RESUMEN
Prostate stem cell antigen (PSCA) is expressed in all stages of prostate cancer, including in advanced androgen-independent tumors and bone metastasis. PSCA may associate with prostate carcinogenesis and lineage plasticity in prostate cancer. PSCA is also a promising theranostic marker for a variety of other solid tumors, including pancreatic adenocarcinoma and renal cell carcinoma. Here, we identified a novel fully human PSCA antibody using phage display methodology. The structure-based affinity maturation yielded a high-affinity binder, F12, which is highly specific and does not bind to 6,000 human membrane proteins based on a membrane proteome array assay. F12 targets PSCA amino acids 63-69 as tested by the peptide scanning microarray, and it cross-reacts with the murine PSCA. IgG1 F12 efficiently internalizes into PSCA-expressing tumor cells. The antimitotic reagent monomethyl auristatin E (MMAE)-conjugated IgG1 F12 (ADC, F12-MMAE) exhibits dose-dependent efficacy and specificity in a human prostate cancer PC-3-PSCA xenograft NSG mouse model. This is a first reported ADC based on a fully human PSCA antibody and MMAE that is characterized in a xenograft murine model, which warrants further optimizations and investigations in additional preclinical tumor models, including prostate and other solid tumors.
Asunto(s)
Antígenos de Neoplasias , Proteínas Ligadas a GPI , Inmunoconjugados , Proteínas de Neoplasias , Neoplasias de la Próstata , Ensayos Antitumor por Modelo de Xenoinjerto , Humanos , Masculino , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/inmunología , Inmunoconjugados/farmacología , Animales , Antígenos de Neoplasias/inmunología , Ratones , Proteínas Ligadas a GPI/inmunología , Proteínas de Neoplasias/inmunología , Proteínas de Neoplasias/antagonistas & inhibidores , Línea Celular Tumoral , Oligopéptidos/inmunología , Oligopéptidos/farmacología , Inmunoglobulina G/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacologíaRESUMEN
Resumen Introducción. La obtención de anticuerpos específicos capaces de detectar alérgenos del grupo 1 de ácaros del polvo doméstico representa una estrategia potencial de salud pública para reducir la exposición y la sintomatología clínica asociada con el asma y la rinitis alérgica. Objetivo. Producir y purificar anticuerpos aviares antialérgenos específicos del grupo 1 de los ácaros Dermatophagoides sp.y Blomia tropicalis utilizando la tecnología IgY. Materiales y métodos. Se diseñaron y sintetizaron oligopéptidos que evidenciaran epítopes inmunogénicos de los alérgenos Der p1, Der f1 y Blo t1 empleados posteriormente para producir anticuerpos IgY policlonales en gallinas Hy Line Brown. Las IgY presentes en las yemas de los huevos se purificaron mediante cromatografía tiofílica. Su inmunorreactividad y especificidad se determinaron mediante un inmunoensayo ELISA indirecto y Dot Blot. Resultados. Se obtuvo una reactividad elevada de las IgY contra epítopes de alérgenos presentes en extractos de cuerpo entero de D. farinae, D. pteronyssinus y B. tropicalis. Los niveles más altos de IgY se produjeron entre los días 32 y 40 de inmunización. Los anticuerpos mostraron mayor inmunorreactividad y especificidad en el reconocimiento de proteínas de D. farinae, con un límite de detección mayor de 0,03 µg de proteína total delcaroajo las condiciones experimentales analizadas. Las IgY purificadas no mostraron reactividad significativa frente al extracto de Periplaneta americana. Conclusión. La tecnología IgY permitió la producción de anticuerpos específicos contra alérgenos del grupo 1 de los ácaros del polvo al utilizar oligopéptidos sintéticos no glicosilados. Hasta donde se sabe, esta es la primera vez que se usan estos reactivos inmunológicos para la detección de ácaros de importancia médica.
Abstract Introduction: The use of specific antibodies capable of detecting allergens of the group 1 of house dust mites represents a potential strategy to reduce exposure and clinical symptomatology associated with asthma and allergic rhinitis. Objective: To produce and purify chicken antibodies specific for the dust mites Dermatophagoides sp. and B. tropicalis using the IgY technology. Materials and methods: We designed and synthesized oligopeptides showing immunogenic epitopes of Der p1, Der f1, and Blo t1. These were used to produce IgY antibodies in Hy Line Brown chickens. IgY were extracted from egg yolk using thiophilic chromatography. The immunogenicity and specificity were assayed by indirect ELISA and Dot Blot. Results: We obtained high reactivity of IgY antibodies against epitopes of allergens present in whole body mites extracts of D. farinae, D. pteronyssinus, and B. tropicalis. The highest IgY levels were registered between days 32 and 40 after immunization. The antibodies showed high immunoreactivity and specificity towards D. farinae proteins with detection limits above 0.03 µg of mite proteins under the experimental conditions used. Purified IgY did not show significant reactivity when binding to Periplaneta americana extract. Conclusion: The IgY technology allowed the production of specific antibodies against house dust mites group 1 allergens using non-glycosylated synthetic peptides. To our knowledge, this is the first time that this immunochemicals are used in the detection of mites of medical relevance.
Asunto(s)
Animales , Oligopéptidos/inmunología , Inmunoglobulinas/inmunología , Pyroglyphidae/inmunología , Antígenos Dermatofagoides/inmunología , Anticuerpos/inmunología , PollosRESUMEN
INTRODUCTION: Minor histocompatibility antigen HA-1 (MiHAg-HA-1) disparity between a patient and his or her human leukocyte antigen (HLA) genoidentical donor has been widely associated with an increased risk of graft-versus-host disease following allogeneic hematopoietic stem cell transplantation. OBJECTIVE: To examine the effect of HA-1 disparity on the incidence of both acute and chronic graft-versus-host disease in Tunisian recipients of hematopoietic stem cells. METHODS: A total of 60 patients and their 60 respective sibling hematopoietic stem cell donors were enrolled in this study. All patients prophylactically received cyclosporine A and/or methotrexate for graft-versus-host disease. An HA-1 genotyping assay was performed with the SSP-PCR method, and HLA-A*0201- and/or HLA-A*0206-positive samples were identified using the Luminex HLA typing method. RESULTS: The Luminex HLA typing assay showed that 54 patients were positive for either the HLA-A*0201 or HLA-A*0206 alleles. Among these cases, six pairs were mismatched for MiHAg-HA-1. Both acute and chronic graft-versus-host disease occurred in four mismatched patients (Fisher's p-values were 0.044 and 0.170, respectively). A univariate logistic regression model analysis showed that only acute graft-versus-host disease may be affected by recipient MiHAg-HA-1 disparity (p: 0.041, OR: 6.727), while chronic graft-versus-host disease correlates with both age and recipient/donor sex mismatch (p: 0.014, OR: 8.556 and p: 0.033, OR: 8.664, respectively). CONCLUSION: Our findings support previously reported data suggesting a significant association between HA-1 disparity and the risk of acute graft-versus-host disease following hematopoietic stem cell transplantation.
Asunto(s)
Adolescente , Adulto , Niño , Preescolar , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto Joven , Enfermedad Injerto contra Huésped/inmunología , Trasplante de Células Madre Hematopoyéticas , Antígenos HLA/inmunología , Antígenos de Histocompatibilidad Menor/inmunología , Oligopéptidos/inmunología , Alelos , Prueba de Histocompatibilidad , Modelos Logísticos , Antígenos de Histocompatibilidad Menor/genética , Oligopéptidos/genética , Reacción en Cadena de la Polimerasa , Factores de Riesgo , Factores Sexuales , TúnezRESUMEN
The organic anion transporters (OATs) are expressed in various tissues, primarily in the kidney and liver, but they are also expressed in the placenta, small intestine, and the choroid plexus, which are all epithelial tissues that transport xenobiotics. Six isoforms of OATs are currently known. Considering the variety of organic anionic compounds, other OATs isoforms can be assumed. In this connection, we have searched for a new isoform in the expressed sequence tag (EST) database. We found the new candidate clone AK052752 in the mouse kidney cDNA library and we named it mouse organic anion transporter like protein 1 (mOATLP1). The mOATLP1 cDNA consisted of 2221 base pairs that encoded a 552 amino acid residue protein with 12 putative transmembrane domains. The deduced amino acid sequence of mOATLP1 showed 37 to 63% identity to other members of the OAT family. According to the tissue distribution based on Northern blot analysis, 2.7 kb and 2.9 kb mOATLP1 transcripts (approximate sizes) were observed in the kidney and liver. An 85-kDa band (approximate) was detected using Western blot analysis of mouse kidney performed with a synthesized oligopeptide-induced mOATLP1 antibody. Immunohistochemical results showed mOATLP1 was stained in the blood vessels, glomeruli (the parietal epithelial cells and podocytes), distal convoluted tubules, connecting tubules, and inner medullary collecting tubules. mOATLP1 appears to be a novel candidate for an organic anion transporter isoform identified in the kidney.
Asunto(s)
Conejos , Ratones , Animales , Distribución Tisular , Homología de Secuencia de Aminoácido , Estructura Terciaria de Proteína , Isoformas de Proteínas/aislamiento & purificación , Filogenia , Transportadores de Anión Orgánico/aislamiento & purificación , Oligopéptidos/inmunología , Familia de Multigenes , Datos de Secuencia Molecular , Riñón/metabolismo , Inmunohistoquímica , Clonación Molecular , Western Blotting , Secuencia de AminoácidosRESUMEN
The stabilizing effects of staphylococcal nuclease (Nuc) and of a synthetic propeptide (LEISSTCDA, hereafter called LEISS) on the production of a model food allergen, bovine ß-lactoglobulin (BLG), in Lactococcus lactis were investigated. The fusion of Nuc to BLG (Nuc-BLG) results in higher production and secretion of the hybrid protein. When LEISS was fused to BLG, the production of the resulting protein LEISS-BLG was only slightly improved compared to the one obtained with Nuc-BLG. However, the secretion of LEISS-BLG was dramatically enhanced (~10- and 4-fold higher than BLG and Nuc-BLG, respectively). Finally, the fusion of LEISS to Nuc-BLG resulting in the protein LEISS-Nuc-BLG led to the highest production of the hybrid protein, estimated at ~8 æg/ml (~2-fold higher than Nuc-BLG). In conclusion, the fusions described here led to the improvement of the production and secretion of BLG. These tools will be used to modulate the immune response against BLG via delivery of recombinant lactococci at the mucosal level, in a mouse model of cow's milk allergy.