RESUMO
Two humanized monoclonal antibody constructs bearing the same variable regions of an anti-CD3 monoclonal antibody, whole IgG and FvFc, were expressed in CHO cells. Random and site-specific integration were used resulting in similar expression levels. The transfectants were selected with appropriate selection agent, and the surviving cells were plated in semi-solid medium for capture with FITC-conjugated anti-human IG antibody and picked with the robotic ClonePix FL. Conditioned media from selected clones were purified by affinity chromatography and characterized by SDS-PAGE, Western-blot, SEC-HPLC, and isoelectric focusing. Binding to the target present in healthy human mononuclear cells was assessed by flow cytometry, as well as by competition between the two constructs and the original murine monoclonal antibody. The humanized constructs were not able to dislodge the murine antibody while the murine anti-CD3 antibody could dislodge around 20% of the FvFc or IgG humanized versions. Further in vitro and in vivo pre-clinical analyses will be carried out to verify the ability of the humanized versions to demonstrate the immunoregulatory profile required for a humanized anti-CD3 monoclonal antibody.
Assuntos
Anticorpos Monoclonais/química , Complexo CD3/imunologia , Fragmentos de Imunoglobulinas/química , Região Variável de Imunoglobulina/química , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/imunologia , Ligação Competitiva , Western Blotting , Complexo CD3/metabolismo , Células CHO , Cricetinae , Cricetulus , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Fragmentos de Imunoglobulinas/biossíntese , Fragmentos de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/metabolismo , Ponto Isoelétrico , Camundongos , Peso MolecularRESUMO
Two humanized monoclonal antibody constructs bearing the same variable regions of an anti-CD3 monoclonal antibody, whole IgG and FvFc, were expressed in CHO cells. Random and site-specific integration were used resulting in similar expression levels. The transfectants were selected with appropriate selection agent, andthe surviving cells were plated in semi-solid medium for capture with FITC-conjugated anti-human IG antibody andpicked with the robotic ClonePix FL. Conditioned media from selected clones were purified by affinity chromatographyand characterized by SDS-PAGE, Western-blot, SEC-HPLC, and isoelectric focusing. Binding to the targetpresent in healthy human mononuclear cells was assessed by flow cytometry, as well as by competition between thetwo constructs and the original murine monoclonal antibody. The humanized constructs were not able to dislodgethe murine antibody while the murine anti-CD3 antibody could dislodge around 20% of the FvFc or IgG humanizedversions. Further in vitro and in vivo pre-clinical analyses.
