Evaluation of IRES-mediated, cell-type-specific cytotoxicity of poliovirus using a colorimetric cell proliferation assay.

PVS-RIPO is a recombinant oncolytic poliovirus designed for clinical application to target CD155 expressing malignant gliomas and other malignant diseases. PVS-RIPO does not replicate in healthy neurons and is therefore non-pathogenic in rodent and non-human primate models of poliomyelitis. A tetrazolium salt dye-based cellular assay was developed and qualified to define the cytotoxicity of virus preparations on susceptible cells and to explore the target cell specificity of PVS-RIPO. In this assay, PVS-RIPO inhibited proliferation of U87-MG astrocytoma cells in a dose-dependent manner. However, HEK293 cells were much less susceptible to cell killing by PVS-RIPO. In contrast, the Sabin type 1 live attenuated poliovirus vaccine strain (PV(1)S) was cytotoxic to both HEK293 and U87-MG cells. The correlation between expression of CD155 and cytotoxicity was also explored using six different cell lines. There was little or no expression of CD155 and PVS-RIPO-induced cytotoxicity in Jurkat and Daudi cells. HEK293 was the only cell line tested that showed CD155 expression and resistance to PVS-RIPO cytotoxicity. The results indicate that differential cytotoxicity measured by the colorimetric assay can be used to evaluate the cytotoxicity and cell-type specificity of recombinant strains of poliovirus and to demonstrate lot to lot consistency during the manufacture of viruses intended for clinical use.

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

The HuMikbeta(1), a humanized IgG1 monoclonal antibody directed toward the IL-2/IL-15 receptor beta-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 HuMikbeta(1) monoclonal antibody to a transfected 32D mouse cell line (32Dbeta) expressing IL-2Rbeta antigen on the cell surface. There is specific binding of HuMikbeta(1) to the transfected cell line, titrating out in the concentration range of 1-1,000 ng/ml. Under identical conditions, there was no binding of HuMikbeta(1) to the parent cell line 32D. Satisfactory binding curves with HuMikbeta(1) were obtained with 32Dbeta cells grown between 3 and 19 passages in culture and at seed densities of 2 x 10(5)-4 x 10(6) cells/well. The binding was specific for Mikbeta antibodies recognizing the IL-2/IL-15 receptor beta subunit as demonstrated by binding of HuMikbeta(1), Mikbeta(2) and Mikbeta(3) antibodies, and lack of binding of irrelevant humanized and chimeric antibodies and isotype-matched human IgG1 to the 32Dbeta cell. Also, the human IgG1 and irrelevant humanized and chimeric antibodies did not interfere with the HuMikbeta(1) binding. The assay could detect changes in binding activity of HuMikbeta(1) 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 32Dbeta cells with HuMikbeta(1). In conclusion, the cell-based ELISA method represents a simple, reproducible accurate quantitative assay for monitoring HuMikbeta(1) activity and could be used as a potency marker assay for monitoring the lot-lot consistency and functional stability of HuMikbeta(1) product.