The Development of a qPCR Assay for the Evaluation of the Dendritic Cell Chimeric Antigen Receptor Transcriptome

Chronic lymphocytic leukemia (CLL) is a hematological malignancy of B cells that accounts for a third of all new leukemia cases, but is incurable with standard first-line chemotherapies. Hematopoietic stem cell transplants offer the only curative treatment but are restricted to those with a matching donor. The recent advent of adoptive transfer therapy utilizing Chimeric Antigen Receptor (CAR) T cells, which are synthetic receptors that combine the target-specificity of antibodies to the effector capabilities of T cells, have emerged as a promising anticancer therapy due to its targeted tumoricidal activity. Clinical trials utilizing CD19-targeted CAR T cells have demonstrated strong efficacy against acute B-cell leukemia, but those performed in CLL have not been as successful for unclear reasons. This may be due to the dysfunctional tumor microenvironment which renders key immune effectors ineffective. Dendritic cells (DCs), a potent antigen presenting cell that functions as a bridge between the innate and adaptive immune systems, are rendered functionally and quantitatively deranged with an impaired ability to present antigen and thereby stimulate immunogenic responses. These limitations can potentially be rectified through an effective recruitment of an endogenous antitumor immune response and thus we hypothesize that genetically modified DCs should be capable of repolarizing the tolerogenic microenvironment induced by CLL toward one of immunogenicity to promote tumor eradication. In this study, we developed a qPCR assay to assess the DC CAR transcriptome. Because our CD19-targeted DC CARs with a CD40 intracellular domain signal via NF-κB to activate downstream elements, we needed to first assess the fidelity of our assay. Using the immortalized mouse DC cell line JAWS II, which lacks CD40, we used a potent TLR4 agonist, MPL, to stimulate our DCs through MyD88, an alternative pathway that also activates NF-κB. We saw elevated expression of downstream NF-κB elements, including IL-6, IL-10, IRF3, and IRF7. Establishing the sensitivity of our assay, we next evaluated the transcriptome of DC CARs by coculturing our CD19-targeted DC CARs containing a CD40 intracellular domain with HEK 293 cells, a human embryonic kidney cell line, retrovirally transduced with a human CD19 antigen to activate our CAR DCs and assess downstream gene expression. We observed expected elevation of downstream NF-κB elements including IL-6, IL-10, NOS2, IRF3, IFN-γ, TNFα, IL12p40, and TGF-β, indicating the viability of this assay. These positive results confirm the multidisciplinary potential of our assay as a promising technology that will allow us to better augment future CARs.