RESUMO
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).
Assuntos
Anticorpos Monoclonais/biossíntese , Retroviridae/genética , Animais , Anticorpos Monoclonais/genética , Linfócitos B/metabolismo , Criopreservação , Avaliação Pré-Clínica de Medicamentos , Citometria de Fluxo , Variação Genética , Vetores Genéticos , Células HEK293 , Humanos , Cadeias Pesadas de Imunoglobulinas/biossíntese , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/biossíntese , Cadeias Leves de Imunoglobulina/genética , Separação Imunomagnética , Biblioteca de Peptídeos , Ligação ProteicaRESUMO
Interleukin (IL)-1α is a potent proinflammatory cytokine that has been implicated in the development of atherosclerosis. We investigated whether a vaccine inducing IL-1α neutralizing antibodies could interfere with disease progression in a murine model of atherosclerosis. We immunized Apolipoprothin E (ApoE)-deficient mice with a vaccine (IL-1α-C-Qß) consisting of full-length, native IL-1α chemically conjugated to virus-like particles derived from the bacteriophage Qß. ApoE(-/-) mice were administered six injections of IL-1α-C-Qß or nonconjugated Qß over a period of 160 days while being maintained on a western diet. Atherosclerosis was measured in the descending aorta and in cross-sections at the aortic root. Macrophage infiltration in the aorta was measured using CD68. Expression levels of VCAM-1, ICAM-1, and MCP-1 were quantified by RT-PCR. Immunization against IL-1α reduced plaque progression in the descending aorta by 50% and at the aortic root by 37%. Macrophage infiltration in the aorta was reduced by 22%. Inflammation was also reduced in the adventitia, with a decrease of 54% in peri-aortic infiltrate score and reduced expression levels of VCAM-1 and ICAM-1. Active immunization targeting IL-1α reduced both the inflammatory reaction in the plaque as well as plaque progression. In summary, vaccination against IL-1α protected ApoE(-/-) mice against disease, suggesting that this may be a potential treatment option for atherosclerosis.