Background Conventional dendritic cells (cDCs) are essential in mounting and maintaining antitumor T cell immunity, but their low prevalence in tumors constitutes a limiting factor to the efficacy of immunotherapies. Anti-tumor immunity may be enhanced by therapeutic agents that promote dendritic cell expansion and differentiation. To better characterize the pharmacology of these therapies, in vitro models are needed that recapitulate physiologically relevant human DC subsets. FMS-like tyrosine kinase 3 ligand (FLT3L) is a key growth factor regulating the development of cDCs in the bone marrow. We used human stem cell derived DCs to characterize the potency of a FLT3L-Fc fusion protein and novel therapeutic modality.
Methods In vitro potency of wild-type human FLT3L and FLT3L-Fc was assessed in an OCI-AML5 cell proliferation assay. A human cDC1 differentiation assay was established to characterize FLT3L-Fc activity in a physiologically relevant context. In addition, we established an in vitro screening platform with stem cell derived DCs to evaluate innate agonist mediated activation of cDC1 and cDC2 subsets by flow cytometry and cytokine release. Progenitor cells were expanded, cryopreserved, and subsequently differentiated into DCs, thereby facilitating repeated screening assays with the same donor.
Results FLT3L-Fc and FLT3L induced equivalent dose dependent proliferation of OCI-AML5 cells with single digit pM EC50 potency. FLT3L-Fc induced differentiation of CD11clow CD141high cDC1 from primary human CD34+ cord blood progenitors in vitro; potency was comparable between donors. Cryopreserved progenitor cells were differentiated into a mixed cell culture containing cDC1 and cDC2. PolyI:C stimulation activated both subsets as measured by upregulation of cell surface markers including CD86, CD83, and HLA-DR, and secretion of CXCL10. This workflow enabled us to characterize PolyI:C potency across multiple donors, and with reproducible findings among experiments using progenitors from the same donor.
Conclusions FLT3L-Fc is a novel therapeutic modality to enhance DC expansion and differentiation and promote antitumor immunity in the context of combination immunotherapy. FLT3L-Fc has comparable in vitro potency to FLT3L and induces cDC1 differentiation from human cord blood progenitors. The physiologically relevant stem cell derived DC model also provides a robust method to assess potency of innate agonists in vitro.
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