Development of Potency Assays for Quality Assessment of an Advanced Therapy Medicinal Product: Mitomycin C-Induced Peripheral Blood Mononuclear Cell (MIC) Product

Introduction:

Quality assessment in terms of product specificity and product potency is obligatory and strictly required by EMA and FDA to provide a platform for analysis of product comparability, stability and compatibility, and for clinical development to help predict product clinical efficacy. Mitomycin-treated donor peripheral blood mononuclear cells (MICs) could induce donor-specific tolerance via generation of recipient-derived tolerogenic dendritic cells (tDCs). To evaluate the immunomodulating capacity of the MIC product, we assessed the phenotype of tDCs, the immunosuppressive capacity of tDCs on allo-reactive CD4⁺ and CD8⁺ T cells as well as on CMV-specific CD8⁺ T cells.

Methods:

For standardization of the potency assay, third-party PBMCs positive for HLA-A2 and anti-CMV IgG were used. Immature DCs (iDCs) were generated 3 days before MIC production. MICs were generated under Good Manufacturing Practice (GMP) conditions. Each batch of MICs was introduced to iDCs at different ratios (1:0, 1:1, 1:10, 1:20) for a two-hour interaction followed by adding a DC maturation cocktail for overnight co-culture. Thereafter tDCs were purified by magnetic negative separation. The morphology of tDCs was observed by microscopy. The expression of HLA-DR, CD80, CD83, CD86 and CD103 was analyzed by flow cytometry. The inhibitory capacity on allo-reactive T cells and virus specific T cells was determined by mixed lymphocyte reaction (MLR) assay, by ELISpot assay and by Tetramer staining assay after one week of expansion in mixed lymphocyte peptide culture (MLPC).

Results:

In light microscopy (magnification: x 40), tDCs showed a relative smooth membrane surface. While the conventional mDCs were significantly larger in size with rough surface, richer ruffles on the cell membrane, and bigger, longer protrusions or pseudopodia. MIC products could inhibit the expression of costimulatory molecules on tDCs with an inhibition of 41% of CD80, 27% of CD83 and 23% of CD86. In parallel, the inhibitory marker CD103 was up-regulated about 65% on tDCs. Functionally, both tDCs and MICs could inhibit the IFN-γ secretion by CMV-specific CD8⁺ T cells, respectively. Moreover, the proliferation of allo-reactive T cells and CMV-specific T cells could be inhibited by tDCs and MICs.

Conclusions:

In summary, the potency assays, including the measurement of physicochemical parameters, the morphology and marker expression of tDCs, as well as the biologic characterization, the functionally immunosuppressive capacity of tDCs, comprise valuable parameters for the evaluation of clinically used advanced therapeutic cellular products.