RESUMEN
Chromosomal instability (CIN) is a hallmark of cancer aggressiveness, providing genetic plasticity and tumor heterogeneity that allows the tumor to evolve and adapt to stress conditions. CIN is considered a cancer therapeutic biomarker because healthy cells do not exhibit CIN. Despite recent efforts to identify therapeutic strategies related to CIN, the results obtained have been very limited. CIN is characterized by a genetic signature where a collection of genes, mostly mitotic regulators, are overexpressed in CIN-positive tumors, providing aggressiveness and poor prognosis. We attempted to identify new therapeutic strategies related to CIN genes by performing a drug screen, using cells that individually express CIN-associated genes in an inducible manner. We find that the overexpression of targeting protein for Xklp2 (TPX2) enhances sensitivity to the proto-oncogene c-Src (SRC) inhibitor dasatinib due to activation of the Yes-associated protein 1 (YAP) pathway. Furthermore, using breast cancer data from The Cancer Genome Atlas (TCGA) and a cohort of cancer-derived patient samples, we find that both TPX2 overexpression and YAP activation are present in a significant percentage of cancer tumor samples and are associated with poor prognosis; therefore, they are putative biomarkers for selection for dasatinib therapy.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Neoplasias de la Mama , Proteínas de Ciclo Celular , Dasatinib , Proteínas Asociadas a Microtúbulos , Proto-Oncogenes Mas , Transducción de Señal , Factores de Transcripción , Proteínas Señalizadoras YAP , Dasatinib/farmacología , Humanos , Neoplasias de la Mama/genética , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Neoplasias de la Mama/metabolismo , Femenino , Proteínas Señalizadoras YAP/metabolismo , Proteínas Señalizadoras YAP/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Transducción de Señal/efectos de los fármacos , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Resistencia a Antineoplásicos/genética , Resistencia a Antineoplásicos/efectos de los fármacosRESUMEN
OBJECTIVES: Individual immunoglobulins expressed by B-cell lymphomas represent tumor-specific antigens ('idiotypes'). Immunization with idiotype in follicular lymphoma patients may induce specific immune responses, sustained progression-free survival, and disappearance of minimal residual disease. Manufacturing of idiotype vaccines has mostly relied on heterohybridomas established from viable lymphoma cells. This paper describes the feasibility of production of GMP-grade idiotype vaccines as recombinant Fab fragments in Escherichia coli. METHODS: IgH and IgL transcripts were analyzed by anchored PCR from 106 lymphoma and nine control biopsies. Lymphoma-derived V segments were inserted into prokaryotic expression plasmids. Recombinant idiotype Fab fragments were expressed in E. coli in a fermentation system. RESULTS: Idiotype IgH and IgL transcripts were identified in 95% of 106 lymphoma biopsies according to stringent clonality criteria. Large-scale idiotype expression was successful in 69 of 78 cases (89%) and yielded a median of 17 mg (range: 1.2-250 mg) recombinant Fab protein. After affinity chromatography, median vaccine purity was 99% heterodimeric Fab protein (range: 72-100%). Bacterial protein contamination was detectable in one vaccine only. Fab proteins with IgL lambda chains had a tendency for inferior yield and lesser purity than kappa-type Fabs. Among other structural idiotype features (isotype, V family usage, somatic hypermutation pattern, novel glycosylation sites, CDR III net charge), no consistent influences on Fab yield or purity were detected. CONCLUSIONS: Anchored PCR cloning and subsequent protein expression in E. coli provides a reliable technological basis for clinical idiotype vaccination trials.
Asunto(s)
Vacunas contra el Cáncer/genética , Fragmentos Fab de Inmunoglobulinas/genética , Cadenas Pesadas de Inmunoglobulina/genética , Idiotipos de Inmunoglobulinas/genética , Cadenas Ligeras de Inmunoglobulina/genética , Linfoma de Células B/genética , Linfoma Folicular/genética , Vacunas contra el Cáncer/aislamiento & purificación , Vacunas contra el Cáncer/uso terapéutico , Escherichia coli/genética , Humanos , Fragmentos Fab de Inmunoglobulinas/aislamiento & purificación , Fragmentos Fab de Inmunoglobulinas/uso terapéutico , Cadenas Pesadas de Inmunoglobulina/aislamiento & purificación , Cadenas Pesadas de Inmunoglobulina/uso terapéutico , Idiotipos de Inmunoglobulinas/aislamiento & purificación , Idiotipos de Inmunoglobulinas/uso terapéutico , Cadenas Ligeras de Inmunoglobulina/aislamiento & purificación , Cadenas Ligeras de Inmunoglobulina/uso terapéutico , Linfoma de Células B/terapia , Linfoma Folicular/terapia , Reacción en Cadena de la Polimerasa/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/uso terapéutico , VacunaciónRESUMEN
The immunoglobulin receptor of B-cell lymphomas constitutes a specific tumor antigen (idiotype) and a target for active immunotherapy. Encouraging results have been reported in phase II trials after s.c. vaccination of follicular lymphoma patients during clinical remission with idiotype produced from eukaryotic cell lines and coupled to an immunogenic carrier macromolecule. We have developed a good manufacturing protocol for rapid expression of idiotype vaccines as recombinant Fab fragments in Escherichia coli. The objectives of this trial were to show safety and feasibility of intradermal immunization with this vaccine and to investigate whether immune responses were induced by this immunization route. Patients (n = 18) with advanced B-cell malignancies received repetitive intradermal vaccinations with 0.5 to 1.65 mg recombinant idiotype Fab fragment mixed with lipid-based adjuvant in combination with 150 mug granulocyte macrophage colony-stimulating factor s.c. at the same location. The patients' immune status was assessed by flow cytometry of peripheral blood lymphocytes and concomitant hepatitis B vaccination. Cellular and humoral immune responses to the vaccine were assessed by enzyme-linked immunospot and ELISA. Side effects of a total of 65 vaccinations were mild and did not affect the immunization schedule. No patient developed hepatitis B surface antibodies (anti-HBs) after two hepatitis B immunizations. Of 17 evaluable patients, five developed specific anti-vaccine antibodies, and eight developed anti-Fab T-cell responses. T-cell reactivity was independent of the cellular immune status and was idiotype specific as shown by statistical regression analysis (P = 0.0024) and epitope mapping studies. Intradermal administration of uncoupled recombinant idiotype with appropriate adjuvants may overcome profound clinical immunosuppression and induce specific immune responses.
Asunto(s)
Vacunas contra el Cáncer/uso terapéutico , Linfoma de Células B/inmunología , Linfoma de Células B/terapia , Vacunación/métodos , Adulto , Anciano , Secuencia de Aminoácidos , Linfocitos B/inmunología , Vacunas contra el Cáncer/efectos adversos , Vacunas contra el Cáncer/inmunología , Epítopos de Linfocito T , Femenino , Humanos , Idiotipos de Inmunoglobulinas/inmunología , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Linfocitos T/inmunología , Resultado del TratamientoRESUMEN
Las células dentríticas (CD) son consideradas las más eficientes células presentadoras de antígenos. Las CD derivan de células progenitoras de la médula ósea y circulan por el torrente sanguíneo como procursores inmaduros. Bajo un estímulo apropiado, las CD maduran y migran a los órganos linfáticos secundarios donde presentan antígenos a linfocitos T e inducen una respuesta inmune. Las CD pueden ser generadas y manipuladas in vitro para presentar antígenos específicos de tumor o de patógenos. Una vez activadas pueden ser reinfundidas en el paciente y actuar como vacunas celulares, constituyendo la base de numerosos ensayos clínicos de inmunoterapia del cáncer
Asunto(s)
Humanos , Células Dendríticas/inmunología , InmunoterapiaRESUMEN
Las células dentríticas (CD) son consideradas las más eficientes células presentadoras de antígenos. Las CD derivan de células progenitoras de la médula ósea y circulan por el torrente sanguíneo como procursores inmaduros. Bajo un estímulo apropiado, las CD maduran y migran a los órganos linfáticos secundarios donde presentan antígenos a linfocitos T e inducen una respuesta inmune. Las CD pueden ser generadas y manipuladas in vitro para presentar antígenos específicos de tumor o de patógenos. Una vez activadas pueden ser reinfundidas en el paciente y actuar como vacunas celulares, constituyendo la base de numerosos ensayos clínicos de inmunoterapia del cáncer (AU)