , Factores Inmunológicos/metabolismo , Macaca mulatta , Masculino , Ratones , Ratas Wistar , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Prostate cancer (PCa) is the most commonly diagnosed malignancy and the second leading cause of cancer related death in men. The early diagnosis and treatment of PCa are still challenging due to the lack of efficient tumor targeting agents in traditional managements. Prostate specific membrane antigen (PSMA) is highly expressed in PCa, while only has limited expression in other organs, providing an ideal target for the diagnosis and therapy of PCa. The antibody library technique has opened the avenue for the discovery of novel antibodies to be used in the diagnosis and therapy of cancer. In this paper, by screening a large yeast display naive human single chain antibody fragment (scFv) library, we obtained a high affinity scFv targeting PSMA, called gy1. The gy1 scFv was expressed in E.coli and purified via a C terminal 6His tag. The binding affinity of gy1 was shown to be at the nanomolar level and gy1 can specifically bind with PSMA positive cancer cells, and binding triggers its rapid internalization through the endosome-lysosome pathway. The specific targeting of gy1 to PSMA positive tumor tissues was also evaluated in vivo. We showed that the IRDye800CW labeled gy1 can efficiently target and specifically distribute in PSMA positive tumor tissues after being injected into xenograft nude mice. This study indicated that the novel antibody gy1 could be used as a great tool for the development of PSMA targeted imaging and therapy agents for PCa.
Asunto(s)
Antígenos de Superficie/inmunología , Glutamato Carboxipeptidasa II/inmunología , Neoplasias de la Próstata/diagnóstico , Anticuerpos de Cadena Única/metabolismo , Animales , Línea Celular Tumoral , Diagnóstico por Imagen , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias de la Próstata/inmunología , Anticuerpos de Cadena Única/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Human acute premyeloid leukemia cell cDNA expression library was constructed to screen acute premyeloid leukemia tumor antigen. Total RNA and purified mRNA were extracted from human premyeloid cell line NB4. First and second strands of cDNA were synthesized by reverse transcription. After blunting, the cDNA fragments were ligated with EcoR I adapters. Then the cDNAs were digested with Xho I, and less than 400 bp cDNA fragment was removed by Sephacryl-S400 spin column, the remaining were ligated with lambdaZAP vector. The recombinants were packaged in vitro, and a small portion of packaged phage was used to infect E. coli XL1-Blue-MRF' for titration. The recombinants were examined by color selection. In order to evaluate the size of cDNA inserts and the diversity of library, the pBK-CMV phagemid was excised from the ZAP express vector by using ExAssist helper phage with XLOLR strain, and then the pBK-CMV phagemid was digested by Xho I and EcoR I. The results showed that the NB4 cell line cDNA library consisting of 1.65 x 10(6) recombinant bacteriophages was constructed with the recombinant ratio of 99.6%. The average length of the recombinant exogenous inserts was about 1.7 kb. It was concluded that the constructed cDNA library are deserved to screen target clones.