PB0197 - FVIII stimulation causes a variable response in FVIII-specific third generation chimeric antigen receptor (CAR)-transduced T cells.

Background: About one third of severe HA patients develop inhibitory antibodies when treated by protein replacement therapy with FVIII due to a lack of tolerance to FVIII. Solutions to avoid or reverse inhibitor formation are urgently needed.

Aims: The main aim of this study is to develop and characterise third-generation FVIII-specific CAR constructs and compare them to FVIII-specific second-generation CARs, which have been shown to suppress T and B cell responses to FVIII, when transduced into regulatory T cells (Tregs).

Methods: Naïve CD4+ T cells and Tregs were purified from PBMCs by magnetic isolation. The designed second- and third-generation FVIII-specific CAR constructs have been cloned into lentiviral vectors to infect naïve CD4+ T cells and Tregs. CAR protein surface expression was studied in both cell types.
Activation marker expression upon FVIII stimulation was analysed in transduced cells to verify a FVIII-specific response. Comparison between second- and third-generation CAR transduced cells was based on their staining profiles.

Results: Surface expression of the second-generation CAR was highest while third-generation constructs differed considerably in their amount of CAR extracellular expression. To check whether low surface expression of CARs was caused by surface trafficking problems we measured intracellular CAR retainment in transduced Jurkat cells, but no intracellular CAR was detected.
Next, we investigated the response of transduced cells to FVIII. Activation marker expression was diverse in transduced cells with different CAR constructs, showing a specific but variable response towards FVIII. This might be a consequence of variable CAR surface expression or the combination of costimulatory domains that impact antigen-specific responses.

Conclusion(s): Our studies showed large differences in terms of surface expression of the CAR proteins and cellular responses towards FVIII. Further experiments will be needed to identify CAR characteristics associated with a robust FVIII-specific response.