Background Chimeric antigen receptor (CAR) T therapy has shown remarkable success in treating liquid tumours but current approved therapies rely on autologous T cells which are expensive, difficult to manufacture and not readily available for patients whose disease progress rapidly. The production of safe and effective allogeneic CAR-T cells is needed to increase accessibility of CAR-T therapy and broaden its application. The main approach to generate allogeneic CAR-T therapy is by disrupting T cell receptor (TCR) expression to minimize Graft-versus-Host Disease (GVHD) mediated through the TCR of donor cells against the recipient’s major histocompatibility complex (MHC). However, the TCR disruption approach has shown limited persistence in vivo  and in clinical trials  unlike the long term durability of autologous CAR-T cells. Here, we propose a novel platform for allogeneic CAR-T therapy that retains the TCR but inhibits TCR signalling by knocking out the Lymphocyte-specific protein tyrosine kinase (LCK) – a well-established kinase for proximal TCR activation. This builds on the discovery that our second generation CD28-CAR can be activated independently of LCK unlike the endogenous TCR.
Methods We utilise the CRISPR-Cas9 system to knockout LCK and TCR in both mouse and human primary T cells. We show how this difference in CAR and TCR signalling can be exploited to generate LCK knockout CAR-T cells that showed similar or enhanced in vitro and in vivo efficacy against tumour cells compared to conventional CAR-T cells of both human and mouse T cell origin.
Results LCK knockout T cells have reduced proliferation and intracellular cytokine staining against allogeneic PBMCs compared to T cells, suggesting comparable suppression of TCR-mediated alloreactivity to TCR knockout T cells. In the immunodeficient mouse model where human T cells cause xenogeneic GVHD, LCK knockout T cells showed reduced xenogeneic GVHD comparable to TCR knockout T cells. Murine LCK knockout T cells showed the same suppression of TCR signalling as TCR knockout T cells in vitro. In murine major mismatched allogeneic models, murine LCK knockout T cells showed a reduction in GVHD symptoms compared to wild-type T cells. Compared to TCR knockout T cells, the LCK knockout T cells showed superior persistence and higher engraftment in allogeneic recipient mice.
Conclusions Our study suggests LCK knockout CAR-T cells inhibits TCR-mediated alloreactivity and retains the TCR for improved persistence while maintaining CAR activation potential which results in a superior allogeneic CAR-T therapy compared to TCR knockout CAR-T cells.
Stenger, D, Stief, T A, Kaeuferle, T, Willier, S, Rataj, F, Schober, K, Vick, B, Lotfi, R, Wagner, B, Grünewald, T G P, Kobold, S, Busch, D H, Jeremias, I, Blaeschke, F, & Feuchtinger, T. Endogenous TCR promotes in vivo persistence of CD19-CAR-T cells compared to a CRISPR/Cas9-mediated TCR knockout CAR. Blood. 2020;136(12):1407-1418.
Wang, Z, Li, N, Feng, K, Chen, M, Zhang, Y, Liu, Y, Yang, Q, Nie, J, Tang, N, Zhang, X, Cheng, C, Shen, L, He, J, Ye, X, Cao, W, Wang, H, & Han, W. Phase I study of CAR-T cells with PD-1 and TCR disruption in mesothelin-positive solid tumors. Cell Mol Immunol. 2021;18(9):2188-2198.
Ethics Approval Animal Protocols were approved by NUS IACUC (R20-1303). Use of human primary T cells was approved by Institutional Review Board (IRB) (H-19-026).
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