Evaluation of strategies to increase safety of adoptively transferred HBV-specific T cells

Hepatitis B is a liver disease with major consequences such as liver cirrhosis and hepatocellular carcinoma leading to death of estimated 887.000 people each year (WHO, 2017). Current medical treatment options for hepatitis B do not cure the infection but only suppress the replication of the virus. A self-limiting, acute infection with HBV is associated with a strong T-cell response. Hence, adoptive T-cell therapy with T cells that express either an HBV-specific T-cell receptor (TCR) or a chimeric antigen receptor (S-CAR) represents a promising therapeutic strategy in order to eradicate HBV and induce functional cure. However, side effects such as hepatotoxicity or cytokine release syndrome, and malignant transformation due to genetic manipulation are not excluded. The evaluation of different safeguard mechanisms, which allow depletion of transferred T cells on demand, is thus important to control collateral damage and to make T-cell therapy safer.

Here, the S-CAR or HBV-specific TCRs, which both recognize HBV-infected hepatocytes were expressed on T cells after retroviral transduction. Besides these receptors, the T cells were equipped with different safeguard mechanisms that can be expressed intra- or extracellularly and can be activated in case of the above-listed side effects. The T cells were then co-cultured with HBV- HepG2 or HBV+ HepG2.2.15 cells and analyzed for their functionality through target cell viability assays (xCELLigence, XTT) and IFNγ ELISA.

The first safeguard mechanism tested was an intracellularly expressed inducible caspase 9 system (iCASP9). When co-cultured with HBV+ HepG2.2.15 cells, the HBV-specific T cells were specifically activated to secret IFNγ and to kill HBV+ target cells, whereas no effect was observed in presence of HBV- target cells. Co-expression of the safeguard mechanism in T cells did not alter their function. After 20 hours, a dimerizer was added to activate iCASP9 in transduced T cells. Activation of iCASP9 reduced or even stopped the killing by HBV-specific T cells in less than 15 minutes.

In conclusion, HBV-specific CARs and TCRs efficiently recognize and kill HBV-replicating cells. iCASP9 shows good efficiency in vitro and leads to a strong T-cell depletion of transduced T cells.

As an outlook, two other safeguard mechanisms, which are based on expression of a truncated CD20 or the HSV-thymidine-kinase, will be first characterized in vitro. The evaluation of all three safeguard mechanisms in vivo is planned.