Laboratorio de Biofarmacos em Celulas Animais, Instituto Butantan, Brasilia , SP 05503-900, Brazil.
Mol Biotechnol. 2010 Jul;45(3):218-25. doi: 10.1007/s12033-010-9269-2.
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.
两种携带抗 CD3 单克隆抗体相同可变区的人源化单克隆抗体构建体，完整 IgG 和 FvFc，在 CHO 细胞中表达。使用随机和定点整合，导致相似的表达水平。转染子用适当的选择剂选择，存活细胞铺在半固体培养基中，用 FITC 标记的抗人 IG 抗体捕获，并用机器人 ClonePix FL 挑选。用亲和层析从选定的克隆中纯化条件培养基，并通过 SDS-PAGE、Western-blot、SEC-HPLC 和等电聚焦进行表征。通过流式细胞术评估在健康人单核细胞中存在的靶标与两种构建体和原始鼠单克隆抗体之间的竞争结合。人源化构建体不能置换鼠抗 CD3 抗体，而鼠抗 CD3 抗体可以置换大约 20%的 FvFc 或 IgG 人源化版本。将进一步进行体外和体内临床前分析，以验证人源化版本展示所需的免疫调节谱的能力。
