(T1430-05-31) ddPCR-Based Assessment of Dendritic Cell Activation for Immunogenicity Evaluation

Purpose: Immunogenicity risk assessment of therapeutic products is a critical component of bioanalytical analysis. At non-clinical/discovery stages, multiple drug constructs may be in evaluation, quick and reliable immunogenicity screening is essential. A variety of assay platforms are used to evaluate immunogenicity risk, including ligand-binding based assays, mass spectrometry, and cell-based assays [1,2]. These approaches enable detection of anti-drug antibodies, identification of potential immunogenic epitopes, and neutralizing antibody binding to better predict and mitigate immunogenicity risk in therapeutic development. Dendritic cells (DCs) are key antigen-presenting cells that initiate immune responses by presenting processed antigens on MHC class II molecules to CD4⁺ T cells. While immature DCs have limited T cell activation capacity, maturation signals trigger upregulation of MHC-II and co-stimulatory molecules (e.g., CD40, CD86). These characteristics make DCs a valuable model for evaluating immunogenicity during therapeutic development [3]. This study aimed to evaluate the potential application of ddPCR analysis to effectively detect upregulation of key activation markers (CD25, CD40, CD80, and CD86) [4] in monocyte-derived human DCs following stimulation with known immunogenic agents (Pasotuxizumab, LPS, Zymosan, KLH) providing a sensitive approach to monitor dendritic cell activation. The data will be compared with results from established methods, including flow cytometry analysis of DC activation, the DC: T cell co-culture assay, and cytokine secretion profiling, to assess consistency and validate the sensitivity of ddPCR in detecting dendritic cell activation.

Methods: Cell culture: CD14⁺ monocytes were isolated from frozen PBMCs (Lonza) using a STEMCELL Technologies kit (#19359) and cultured with a dendritic cell differentiation supplement (#10988) for 3 days at 37°C with 5% CO₂. On day 4, the resulting MoDCs were stimulated with antigen in the presence of a maturation supplement to induce upregulation of co-stimulatory molecules (CD80, CD83, CD86, and MHC class II) indicative of a mature dendritic cell phenotype. ddPCR: Total RNA was extracted using the MagMAX mirVana Total RNA Isolation Kit (Thermo Fisher, A27828) and reverse transcribed into cDNA with the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher, #4368814). cDNA was either stored at −20°C or immediately used for droplet digital PCR (ddPCR). ddPCR was performed following Bio-Rad’s standard workflow: a 20 µL reaction was partitioned into ~20,000 nanoliter droplets, thermocycled for target amplification, and analyzed using a droplet reader. For dendritic cell activation analysis by flow cytometry, cells were labeled with fluorophore-conjugated antibodies targeting surface activation markers. Cytokine levels were measured using the MSD 10-Plex Proinflammatory Panel (Cat #K15049D) on supernatants collected after centrifugation and stored at −20°C or analyzed immediately. In the DC:CD4+T Cell Proliferation Assay, monocyte-derived dendritic cells (MoDCs) were incubated with the test articles for 24-48 hours. After the test articles were removed from the culture medium, the matured MoDCs were then co-cultured with autologous CD4+ T cells for 7 days. T cell activation and proliferation were then evaluated as indicators of the immune response. Flow cytometry data analysis: FCS files obtained from the flow cytometer were analyzed using the FlowJo software. Single-color compensation controls were acquired for each run. Compensation matrices were generated using data from approximately 10,000 events per sample. Data were cleaned using gating to exclude debris, doublets, and dead cells identified through fluorescence and forward/side scatter profiles.

Results: We evaluated the immune response of Pasotuxizumab, LPS, Zymosan, and KLH on monocyte-derived dendritic cells (moDCs) generated from five healthy donors. CD11c expression was used to confirm the differentiation of CD14⁺ monocytes into moDCs. Expression of the co-stimulatory molecules CD40, CD80, and CD86 was quantified by droplet digital PCR (ddPCR) at 24- and 48-hours post-stimulation. Each stimulus induced upregulation of these markers, indicating differences in dendritic cell activation. Importantly, expression profiles varied across donors and stimuli, highlighting donor-specific responses and distinct MOA for each compound. ddPCR demonstrated a consistent and modest increase in sensitivity relative to flow cytometry, with results closely matching those obtained through flow analysis. Activation marker expression and cytokine secretion increased after 48 hours of treatment.

Conclusion: The data shows that dendritic cell activation can be effectively monitored using ddPCR method. When used alongside established assays, ddPCR improves the immunogenicity risk assessment of drug candidates at early stages as it’s known to be more sensitive and provides better precision compared to flow cytometry. Moreover, increasing the donor pool will be important to improve statistical power and address the variability in immunogenic responses driven by both donor and antigen/drug differences.

References: 1. Smeijsters, E. H., et al. (2023). Optimization of a quantitative anti-drug antibodies against infliximab assay with the liquid chromatography-tandem mass spectrometry: A method validation study and future perspectives. Pharmaceutics, 15(5), 1477.
2. Collet-Brose, J., et al. (2016). Evaluation of multiple immunoassay technology platforms to select the anti-drug antibody assay exhibiting the most appropriate drug and target tolerance. Journal of Immunology Research, 2016, Article 5069678.
3. Walseng, E., et al. (2010). Dendritic cell activation prevents MHC class II ubiquitination and promotes MHC class II survival regardless of the activation stimulus. Journal of Biological Chemistry, 285(53), 41749–41754.
4. Hellman, P., & Eriksson, H. (2007). Early activation markers of human peripheral dendritic cells. Human Immunology, 68(5), 324–333.
5. Dai, Y., Adusumilli, A., Adeel, F., Kozhich, A., & Jawa, V. (2025). Monitoring antigen-specific cellular immune response using an RT-ddPCR-based assay for IFN-γ gene expression. Journal of Pharmaceutical Sciences, 114(2), 1017–1023.

Acknowledgements: This study was sponsored and funded by Takeda Development Center Americas, Inc. JP.M., J.R., X.Z. and M.Q. are employees of Takeda Development Center Americas, Inc., Cambridge, MA, USA.