Allogeneic CAR-T Therapy Enabled By Base Editing of Lck to Resist Dasatinib Used to Prevent Rejection Mediated through Both T and NK in Host

We are reporting a new strategy to overcome the rejection of the universal CAR-T cells by host through inhibiting host T and NK cells with dasatinib, while preserving the function of UCAR-T through base editing of LCK, a critical tyrosine kinase for T and NK cell activation. Autologous chimeric antigen receptor (CAR) T cells have changed the therapeutic landscape in hematological malignancies. The universal CAR-T (UCAR-T) therapies derived from healthy donor hold many promises, such as better function of CAR-T cells, off-shelf in availability, reduced variation in product quality and decreased cost. However, one challenge facing UCAR-T therapy is rapidly elimination of allogeneic cells by the host immune system. Although many strategies, such as clearance of host T/NK cells with alemtuzumab and knockout of B2M gene, have been developed and tested with some success in patients, novel ways to circumvent the surveillance of the host immune system against allogeneic UCAR-T cells are still under intensive investigation. The threonine (T316) in LCK, similar to T315 in Abl kinase, is a gatekeeping amino acid in ATP pocket. The T315I mutation of ABL kinase is the most prevalent mutation in imatinib resistant patients without affecting its kinase function. the LCK-T316M mutation has previously been shown to render T cells resistant to dasatinib. Therefore, we hypothesized that T316I mutation, achievable with cytosine base editing, can also make LCK resistant to dasatinib. In vitro experiments confirmed that in the presence of dasatinib, T cells with LCK-T316I point mutation could still be stimulated by anti-CD3/CD28 DynaBeads and expressed higher level of CD25, CD69, and 4-1BB while the wildtype T and NK cells were completely inhibited. CD19 targeting UCAR-T (Km UCAR-T) were generated by knocking out TRAC, B2M and base-editing of LCK simultaneously. Mixed lymphocyte reactions (MLRs) were performed by the incubation of Km UCAR-T and PBMC from HLA mismatched donors upon dasatinb or DMSO treatment. It was demonstrated that only the Km UCAR-T cells could survive and expand under the action of dasatinib. Besides, higher dasatinib concentrations could increase the proportion of Km UCAR-T cells even more. Dasatinib is an approved small molecule drug, with well understood safety profile in patients. Furthermore, dasatinib had no effect on the proliferation of T and NK cell, implying that the immune function can be quickly restored after withdrawing. Allogeneic CAR-T cells have the potential to overcome the limitations of autologous CAR-T cells and Km UCAR-T cells will be a new orthogonal approach that can be combined with other strategies to achieve the goal. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal