Epigenetic Priming of Bladder Cancer Cells With Decitabine Increases Cytotoxicity of Human EGFR and CD44v6 CAR Engineered T-Cells

Abstract

BackgroundTreatment of B-cell malignancies with CD19-directed chimeric antigen receptor (CAR) T-cells marked a new era in immunotherapy, which yet has to be successfully adopted to solid cancers. Epigenetic inhibitors of DNA methyltransferases (DNMTi) and histone deacetylases (HDACi) can induce broad changes in gene expression of malignant cells, thus making these inhibitors interesting combination partners for immunotherapeutic approaches.MethodsUrothelial carcinoma cell lines (UCC) and benign uroepithelial HBLAK cells pretreated with the DNMTi decitabine or the HDACi romidepsin were co-incubated with CAR T-cells directed against EGFR or CD44v6, and subsequent cytotoxicity assays were performed. Effects on T-cell cytotoxicity and surface antigen expression on UCC were determined by flow cytometry. We also performed next-generation mRNA sequencing of inhibitor-treated UCC and siRNA-mediated knockdown of potential regulators of CAR T-cell killing.ResultsExposure to decitabine but not romidepsin enhanced CAR T-cell cytotoxicity towards all UCC lines, but not towards the benign HBLAK cells. Increased killing could neither be attributed to enhanced target antigen expression (EGFR and CD44v6) nor fully explained by changes in the T-cell ligands PD-L1, PD-L2, ICAM-1, or CD95. Instead, gene expression analysis suggested that regulators of cell survival and apoptosis were differentially induced by the treatment. Decitabine altered the balance between survival and apoptosis factors towards an apoptosis-sensitive state associated with increased CAR T-cell killing, while romidepsin, at least partially, tilted this balance in the opposite direction. Knockdown experiments with siRNA in UCC confirmed BID and BCL2L1/BCLX as two key factors for the altered susceptibility of the UCC.ConclusionOur data suggest that the combination of decitabine with CAR T-cell therapy is an attractive novel therapeutic approach to enhance tumor-specific killing of bladder cancer. Since BID and BCL2L1 are essential determinants for the susceptibility of a wide variety of malignant cells, their targeting might be additionally suitable for combination with immunotherapies, e.g., CAR T-cells or checkpoint inhibitors in other malignancies.

A novel CD34-derived hinge for rapid and efficient detection and enrichment of CAR T cells

“…Interestingly, extending the C6 hinge by 80 additional amino acids (C7) was not possible, as we observed non-specific binding of C7-hinged CD19 and R12 CARs to AML-M4/M5 cell lines, similarly to what has been described for CARs containing CH 2 CH 3 hinges from human immunoglobulins. 14 Moreover, we recently demonstrated that our C6 hinge worked in CAR constructs recognizing solid tumor-associated antigens 49 , 50 and also here did not result in off-target activation of the genetically modified T cells, thus suggesting again that the C6 hinge seems to be well suited for CAR constructs for clinical products.…”

Genetic Engineering and Enrichment of Human NK Cells for CAR-Enhanced Immunotherapy of Hematological Malignancies

“…After the establishment of an optimized transduction protocol, primary human NK cells with low CD33 expression were transduced with 5-fold concentrated BaEV-Rless-pseudotyped lentiviral particles encoding BFP in cis with CD19, CD33, CD123, or EGFR CARs, harboring our CD34-derived hinge ( 20 , 29 ), on Retronectin ( Figures 2A, B ). Three to four days after transduction, the expression of the CARs was analyzed after staining of the C6 hinge region with the QBend-10 antibody and detecting BFP expression by flow cytometry.…”

“…The efficacy of CAR immune effector cells to eliminate their target cells is influenced by several factors, including the affinity of the scFv present in the CAR construct, the expression levels of the CAR itself on the effector cells, and availability of the targeted antigen/epitope on the target cell ( 8 , 29 ). Thus, for CARs with a low affinity or when the target antigen is expressed at low levels, high and stable CAR expression is crucial to ensure excellent antitumor cytotoxicity.…”

“…The CARs were co-expressed via a T2A site with the monomeric tag blue fluorescent protein (BFP from Evrogen, Moscow, Russia) under the control of the myeloproliferative sarcoma virus (MPSV) promoter ( 27 ) and equipped with our CD34-derived hinge C6 ( 28 ), CD28 transmembrane, and co-stimulatory domains as well as the CD3 zeta-chain unit ( 27 ). The CD19, CD33, CD123, and epidermal growth factor receptor (EGFR) (clone cetuximab/C225, from now on referred to as Cetux) CARs were previously described ( 20 , 27 – 29 ). In the ALL xenograft model, CD19 CARs were co-expressed with codon-optimized soluble human IL15 or human IL15 tethered to the IL15 receptor α-chain (IL15-IL15R).…”

Optimized NGFR-derived hinges for rapid and efficient enrichment and detection of CAR T cells in vitro and in vivo

“…Vesicular stomatitis virus G glycoprotein-pseudotyped replication-deficient lentiviral vectors were generated by polyethyleneiminie transfection (Sigma-Aldrich, Darmstadt, Germany) of 6 μg pczVSV-G, 6 μg pCD-NL/BH, and 6 μg vector plasmid into HEK293T cells as described previously. 24 , 62 , 63 Two days after transfection, virus-containing supernatant was harvested, filtered (0.45 μm), and used for transduction of eukaryotic cells. For the transduction of Jurkat or primary human T cells, 5 × 10 5 cells were incubated with 2 mL virus-containing supernatant and 10 μg/mL protamine phosphate (Sigma-Aldrich) for 24 h, replenished with fresh medium, and used for experiments after 48 h.…”