RESUMEN
Over the last nearly three decades in vitro display technologies have played an important role in the discovery and optimization of antibodies and other proteins for therapeutic applications. Here we describe the use of retroviral expression technology for the display of full-length IgG on B lineage cells in vitro with a hallmark of a tight and stable genotype to phenotype coupling. We describe the creation of a high-diversity (>1.0E09 different heavy- and light-chain combinations) cell displayed fully human antibody library from healthy donor-derived heavy- and light-chain gene libraries, and demonstrate the recovery of high affinity target-specific antibodies from this library by staining of cells with a labeled target antigen and their magnetic- and flow cytometry-based cell sorting. The present technology represents a further evolution in the discovery of full-length, fully human antibodies using mammalian display, and is termed Retrocyte Display® (Retroviral B lymphocyte Display).
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Retroviridae/genética , Animales , Anticuerpos Monoclonales/genética , Linfocitos B/metabolismo , Criopreservación , Evaluación Preclínica de Medicamentos , Citometría de Flujo , Variación Genética , Vectores Genéticos , Células HEK293 , Humanos , Cadenas Pesadas de Inmunoglobulina/biosíntesis , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/biosíntesis , Cadenas Ligeras de Inmunoglobulina/genética , Separación Inmunomagnética , Biblioteca de Péptidos , Unión ProteicaRESUMEN
Interleukin 7 (IL-7) plays a crucial role in adult lymphopoiesis, while in fetal life its effect can be partially compensated by TSLP. Whether adult hematopoietic progenitor cells are unresponsive to TSLP or whether TSLP is less available in adult microenvironments is still a matter of debate. Here, we show that increased TSLP availability through transgene (Tg) expression fully restored lymphopoiesis in IL-7-deficient mice: it rescued B-cell development, increased thymic and splenic cellularities, and restored double-negative (DN) thymocytes, alphabeta and gammadelta T-cell generation, and all peripheral lymphoid compartments. Analysis of bone marrow chimeras demonstrated that hematopoietic progenitor cells from adult wild-type mice efficiently differentiated toward B- and T-cell lineages in lethally irradiated IL-7 deficient mice provided TSLP Tg was expressed in these mice. In vitro, TSLP promoted the differentiation of uncommitted adult bone marrow progenitors toward B and T lineages and the further differentiation of DN1 and DN2 thymocytes. Altogether, our results show that adult hematopoietic cells are TSLP responsive and that TSLP can sustain long-term adult lymphopoiesis.