RESUMO
The tubular gland of the chicken oviduct is an attractive system for protein expression as large quantities of proteins are deposited in the egg, the production of eggs is easily scalable and good manufacturing practices for therapeutics from eggs have been established. Here we examined the ability of upstream and downstream DNA sequences of ovalbumin, a protein produced exclusively in very high quantities in chicken egg white, to drive tissue-specific expression of human mAb in chicken eggs. To accommodate these large regulatory regions, we established and transfected lines of chicken embryonic stem (cES) cells and formed chimeras that express mAb from cES cell-derived tubular gland cells. Eggs from high-grade chimeras contained up to 3 mg of mAb that possesses enhanced antibody-dependent cellular cytotoxicity (ADCC), nonantigenic glycosylation, acceptable half-life, excellent antigen recognition and good rates of internalization.
Assuntos
Anticorpos Monoclonais/química , Animais , Southern Blotting , Western Blotting , Células CHO , Varredura Diferencial de Calorimetria , Carboidratos/química , Galinhas , Cricetinae , DNA/metabolismo , Clara de Ovo , Embrião de Mamíferos/citologia , Embrião não Mamífero , Ensaio de Imunoadsorção Enzimática , Feminino , Vetores Genéticos , Genoma , Glicosilação , Humanos , Imunoglobulina G , Imuno-Histoquímica , Focalização Isoelétrica , Camundongos , Camundongos Endogâmicos BALB C , Modelos Genéticos , Monossacarídeos/química , Oligossacarídeos/química , Ovalbumina/genética , Ovalbumina/metabolismo , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas por Ionização por Electrospray , Células-Tronco/citologiaRESUMO
Type I interferons (IFNs) are potent regulators of both innate and adaptive immunity. All type I IFNs bind to the same heterodimeric cell surface receptor composed of IFN-alpha receptor (IFNAR-1) and IFN-alpha/beta receptor (IFNAR-2) polypeptides. This study revealed that type I IFN receptor levels vary considerably on hematopoietic cells, with monocytes and B cells expressing the highest levels. Overnight treatment of peripheral blood mononuclear cells (PBMCs) with IFN-alpha2b or IFN-beta led to increased expression on monocytes and B cells of surface markers commonly associated with activated antigen-presenting cells (APCs), such as CD38, CD86, MHC class I, and MHC class II. Five-day exposure of adherent monocytes to granulocyte-macrophage colony-stimulating factor (GM-CSF) plus IFN-alpha or IFN-beta caused the development of potent allostimulatory cells with morphology similar to that of myeloid dendritic cells (DCs) obtained from culture with GM-CSF and interleukin-4 (IL-4) but with distinct cell surface marker profiles and activity. In contrast to IL-4-derived DCs, IFN-alpha-derived DCs were CD14+, CD1a-, CD123+, CD32+, and CD38+ and expressed high levels of CD86 and MHC class II. Development of these cells was completely blocked by an antibody to IFNAR-1. Furthermore, activity of the type I IFN-derived DC in a mixed lymphocyte reaction (MLR) was consistently more potent than that of IL-4-derived DCs, especially at high responder/stimulator ratios. This MLR activity was abrogated by the addition of anti-IFNAR-1 antibody at the start of the DC culture. In contrast, there was no effect of anti-IFNAR-1 on IL-4-derived DCs, indicating that this is a distinct pathway of DC differentiation. These results suggest a potential role for anti-IFNAR-1 immunotherapy in autoimmune diseases, such as systemic lupus erythematosus (SLE), in which the action of excessive type I IFN on B cells and myeloid DCs may play a role in disease pathology.