In vitro toxicological support to establish specification limit for anti-CD3 monospecific impurity in a bispecific T cell engager drug, ERY974.

An emerging structure for anti-tumor antibody drugs utilizes a bispecific antibody (BiAb) that recognizes a tumor surface antigen and CD3 on T cells. An impurity that commonly contaminates these BiAb products is an anti-CD3 monoclonal antibody (mAb). The most plausible cause of toxic activity by an anti-CD3 mAb is the induction of cytokines via T cell activation. In this in vitro study, we compared cytokine induction and T cell activation after treatment with an anti-glypican-3/CD3 BiAb (ERY974), anti-CD3 mAb impurity (aCD3), or ERY974 spiked with 5% aCD3. We found that contamination with up to 5% aCD3 did not affect cytokine release by ERY974. Cytokine levels induced by ERY974 in the presence of target cells were significantly higher than those induced by aCD3, but were very similar to those by the spiked treatment. The results supported the specification of a 5% limit for aCD3. OKT-3 had much higher activity to induce cytokines from peripheral blood mononuclear cells in an in vitro assay than aCD3. This suggests that specification limit should be decided for each type of anti-CD3 impurity that affects T cell-activating BiAb drug products. In vitro cytokine assays can provide useful information for determining these specification limits.