GMP-compliant sorting of dendritic cells for CCR7 expression improves therapeutic efficacy in cancer by enhancing lymph node migration and antigen-specific T cell activation

Abstract

Background & Aim Dendritic cells (DCs) are a potentially powerful cell therapy due to their ability to induce T cell activation to defined antigens; however, this potential has not yet been realised in clinical trials. Monocyte-derived DCs (moDCs) are most commonly used in cancer therapy, although the rare blood DC subsets have also been explored. Crucially, cell function requires the chemokine receptor CCR7, which guides DCs from the site of antigen uptake to the lymph nodes. CCR7 expression, and therefore migration, is variable after isolation of DCs from blood or ex vivo culture, and may account for the limited efficacy seen in clinical trials. We hypothesised that selection of CCR7+ DCs by sorting would to improve cell function and therapeutic use of DCs. Methods, Results & Conclusion We have previously shown that murine CCR7+ bone marrow DCs (BMDCs) can be sorted using a novel chemokine-based system and show enhanced lymph node migration. This CCL19-fluorophore construct was shown to also detect expression of CCR7 on human DCs, and significantly enrich CCR7+ cells to high purity and viability (>90%) using magnetic bead and fluorescence-based sorting methods. Both murine and human CCR7+ DCs were more mature than CCR7- DCs by expression of CD80 and CD86 and production of T cell chemoattractants such as CCL17 and CCL22. In vitro, CCR7+ DCs were also most effective in stimulating antigen-specific T cells, especially memory T cells producing IFNγ, TNFα and IL-2. In vivo, sorted murine CCR7+ BMDCs, but not unsorted DCs, were sufficient to reduce subcutaneous and metastatic tumour growth in the B16F10.ova mouse model. Increased migration of injected DCs to lymph nodes led to an elevated overall number of tumour-specific T cells in CCR7+ DC-receiving mice, but this was not seen in those receiving unsorted DCs. In agreement with in vitro data, CCR7+ DCs increased production of mature tumour-specific T cells, particularly with the desirable memory (CD62L+CD44+) phenotype, suggesting their potency in a human therapeutic context. As production of this chemokine construct is possible at GMP grade, this protocol is amenable to GMP translation using the MACSQuant Tyto cell sorter. Purity and viability of CCR7+ DCs remained consistently high between the original and GMP-ised protocols, with an improved cell yield versus bead sorting. Overall, this novel sorting process is compliant with current GMP manufacture and creates a realistic DC product with enhanced function and potential therapeutic benefit.