The Universal ON/OFF Switch for Chimeric Antigen T (CAR-T) Cells

Introduction:: CAR-T therapy, a type of immunotherapy, employs genetically engineered T-cells to identify and eliminate cancer cells. While it has demonstrated encouraging outcomes in addressing specific blood cancer varieties, controlling the modified T-cells' activity and managing their response are challenges inherent to CAR-T therapy. One concern involves the possibility of "on-target, off-tumor" effects, where the altered T-cells mistakenly attack healthy cells that express the same target protein. This can lead to severe side effects, such as cytokine release syndrome and tumor lysis syndrome. Consequently, enhancing the safety and effectiveness of CAR-T therapy is essential. To overcome these limitations, we have designed an ON/OFF switch for CAR-T therapy that controls T cell receptor activity via the phosphorylation of C-terminal Src kinase (CSK). This switch comprises a DrmB homo-dimerizer and a CSK kinase domain. Upon the introduction of rimiducid, CSK is phosphorylated, activating the inhibitory tyrosine residue of LCK (Y505). As a result, when the switch is activated, there is a decrease in the production of intracellular cytokines, such as IFNγ and TNFα, as well as a reduction in IL2 secretion by T cells. This innovative approach aims to improve the safety and efficacy of CAR-T therapy by providing better control over the modified T cells' activity.


Materials and Methods:: Our approach involves incorporating the switch into Jurkat T-cells, CD8+ and CD4+ T-cells, and IL-13R CAR-T cells. We will assess the activity of tyrosine Y394 (LCK activation tyrosine residue) and Y505 (LCK inhibition tyrosine residue) of LCK (CSK downstream signal) using western blot and flow cytometry analysis. We will confirm that forced dimerization of CSK will result in its phosphorylation. Cytokine secretion in cell supernatants, after the addition (CSK phosphorylation) and removal of rimiducid, will be determined through a high-sensitivity multiplex assay and ELISA. Intracellular cytokines secretion, including IFNγ and TNFα, will be examined using flow cytometry (FACS) after performing intracellular cytokine staining.

The Switch and CAR receptors will be introduced into T cells using the piggyBac transposon system. Subsequently, we will establish a killing assay for IL13R-CAR-T cells, which will be conducted after adding the inducer (switch is ON) and again following its removal (switch is OFF). Lastly, we will evaluate the proliferation of CD8+ and CD4+ T-cells, as well as the elimination of IL13R+ cells in the A375 melanoma cell line in a killing assay using FACS staining techniques. This comprehensive methodology will allow us to thoroughly analyze the performance of the ON/OFF switch in controlling CAR-T cell activity and its potential to enhance the safety and efficacy of CAR-T therapy.


Results, Conclusions, and Discussions:: Through Western blot and flow cytometry analyses, we verified that the forced dimerization of CSK led to its phosphorylation. As a consequence, the inhibitory tyrosine of LCK (Y505) was activated, while the activation tyrosine Y394 was inhibited. ELISA results demonstrated that IL2 secretion by Jurkat and T cells was suppressed when the CSK switch was ON (following the addition of rimiducid). Removal of rimiducid (switch is OFF) restored IL2 secretion, confirming the switch's reversible activity. Intracellular cytokine secretion, including IFNγ and TNFα by T cells and CAR-T cells, was also reduced after the inducer was added.

Incorporating the CSK switch into CAR-T cells has the potential to enhance treatment safety. The activity of CAR-T cells can be regulated by adding rimiducid when patients experience cytokine release syndrome (CRS) or tumor lysis syndrome (TLS). The herpes simplex virus thymidine kinase (HSV-TK) gene is the most commonly used suicide gene; it encodes an enzyme that converts the prodrug ganciclovir into a toxic metabolite that eliminates CAR-T cells. However, introducing this gene into CAR-T cells carries substantial risk, as it is a viral gene that can trigger an immunogenic response.

In contrast, the CSK switch is derived from an endogenous human protein and relies on a safe, clinical-grade inducer for activation. When activated, the CSK switch specifically affects the CAR-T cell population without impacting unmodified T cells. This innovative approach offers a more controlled and safer alternative to existing methods, paving the way for improved CAR-T therapy outcomes with fewer side effects.


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