Leukemia-Reactive Virus-Specific T Cells Generated by T Cell Receptor (TCR) Transfer Preserve Their Dual Specificity upon Repetitive Stimulation of the Endogenous TCR.
Authors: van Loenen M.M. , Hagedoorn R.S. , Hoogeboom M. , Kester M.G.D. , Willemze Roelof , Falkenburg J.H. Frederik , Heemskerk M.H.M. 
Published: April 2020

Abstract
TCR-transfer to engineer tumor-specific T cells may be a strategy for adoptive immunotherapy. For complete eradication of leukemic cells and to achieve long-term protection, potent effector T cell function and long-term T cell persistence are necessary. Therefore, we propose to use virus specific T cells for TCR transfer since such engineered dual specific T cells can be triggered via their endogenous TCR by latent presence of viral antigens, improving their long-term persistence. We have previously shown that virus specific T cells can be redirected towards anti-leukemic reactivity by transfer of the hematopoietic minor histocompatibility antigen HA-2 specific TCR (HA-2-TCR). The TCR-transferred virus specific T cells showed differences in TCR cell surface make up, which was stable for months after repetitive non-specific TCR triggering. The T cells expressed either both TCRs intermediately at the cell surface, or the endogenous TCR was highly expressed with a low expression of the introduced TCR, or the introduced TCR was highly expressed with a low expression of the endogenous TCR. It may be anticipated that frequent encounter with viral antigens in vivo leads to selective outgrowth of TCR-transferred dual specific T cells with high expression of the endogenous viral specific TCR but low expression of the introduced tumor specific TCR, resulting in reduced anti-leukemic reactivity. To address this issue, we generated CMVA2-specific T cells transduced with the HA-2-TCR. This resulted in dual specific cells with different TCR cell surface make up. The dual specific T cells were repetitively stimulated specifically either via their endogenous virus specific TCR or via the introduced HA-2 specific TCR. In time, the cell surface expression of the endogenous and introduced TCRs as measured with CMVA2 and HA-2A2 tetramers diverged. Repetitive stimulation of the endogenous TCR skewed the dual specific T cells towards a cell population that predominantly expressed the endogenous TCR. In contrast, repetitive stimulation of the introduced TCR skewed the cells towards T cells that predominantly expressed the introduced TCR. However, this divergence in tetramer stainings was shown to quickly revert after a single stimulation via the other TCR. To study whether this divergence was the result of a difference in TCR cell surface distribution or of selective outgrowth of different T cells, T cells were sorted that predominantly expressed either the endogenous or the introduced TCR. These cells were subsequently stimulated on the endogenous or introduced TCR, and compared regarding TCR cell surface expression and functional activity. Directly after sorting dual specific T cells preferentially expressing the endogenous TCR were still reactive against HA-2+ target cells, although the reactivity was reduced compared to cells preferentially expressing the introduced TCR. However, when restimulated on the introduced HA-2-TCR, the dual specific T cells expanded antigen specifically, and reverted within several days into cells with high expression of the introduced TCR that exerted potent HA-2 specific anti-leukemic effector functions. In conclusion, we demonstrate that these dual specific T cells are likely to persist in vivo due to repetitive encounter with viral antigens with preservation of anti-leukemic effector function. Moreover, in vivo exposure to the tumor associated antigen will further enhance the relevant specificity.