CAR-T Cells Targeting gp350 Recognize Immortalized Cells Latently Infected with EBV

Objectives: Epstein-Barr virus (EBV) is associated with lymphoproliferative disease in immunocompromised hosts and with human B-cell lymphomas and carcinomas. Adoptive transfer of virus-specific T cells is not practical when memory T cells from HLA-matched donors are not available. Hence, we designed T cells expressing EBV-specific chimeric antigen receptors (CARs) to bypass the need of matched memory T cells. The surface-bound glycoprotein 350 (gp350) was used as target, because it is abundantly expressed during lytic EBV replication and it can also be detected in EBV-immortalized cells.

Methods: gp350-CARs were constructed by fusion of single-chain variable fragments of two high affinity gp530-specific human mABs (7A1 and 6G4) to CAR-backbones containing the CD28/CD3ζ domains. Transduction of human T cells from PBMC and cord blood with γ-retroviral vectors showed higher expression levels of 7A1-gp350-CAR than 6G4-gp350-CAR.

Results: We used 293T cells expressing gp350 and B95-8 immortalized cells from a tamarin monkey (6-10% gp350+) and human B cells immortalized with the EBV laboratory strain M81 (30% gp350+) in order to compare the potency of gp350-CAR T cells in vitro. Both 7A1-gp350-CAR and 6G4-gp350-CAR were activated and proliferated in the presence of gp350+ cells, inducing cytotoxicity of the target cells. Pilot experiments in a preclinical humanized mouse model consisting of Nod.Rag mice transplanted with human cord-blood (CB) stem cells and infected with an EBV/fLUC strain, we could confirm persistence of CB-matched 7A1-gp350-CAR T cells in spleen, bone marrow and lung for up to 6 weeks. In some animals, this was correlated with lower EBV dissemination measured by optical imaging and PCR.

Conclusions: We showed that EBV-specific CARs can reprogram naïve or memory T cells from PBMC or CB to react against EBV infected cells in an HLA-independent manner. This approach can be translated in the future for the generation of anti-EBV-CAR T cells for patients in Need.