Non-viral TRAC-knocked-in CD19KICAR-T and gp350KICAR-T cells tested against Burkitt lymphomas with type 1 or 2 EBV infection: In vivo cellular dynamics and potency
Tobias Braun, Alina Pruene, Milita Darguzyte, Alexander F. vom Stein, Phuong‐Hien Nguyen, Dimitrios L. Wagner, Jonas Kath, Alicia Roig‐Merino, Michael Heuser, Lucas L. Riehm, Andreas Schneider, Sabine Awerkiew, Steven R. Talbot, André Bleich, Constança Figueiredo, Martin Bornhäuser, Renata Stripecke
Abstract
Introduction The ubiquitous Epstein–Barr virus (EBV) is an oncogenic herpes virus associated with several human malignancies. EBV is an immune-evasive pathogen that promotes CD8 + T cell exhaustion and dysregulates CD4 + T cell functions. Burkitt lymphoma (BL) is frequently associated with EBV infections. Since BL relapses after conventional therapies are difficult to treat, we evaluated prospective off-the-shelf edited CAR-T cell therapies targeting CD19 or the EBV gp350 cell surface antigen. Methods We used CRISPR/Cas9 gene editing methods to knock in (KI) the CD19CAR.CD28z or gp350CAR.CD28z into the T cell receptor (TCR) alpha chain ( TRAC ) locus. Results Applying upscaled methods with the ExPERT ATx ® MaxCyte system, KI efficacy was ~20% of the total ~2 × 10 8 TCR-knocked-out (KO) generated cells. KO TCR KI CAR-T cells were co-cultured in vitro with the gp350 + CD19 + BL cell lines Daudi (infected with type 1 EBV) or with Jiyoye (harboring a lytic type 2 EBV). Both types of CAR-T cells showed cytotoxic effects against the BL lines in vitro . CD8 + KI CAR-T cells showed higher persistency than CD4 + KI CAR-T cells after in vitro co-culture with BL and upregulation of the activation/exhaustion markers PD-1, LAG-3, and TIM-3. Two preclinical in vivo xenograft models were set up with Nod.Rag.Gamma mice injected intravenously (i.v.) with 2 × 10 5 Daudi/fLuc-GFP or with Jiyoye/fLuc-GFP cells. Compared with the non-treated controls, mice challenged with BL and treated with CD19 KI CAR-T cells showed delayed lymphoma dissemination with lower EBV DNA load. Notably, for the Jiyoye/fLuc-GFP model, almost exclusively CD4 + CD19 KI CAR-T cells were detectable at the endpoint analyses in the bone marrow, with increased frequencies of regulatory T cells (T regs ) and TIM-3 + CD4 + T cells. Administration of gp350 KI CAR-T cells to mice after Jiyoye/GFP-fLuc challenge did not inhibit BL growth in vivo but reduced the EBV DNA load in the bone marrow and promoted gp350 antigen escape. CD8 + PD-1 + LAG-3 + gp350 KI CAR-T cells were predominant in the bone marrow. Discussion The two types of KO TCR KI CAR-T cells showed different therapeutic effects and in vivo dynamics. These findings reflect the complexities of the immune escape mechanisms of EBV, which may interfere with the CAR-T cell property and potency and should be taken into account for future clinical translation.