RESUMEN
RATIONALE: Because of the large molecular weight, the structural complexity and the similarity with endogenous immunoglobulins present in high concentrations, in vivo quantitative studies with therapeutic monoclonal antibodies are particularly challenging. In this work, an UPLC/MRM MS-based methodology is described for the quantification of trastuzumab in human serum by monitoring a novel specific peptide marker located within its heavy chain Complementarity-Determining Region (CDR). METHODS: For maximum sensitivity and selectivity, specific transitions of this diagnostic proteotypic peptide were optimized and monitored at m/z 364.1 â 437.3 (quantitation ion) and m/z 364.1 â 358.0 (confirmation ion). As a proof-of-concept, the methodology was applied to the determination of trastuzumab in human serum over a clinically relevant range from 0.02 to 200 µg/mL. The methodology has been evaluated in terms of specificity, linearity, accuracy, precision, detection and quantitation limits. RESULTS: An excellent linear response has been obtained in the range from 0.036 to 3.6 fmol/µL for the standard peptide and from 0.03 to 285 fmol/µL for the trastuzumab in human serum with typical R2 values of 0.99. The limit of detection (LOD) and limit of quantification (LOQ) are 0.005 fmol/µL and 0.05 fmol/µL, respectively, with mean bias and RSD values of 18% and 1%, respectively, for quality control samples. CONCLUSIONS: The strategy used to set up the UPLC/MRM MS methodology based on monitoring specific peptide markers within CDRs can be potentially applied to the detection and quantification of other humanized or human mAbs in biological fluids. Copyright © 2017 John Wiley & Sons, Ltd.
Asunto(s)
Biomarcadores/sangre , Regiones Determinantes de Complementariedad/sangre , Fragmentos de Péptidos/sangre , Trastuzumab/sangre , Biomarcadores/química , Biomarcadores/metabolismo , Cromatografía Líquida de Alta Presión/métodos , Regiones Determinantes de Complementariedad/química , Regiones Determinantes de Complementariedad/metabolismo , Monitoreo de Drogas , Humanos , Límite de Detección , Modelos Lineales , Espectrometría de Masas/métodos , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Trastuzumab/química , Trastuzumab/metabolismoRESUMEN
We assessed mammaglobin (MMG) gene expression in bone marrow (BM) aspirates from patients with advanced breast cancer who had received a reduced-intensity conditioning and stem cell allografting, in order to detect a graft-versus-tumor effect on micrometastatic disease. Nine patients received a reduced-intensity conditioning with fludarabine, cyclophosphamide, and thiotepa, followed by peripheral blood allografting from HLA-identical sibling donors. Nested RT-PCR analysis with sequence-specific primers for MMG was carried out on a monthly basis on BM samples. Three patients had MMG-positive BM, four patients had MMG-negative BM before allografting, and two were undetermined. In two patients, a clinical response after allografting (partial remission) occurred concurrently with the clearance of MMG expression, at a median of 6 months after allografting, following immune manipulation. In two patients, a prolonged stable disease and negative MMG expression occurred after day +360 from allografting. In two patients, progression of the disease was associated with MMG RT-PCR changing from negative to positive. In one case, a disease response occurring after donor lymphocyte infusion and grade II acute GVHD was heralded by negativization of MMG expression. Although preliminary, these data suggest that a graft-versus-breast cancer effect is detectable on micrometastatic BM disease.
Asunto(s)
Biomarcadores de Tumor/biosíntesis , Neoplasias de la Médula Ósea/metabolismo , Médula Ósea/metabolismo , Neoplasias de la Mama/metabolismo , Regulación Neoplásica de la Expresión Génica , Efecto Injerto vs Tumor , Proteínas de Neoplasias/biosíntesis , Uteroglobina/biosíntesis , Adulto , Médula Ósea/patología , Neoplasias de la Médula Ósea/patología , Neoplasias de la Médula Ósea/secundario , Neoplasias de la Médula Ósea/terapia , Trasplante de Médula Ósea , Neoplasias de la Mama/patología , Neoplasias de la Mama/terapia , Ciclofosfamida/administración & dosificación , Femenino , Supervivencia de Injerto , Humanos , Mamoglobina A , Persona de Mediana Edad , Agonistas Mieloablativos/administración & dosificación , Valor Predictivo de las Pruebas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Tiotepa/administración & dosificación , Acondicionamiento Pretrasplante/métodos , Trasplante Homólogo , Vidarabina/administración & dosificación , Vidarabina/análogos & derivadosRESUMEN
Gene therapy has been proposed as a potential treatment for Wiskott-Aldrich syndrome (WAS), a severe primary immune deficiency characterized by multiple hematopoietic-specific cellular defects. In order to develop an optimal lentiviral gene transfer cassette for this application, we compared the performance of several internal promoters in a variety of cell lineages from human WAS patients. Vectors using endogenous promoters derived from short (0.5 kb) or long (1.6 kb) 5' flanking sequences of the WAS gene, expressed the transgene in T, B, dendritic cells as well as CD34(+) progenitor cells, but functioned poorly in non-hematopoietic cells. Defects of T-cell proliferation and interleukin-2 production, and the cytoskeletal anomalies in WAS dendritic cells were also corrected. The levels of reconstitution were comparable to those obtained following transduction with similar lentiviral vectors incorporating constitutive PGK-1, EF1-alpha promoters or the spleen focus forming virus gammaretroviral LTR. Thus, native regulatory sequences target the expression of the therapeutic WAS transgene to the hematopoietic system, as is naturally the case for WAS, and are effective for correction of multiple cellular defects. These vectors may have significant advantages for clinical application in terms of natural gene regulation, and reduction in the potential for adverse mutagenic events.