Development of an effective direct qPCR method for cell therapy product quantification in non-clinical biodistribution studies

Background Evaluating the non-clinical biodistribution (BD) of cell therapy products (CTPs) is essential to predicting their efficacy and safety in clinical trials. Although BD is typically quantified by qPCR following DNA extraction, this process is labor-intensive and time-consuming, and subject to variability in extraction yield, which complicates normalization to organ weight or blood volume. In contrast, direct qPCR enables the quantification of lysed samples without DNA extraction. Here, we systematically developed an effective direct qPCR method applicable to BD studies of CTPs. Methods We used HLCN061, a genetically modified natural killer (NK) cell derived from human induced pluripotent stem (iPS) cells. Mouse organs and blood containing HLCN061 cells were lysed, diluted, and directly subjected to real-time PCR using a human-specific Alu primer set. PCR reactions employed an inhibitor-tolerant master mix. Assay performance was evaluated by linearity (R 2 ), amplification efficiency, relative error (RE), coefficient of variation (CV), and lower limit of quantification (LLOQ). The validated method was then applied to evaluate the BD of HLCN061 cells over 24 h following intravenous administration to mice. Results Across all tissues and blood samples, R 2 exceeded 0.99, PCR amplification efficiency ranged from 90% to 110%, and RE was within ±25%. CV values were within 25% except for one case. LLOQ was 0.25 cells/mg weight for the liver, lungs, kidneys, and heart; 1 cell/mg weight for the spleen and ovaries; and 0.3 cells/μL for blood. Weight-normalized concentrations were highest in the lung, with the most rapid clearance from blood. Conclusion This direct qPCR method is effective and suitable for non-clinical BD assessment of CTPs.