140 Allogeneic CAR T cells with deoxycytidine kinase knockdown demonstrate resistance to fludarabine

BackgroundClinical outcomes in CAR T therapy correlate with engraftment, expansion, and persistence of CAR T cells. In order to facilitate engraftment and expansion, a lymphodepleting regimen consisting of cyclophosphamide and fludarabine precedes CAR T infusion. This creates niches for infused CAR T cells and stimulates beneficial homeostatic cytokine production. As these compounds are also toxic to CAR T cells, administering the proper doses of both the conditioning drugs and the cell therapies with appropriate timing can be a challenge.MethodsTo protect CAR T cells from fludarabine toxicity, we have knocked down deoxycytidine kinase (dCK), which converts fludarabine from the prodrug form to an active compound. This was accomplished using an RNAi sequence featuring a dCK-specific shRNA sequence embedded into a micro-RNA backbone. The resulting RNAi sequence demonstrated the potency of shRNA and the stability of a microRNA. Using Precision BioSciences’ ARCUS® gene editing technology and AAV-mediated targeted transgene insertion strategy, we disrupted the endogenous T cell receptor and inserted a transgene encoding a CD19-specific CAR and a dCK-specific RNAi sequence. Cells produced in this manner were exposed to CD19+ target cells in vitro and in immune-deficient mice and CAR T proliferation and target killing were monitored in the presence and absence of fludarabine.ResultsWe observed that the inclusion of the RNAi feature resulted in 70% reduction in dCK mRNA abundance, and conferred resistance to fludarabine in vitro. Moreover, treatment of tumor-bearing mice with fludarabine and dCK knockdown CAR T cells resulted in enhanced tumor clearance and survival compared to mice receiving CAR T cells alone or fludarabine plus dCK replete CAR T cells.ConclusionsCAR T cells expressing a dCK-specific RNAi gene exhibited resistance to fludarabine in vitro and in vivo. This drug resistance feature may enable allogeneic CAR-T cells to be simultaneously administered with fludarabine, suppressing rejection of CAR T and improving CAR T engraftment and expansion. This synergy between conditioning and CAR T therapy may improve clinical outcomes by enhancing effector persistence and tumor clearing.AcknowledgementsI would like to thank Aaron J. Martin, PhD and Daniel T. Macleod, PhD for their excellent mentorship and the Precision Biosciences Vivarium team for their support during this study.ReferencesMacleod DT, et al. Integration of a CD19 CAR into the TCR Alpha Chain Locus Streamlines Production of Allogeneic Gene-Edited CAR T Cells. Mol Ther 2017; 25(4):949–961.Fellmann C, et al. An Optimized microRNA Backbone for Effective Single-Copy RNAi. Cell Reports 2013;5:1704–1713.Ethics ApprovalThe animal study conducted was approved by the Institutional Animal Care and Use Committee (IACUC) of Mispro Biotech.

Different Susceptibility of T and B Cells to Cladribine Depends On Their Levels of Deoxycytidine Kinase Activity Linked to Activation Status

Deoxycytidine kinase (dCK) and 5’ deoxynucleotidase (NT5C2) are involved in metabolism of cladribine (2CdA), the immunomodulatory drug for multiple sclerosis; by mediating phosphorylation (activation) or phosphorolysis (deactivation) of 2CdA, respectively, these enzymes promote or prevent its accumulation in the cell, which leads to cell death. In particular, lymphocytes which present with a high intracellular dCK/NT5C2 ratio are more sensitive to 2CdA than other immune cells. We aim at determining if the expression of these enzymes and/or their activity differ in specific progenitor and mature immune cells and are influenced by cellular activation and/or exposure to 2CdA. Flow cytometry analysis showed no difference in dCK/NT5C2 ratio in progenitor and mature immune cells. 2CdA induced apoptosis in stimulated T and B cells and unstimulated B cells. dCK expression was enhanced by 2CdA at mRNA and protein levels in activated T cells and mRNA level in activated B cells. dCK activity, measured through an in-house luminescence release enzyme assay was higher in activated T and B cells, and such an increase was abrogated in activated B cells, but not T cells, upon exposure to 2CdA. These results reveal an important relationship between dCK activity and the effect of 2CdA on B and T cells, according to their activation status. Further study is warranted to evaluate whether dCK activity could, in the future, be a suitable predictive biomarker of lymphocyte response to 2CdA. Graphical Abstract "Image missing"

Deoxycytidine Kinase (DCK) Mutations in Human Acute Myeloid Leukemia Resistant to Cytarabine

Resistance to cytarabine is an important cause of therapy failure in persons with acute myeloid leukemia (AML). Deoxycytidine kinase, encoded by DCK, catalyzes phosphorylation of cytarabine to cytarabine monophosphate, a necessary step for eventual incorporation of cytarabine triphosphate into DNA and for clinical efficacy. Whether DCK mutations make AML cells resistant to cytarabine is controversial. We studied DCK mutations and messenger RNA (mRNA) concentrations in leukemia cells from 10 subjects with AML who received cytarabine-based therapy and relapsed and in 2 artificially induced cytarabine-resistant AML cell lines. DCK mutations were detected in 4 subjects with AML relapsing after achieving a complete remission and receiving high-dose cytarabine postremission therapy. Most mutations were in exons 4–6 and were not present before therapy. DCK was also mutated in cytarabine-resistant but not parental AML cell lines. DCK mRNA concentrations were significantly decreased in cytarabine-resistant K562 and SHI-1 cells compared with cytarabine-sensitive parental cells. Mutation frequency of DCK and mRNA concentration did not correlate with the extent of cytarabine resistance indicating other factors operate. Overexpression of wild-type DCK restored cytarabine sensitivity to previously resistant leukemia cell lines. Our data contribute to the understanding of cytarabine resistance in persons with AML.

Small Molecular Gemcitabine Prodrugs for Cancer Therapy

Gemcitabine as a pyrimidine nucleoside analog anticancer drug has high efficacy for a broad spectrum of solid tumors. Gemcitabine is activated within tumor cells by sequential phosphorylation carried out by deoxycytidine kinase to mono-, di-, and triphosphate nucleotides with the last one as the active form. But the instability, drug resistance and toxicity severely limited its utilization in clinics. In the field of medicinal chemistry, prodrugs have proven to be a very effective means for elevating drug stability and decrease undesirable side effects including the nucleoside anticancer drug such as gemcitabine. Many works have been accomplished in design and synthesis of gemcitabine prodrugs, majority of which were summarized in this review.