RESUMEN
Bispecific antibodies (BsAbs) have emerged as a major class of antibody therapeutics owing to their substantial potential in disease treatment. While several BsAbs have been successfully approved in recent years, ongoing development efforts continue to focus on optimizing various BsAbs tailored to particular antigens and action mechanisms, aiming to achieve favorable physicochemical properties. BsAbs generally encounter challenges due to their unfavorable physicochemical characteristics and poor manufacturing efficiencies, highlighting the need for optimization to achieve reliable productivity and developability. Herein, we describe the development of a novel symmetric BsAb, REGULGENT™ (N-term/C-term), comprising two Fab domains, using a common light chain. The heavy chain fragment encoded two antigen-binding determinants in one chain. The design and production of REGULGENT™ (N-term/C-term) are simple owing to the use of the same light chain, which does not induce heavy and light chain mispairing, frequently observed with the asymmetric BsAb format. REGULGENT™ (N-term/C-term) exhibited high expression and low aggregation characteristics during cell culture and stress treatment under low pH conditions. Differential scanning calorimetric data indicated that REGULGENT™ molecules had high conformational stability, similar to that of stabilized monoclonal antibodies. Surface plasmon resonance data showed that REGULGENT™ (N-term/C-term) could bind to two antigens simultaneously and exhibited a high affinity for two antigens. In summary, the symmetric BsAb format of REGULGENT™ confers its desirable IgG-like physicochemical properties, thus making it an excellent candidate for commercial development. The findings demonstrate a novel BsAb with substantial development potential for clinical applications.
Asunto(s)
Anticuerpos Biespecíficos , Ingeniería de Proteínas , Anticuerpos Biespecíficos/química , Anticuerpos Biespecíficos/inmunología , Anticuerpos Biespecíficos/genética , Humanos , Ingeniería de Proteínas/métodos , Estabilidad Proteica , Cadenas Ligeras de Inmunoglobulina/química , Cadenas Ligeras de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/metabolismo , AnimalesRESUMEN
Health insurance companies have an interest in understanding what factors may lead to positive word-of-mouth (WOM) from consumers. This research identifies factors that influence positive WOM about health insurance firms. Using data collected from 425 insurance policy holders, we find influential factors vary depending on the purchase source. Significant factors identified in the study that influence positive word-of-mouth include service quality, plan satisfaction, and plan type (individual vs. family). Further, claim count, preferred information source, and deductible levels also affect the spread of positive WOM differently among purchase sources. The study concludes with a discussion of implications and future research.
Asunto(s)
Comunicación , Comportamiento del Consumidor , Seguro de Salud , Satisfacción del Paciente , Adulto , Deducibles y Coseguros/economía , Femenino , Humanos , Seguro de Salud/economía , Seguro de Salud/organización & administración , Internet , Masculino , Persona de Mediana Edad , Encuestas y CuestionariosRESUMEN
This study developed the Japanese version of the gerotranscendence scale type 2 (the GST2) and examined reliability and validity of the scale. In Japan, 525 community-dwelling older adults (male = 260, female = 265) answered a questionnaire. An exploratory factor analysis of the Japanese version of the GST2 revealed the same three-factor structure including the Dimensions of the Cosmic, the Coherence, and the Solitude, which had been reported by Tornstam with the omission of item 1. Reliability and construct validity of the Japanese version of the GST2 were confirmed. These findings provide support for use of the Japanese version of the GST2 as a measure of lifespan development among the oldest old in Japan. The GST2 can be applicable not only for older adults in Sweden but also for older adults in Japan.
Asunto(s)
Envejecimiento/psicología , Psiquiatría Geriátrica/métodos , Acontecimientos que Cambian la Vida , Modelos Psicológicos , Anciano , Anciano de 80 o más Años , Análisis Factorial , Femenino , Humanos , Japón , Masculino , Persona de Mediana Edad , Escalas de Valoración Psiquiátrica , Reproducibilidad de los Resultados , Encuestas y CuestionariosRESUMEN
We previously reported the construction and activity of a humanized, bispecific diabody (hEx3) that recruited T cells towards an epidermal growth factor receptor (EGFR) positive tumor. Herein, we describe the construction of a second functional, fully humanized, anti-EGFR bispecific diabody that recruits another subset of lymphocyte effectors, the natural killer cells, to EGFR-expressing tumor cells. After we confirmed that an anti-EGFR × anti-CD16 bispecific diabody (Ex16) consisting of a previously humanized anti-EGFR variable fragment (Fv) and a mouse anti-CD16 Fv had growth inhibitory activity, we designed a humanized anti-CD16 Fv to construct the fully humanized Ex16 (hEx16). However, the humanized form had lower activity for inhibition of cancer growth. To restore its growth inhibitory activity, we introduced mutations into the Vernier zone, which is located near the complementarity-determining regions and is involved in their binding activity. We efficiently prepared 15 different hEx16 mutants by expressing each chimeric single-chain component for hEx16 separately. We then used our in vitro refolding system to select the most functional mutant, which had a growth inhibitory effect comparable with that of the commercially available chimeric anti-EGFR antibody, cetuximab. Our refolding system could aid in the efficient optimization of other proteins with heterodimeric structure.
Asunto(s)
Anticuerpos Biespecíficos/biosíntesis , Anticuerpos Monoclonales Humanizados/biosíntesis , Diseño de Fármacos , Receptores ErbB/antagonistas & inhibidores , Factores Inmunológicos/metabolismo , Receptores de IgG/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Anticuerpos Biespecíficos/química , Anticuerpos Biespecíficos/genética , Anticuerpos Biespecíficos/farmacología , Anticuerpos Monoclonales Humanizados/química , Anticuerpos Monoclonales Humanizados/genética , Anticuerpos Monoclonales Humanizados/farmacología , Línea Celular Tumoral , Proliferación Celular , Células Cultivadas , Técnicas de Cocultivo , Receptores ErbB/metabolismo , Proteínas Ligadas a GPI/antagonistas & inhibidores , Humanos , Hibridomas , Región Variable de Inmunoglobulina/química , Región Variable de Inmunoglobulina/genética , Factores Inmunológicos/química , Factores Inmunológicos/farmacología , Células Asesinas Activadas por Linfocinas/efectos de los fármacos , Células Asesinas Activadas por Linfocinas/inmunología , Células Asesinas Activadas por Linfocinas/metabolismo , Ratones , Datos de Secuencia Molecular , Proteínas Mutantes Quiméricas/biosíntesis , Proteínas Mutantes Quiméricas/química , Neoplasias/inmunología , Neoplasias/metabolismo , Neoplasias/terapia , Replegamiento Proteico , Alineación de SecuenciaRESUMEN
We report the preparation and functional characterization of an Escherichia coli-expressed recombinant fusion protein, 528scFv-TRAIL, specific for the human epidermal growth factor receptor (EGFR) and empowered by the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). The 528scFv-TRAIL, expressed as insoluble inclusion bodies in E. coli, was solubilized and then refolded by using a modified stepwise dialysis method. Treatment with 528scFv-TRAIL resulted in the specific binding to the cell surface of EGFR-positive cells with concomitant deployment of the TRAIL moiety to DR-5 receptor in a manner comparable to a commercially available form of recombinant TRAIL (cTRAIL). 528scFv-TRAIL, prepared by either of three refolding processes described herein, showed potent cytotoxic activity against EGFR-positive TFK-1 cell line and was superior to its parental 528scFv; a recombinant variable fragment with single specificity against human EGFR. Narrow variations in the cytotoxic potential of 528scFv-TRAIL were ascribed to manipulation of redox conditions during the refolding process. Together, our findings point to the potential value of 528scFv-TRAIL for treatment of EGFR-expressing cancers. Furthermore, preparation of 528scFv-TRAIL from insoluble aggregates in a prokaryotic cell based expression system by means of in vitro refolding introduces a feasible cost-benefit, time-efficient approach for industrial-scale production.