Development of a quantitative cell-based ELISA, for a humanized anti-IL-2/IL-15 receptor beta antibody (HuMik beta(1)), and correlation with functional activity using an antigen-transfected murine cell line

The HuMi beta(1), humanized IgG1 monoclonal antibody directed toward the IL-2/IL-15 receptor beta-chain (CD 122), 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 if potential biological activity marker, of the HuMik beta(1) monoclonal antibody to a transfected 32D mouse cell line (32D beta) expressing IL-2R beta antigen on the cell surface. There is specific binding of HuMik beta(1) to the transfected cell line, titrating out in the concentration range of 1-1000ng/ml. Under identical conditions. there was no binding of HuMik beta(1) to the parent cell line 32D. Satisfactory binding curves with HuMik beta(1) were obtained with 32D beta cells grown between 3 and 19 passages in culture and at sced densities of 2 x 10(5) - 4 x 10(6) cells/well. The binding was specific for Mik beta antibodies recognizing the IL-2/IL-15 receptor beta subunit as demonstrated by binding of HuMik beta(1), Mik beta(2), and Mik beta(3) antibodies, and lack of binding of irrelevant humanized and chimeric antibodies and isotype-matched human IgG1 to the 32D beta cell. Also, the human IgG1 and irrelevant humanized and chimeric antibodies did not interfere with the HuMik beta(1) binding. The assay could detect changes in binding activity of HuMik beta(1) antibody under stressful conditions (heat and low pH) and the results paralleled the effect of stress oil the physicochemical characteristics. More importantly, the binding activity shows in apparent correlation to inhibition of IL-15-induced proliferation of 32D beta cells with HuMik beta(1). In conclusion, the cell-based ELISA method represents a simple, reproducible accurate quantitative assay for monitoring HuMik beta(1) activity and could be used as a potency marker assay for monitoring the lot-lot consistency and functional stability of HuMik beta(1) product. (c) 2006 Elsevier B.V. All rights reserved

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