Abstract A043: Anti-CD19 CAR T-cells with a CRISPR/Cas9-mediated T-cell receptor knockout show high functionality in the absence of alloreactivity in vitro
Authors: Stenger Dana , Stief Tanja , Käuferle Theresa , Willier Semjon Manuel , Rataj Felicitas , Schober Kilian , Lotfi Ramin , Wagner Beate , Busch Dirk H. , Kobold Sebastian , Blaeschke Franziska , Feuchtinger Tobias 
Published: February 2019

Abstract
Overall survival of pediatric B-precursor ALL patients reached 90% in recent years. However, the outcome for refractory or relapsed children remains very poor. Anti-CD19 chimeric antigen receptor T-cells (CD19-CAR) showed significant antileukemic activity in relapsed and refractory B-precursor ALL. Especially in children, isolation of a suitable T-cell amount for autologous CAR T-cell manufacturing can be challenging due to low blood volume, low T-cell counts and clinical condition. In this case, the adoptive transfer of CAR T-cells from an unmatched healthy third-party donor provides a promising strategy. In order to prevent life-threatening graft-versus-host disease, a knockout (KO) of the endogenous T-cell receptor (TCR) has to be performed. Here, we generated CD19-CARs with a CRISPR/Cas9 mediated TCR KO, which remain highly functional and show strongly reduced alloreactivity compared to conventional CAR T-cells introduced into third-party T-cells. T-cells were isolated from peripheral blood mononuclear cells (PBMCs) of healthy donors and activated via anti-CD3/anti-CD28 stimulation. Retroviral transduction of a second generation anti-CD19 CAR (containing CD3zeta and 4-1BB stimulatory domains) was performed, followed by CRISPR/Cas9 mediated KO of the T-cell receptor beta chain via electroporation. After eleven days of expansion in the presence of IL-7 and IL-15, cells were purified for TCR KO-CD19-CAR T-cells via magnetic separation. Finally, the cell product was analyzed for cellular characteristics, functionality and alloreactivity by flow cytometry. A mean transduction rate of 37% for CD19-CARs and 40% for TCR KO-CD19-CARs was reached as well as a mean TCR KO rate of 78%. Both CD19-CARs as well as TCR KO-CD19-CARs showed suitable amounts of CD4- (45% vs. 33%) and CD8-T-cells (37% vs. 48%). The phenotype of CD19-CARs and TCR KO-CD19-CARs were comparable with mainly central memory (CM) (38% vs. 40%) and effector memory (EM) (57% vs. 51%) T-cells. The expansion of TCR KO-CD19-CARs was significantly reduced compared to conventional CD19-CARs (54-fold vs. 109-fold). This effect was not mediated by the loss of the TCR, but due to electroporation procedure. While CD19-CARs with or without TCR KO showed almost no background expression of the activation marker CD25 (2% vs 1%), contact with CD19-expressing targeT-cells resulted in a comparable upregulation of CD25 in both groups (95% vs. 94%). Co-culture with a CD19-expressing targeT-cell line led to an increased Interferon-γ secretion compared to unstimulated CARs, which was not significantly altered by the TCR KO (17% CD19-CAR vs. 14% TCR KO-CD19-CAR). CD19-dependent proliferative capacity of CAR T-cells was not influenced by loss of the TCR, as in both cases 97% of the T-cells proliferated after antigen recognition. Both CD19-CARs as well as TCR KO-CD19-CARs showed high, antigen-specific killing of 86% vs. 87% of the CD19-expressing targeT-cells at a 1:1 effector to target ratio. To evaluate the alloreactive potential of those T-cells, T-cells were co-cultured with irradiated PBMCs pooled from six different donors. 20% of TCR-expressing T-cells showed proliferation upon contact with non-HLA-matched PBMCs, whereas T-cells with a TCR KO showed almost no proliferation (&lt;3%), demonstrating significantly reduced alloreactivity of TCR KO T-cells. CD19-CAR T-cells lacking the endogenous TCR show a balanced CD4 to CD8 ratio and high proportion of the favorable CM T-cell phenotype. TCR KO-CD19-CARs remain highly functional and show similar activation, cytotoxicity, proliferative capacity and cytokine secretion as conventional CD19-CARs upon antigen recognition. T-cells with TCR KO do not mediate an alloreactive response to non-HLA-matched PBMCs and therefore are promising candidates for the generation of CAR T-cells derived from nonmatched healthy third-party donors or for use of donor-derived CAR T-cells after haploidentical stem cell transplantation.
Citation Format: Dana Stenger, Tanja Stief, Theresa Käuferle, Semjon Manuel Willier, Felicitas Rataj, Kilian Schober, Ramin Lotfi, Beate Wagner, Dirk H. Busch, Sebastian Kobold, Franziska Blaeschke, Tobias Feuchtinger. Anti-CD19 CAR T-cells with a CRISPR/Cas9-mediated T-cell receptor knockout show high functionality in the absence of alloreactivity in vitro [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A043.