Development of a quantitative cell-based ELISA, for a humanized anti-IL-2/IL-15 receptor β antibody (HuMikβ1), and correlation with functional activity using an antigen-transfected murine cell line

The HuMikβ1, a humanized IgG1 monoclonal antibody directed toward the IL-2/IL-15 receptor β-chain (CD122), inhibits the actions of the inflammatory cytokine IL-15, and may be useful for immunotherapy of an array of autoimmune disorders as well as diseases associated with the retrovirus human T-cell lymphotrophic virus 1 (HTLV-1). In order to facilitate the production of material for clinical investigation, we developed a cell-based ELISA (CbELISA) for measuring the binding activity, as a potential biological activity marker, of the HuMikβ1 monoclonal antibody to a transfected 32D mouse cell line (32Dβ) expressing IL-2Rβ antigen on the cell surface. There is specific binding of HuMikβ1 to the transfected cell line, titrating out in the concentration range of 1–1000 ng/ml. Under identical conditions, there was no binding of HuMikβ1 to the parent cell line 32D. Satisfactory binding curves with HuMikβ1 were obtained with 32Dβ cells grown between 3 and 19 passages in culture and at seed densities of 2 × 105–4 × 106 cells/well. The binding was specific for Mikβ antibodies recognizing the IL-2/IL-15 receptor β subunit as demonstrated by binding of HuMikβ1, Mikβ2 and Mikβ3 antibodies, and lack of binding of irrelevant humanized and chimeric antibodies and isotype-matched human IgG1 to the 32Dβ cell. Also, the human IgG1 and irrelevant humanized and chimeric antibodies did not interfere with the HuMikβ1 binding. The assay could detect changes in binding activity of HuMikβ1 antibody under stressful conditions (heat and low pH) and the results paralleled the effect of stress on the physicochemical characteristics. More importantly, the binding activity shows an apparent correlation to inhibition of IL-15-induced proliferation of 32Dβ cells with HuMikβ1. In conclusion, the cell-based ELISA method represents a simple, reproducible accurate quantitative assay for monitoring HuMikβ1 activity and could be used as a potency marker assay for monitoring the lot–lot consistency and functional stability of HuMikβ1 product.