4'-Ethynyl-2'-Deoxycytidine (EdC) Preferentially Targets Lymphoma and Leukemia Subtypes by Inducing Replicative Stress.

Anticancer nucleosides are effective against solid tumors and hematological malignancies, but typically are prone to nucleoside metabolism resistance mechanisms. Using a nucleoside-specific multiplexed high-throughput screening approach, we discovered 4'-ethynyl-2'-deoxycytidine (EdC) as a third-generation anticancer nucleoside prodrug with preferential activity against diffuse large B-cell lymphoma (DLBCL) and acute lymphoblastic leukemia (ALL). EdC requires deoxycytidine kinase (DCK) phosphorylation for its activity and induced replication fork arrest and accumulation of cells in S-phase, indicating it acts as a chain terminator. A 2.1Å co-crystal structure of DCK bound to EdC and UDP reveals how the rigid 4'-alkyne of EdC fits within the active site of DCK. Remarkably, EdC was resistant to cytidine deamination and SAMHD1 metabolism mechanisms and exhibited higher potency against ALL compared to FDA approved nelarabine. Finally, EdC was highly effective against DLBCL tumors and B-ALL in vivo. These data characterize EdC as a pre-clinical nucleoside prodrug candidate for DLBCL and ALL.

Targeted Association and Intracellular Delivery of Nanocargoes into Primary T Lymphocytes via Interleukin-2 Receptor-Mediated Endocytosis.

Despite the tremendous progress in immunotherapy regimens using T cells, efforts to modulate the functions of T cells are still significantly hampered by the lack of reliable methods to deliver various cargoes into the T cells. This ongoing challenge originates from the intrinsic resistance of T cells in taking up exogenous materials. Here, we strategically aimed to hijack the natural endocytosis of Interleukin-2 (IL2) by the activated T cells for the targeted association and intracellular delivery of cargoes in varying sizes. First, we carefully characterized the fluctuations in the expression levels of IL2 receptor (IL2R) subunits (CD25, CD122, and CD132) during the murine primary T cell cultures over 12 days. We identified the highest fraction of T cells that would express the high-affinity trimeric IL2R on Day 3. By examining the association and uptake efficiencies of IL2 molecules that are biotinylated via either random lysine-targeting chemical reaction (using NHS-PEG4-Biotin) or site-specific enzymatic modification (using Avitag sequence), we demonstrated that the most efficient delivery of cargo can be achieved by C-terminal conjugation. Upon confirmation of successful delivery of a small model cargo, streptavidin, we employed superparamagnetic iron oxide nanoparticles (SPIONs) as bigger model cargoes having core diameters of 50, 100, and 200 nm. We examined the association and intracellular delivery of the IL2-conjugated nanocargoes using flow cytometry, confocal laser scanning microscopy, and transmission electron microscopy. While cargoes of all tested sizes were successfully associated with the IL2R-expressing T cells in comparable efficiencies, the uptake efficiencies were inversely proportional to the sizes of the cargoes. Nevertheless, our current definitive report confirms that nanocargoes with a practical maximum size limit around 100-200 nm can be intracellularly delivered into activated primary T cells using IL2R-mediated endocytosis, which opens a new horizon for engineering and manufacturing of various T cell immunotherapeutics.

Calcium signaling on Jurkat T cells induced by microbeads coated with novel peptide ligands specific to human CD3ε.

CD3ε is expressed on T lymphocytes as a part of the T cell receptor (TCR)-CD3 complex. Together with other CD3 molecules, CD3ε is responsible for the activation of T cells via transducing the event of antigen recognition by the TCR into intracellular signaling cascades. The present study first aims to identify a novel peptide ligand that binds to human CD3ε in a specific manner and to perform an initial evaluation of its biological efficacy on the human T cell line, Jurkat cells. We screened a phage-display peptide library against human CD3ε using a subtractive biopanning process, from which we identified 13 phage clones displaying unique peptide sequences. One dominant phage clone displaying the 7 amino acid sequence of WSLGYTG, which occupied 90% of tested plaques (18 out of 20) after the 5th round of biopanning, demonstrated a superior binding behavior to other clones in the binding assays against recombinant CD3ε on microbeads or Jurkat cells. The synthesized peptide also showed specific binding to Jurkat cells in a dose-dependent manner but not to B cell lymphoma line, 2PK3 cells. Molecular modeling and docking simulation confirmed that the selected peptide ligand in an energetically stable conformation binds to a pocket of CD3ε that is not hidden by either CD3γ or CD3δ. Lastly, magnetic microbeads conjugated with the synthesized peptide ligands showed a weak but specific association with Jurkat cells and induced the calcium flux, a hallmark indication of proximal T cell receptor signaling, which gave rise to an enhancement of IL-2 section and cell proliferation. The novel peptide ligand and its various multivalent forms have a great potential in applications related to T cell biology and T cell immunotherapy.

Calcium enhances polyplex-mediated transfection efficiency of plasmid DNA in Jurkat cells.

Jurkat, an immortalized cell line derived from human leukemic T lymphocytes, has been employed as an excellent surrogate model of human primary T-cells for the advancement of T-cell biology and their applications in medicine. However, presumably due to its T-cell origin, Jurkat cells are very difficult to transfect. Thus, for the genetic modification of Jurkat cells, expensive and time-consuming viral vectors are normally required. Despite many previous efforts, non-viral vectors have not yet overcome the hurdles of low transfection efficiency and/or high toxicity in transfection of Jurkat cells. Here, we report that a simple addition of calcium ions (Ca2+) into culture media at optimal concentrations can enhance the efficiency of the polyplex-mediated transfection using poly(ethylene imine) (PEI) by up to 12-fold when compared to the polyplex-only control. We show that calcium enhances the association between polyplex and Jurkat, which is at least partially responsible for the increase in transmembrane delivery of polyplex and consequential enhancement in expression of transgene. Other cations, Mg2+ or Na+ did not show similar enhancement. Interestingly, addition of Ca2+ was rather detrimental for the transfection of lipoplex on Jurkat cells. Observation of significant enhancement in the transfection of non-viral vectors with a simple and physiologically relevant reagent like Ca2+ in the engineering of hard-to-transfect cells such as Jurkat warrants further investigation on similar strategies.