Comparison of Humanized IgG and FvFc Anti-CD3 Monoclonal Antibodies Expressed in CHO Cells

Abstract

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.

Utilization of non‐AUG initiation codons in a flow cytometric method for efficient selection of recombinant cell lines

“…Although there are several other screening strategies and technologies (Browne and Al-Rubeai, 2007;Caron et al, 2009;Serpieri et al, 2010) specifically designed to overcome the challenge of finding rare high expressing cells in the background of low and non-expressing cells, the versatility of the alternate start reporter strategy gives it several advantages over these systems. For instance, the pivotal role of flow cytometry in this method provides a considerable throughput advantage relative to the immunological detection of secreted product in semi solid media.…”

Improving biophysical properties of a bispecific antibody scaffold to aid developability

“…3B). Subsequently, clones from Pool N were selected using the standard ClonePIX™ platform procedures 52 with the clones serially expanded and screened based on expression titer to arrive at 20 final candidate clones. The productivity and performance in fed-batch cultivation process indicate that stable clones yield greater than 900 mg/L antibody prior to feed and upstream process optimization, comparable to what can be achieved for a conventional mAb at this stage of process development.…”

Generation of monoclonal antibody-producing mammalian cell lines

“…ClonePix™ from Genetix [202,203] and CellCelector from Aviso [204] are two systems that have been used for selecting recombinant protein-producing colonies. Cells expressing mAb are plated in semi-solid media containing a fluorescent detection antibody against the…”