Adoptive Transfer of T Cell Receptor (TCR)-Gene Engineered Allogeneic T Cells: Competitive Downregulation of Alloreactive Endogenous TCRs Reduces the Risk of Graft Versus Host Disease and Preserves Strong Graft Versus Leukemia Effects After MHC-Mismatched Hematopoietic Stem Cell Transplantation (HCT).

Adoptive transfer (AT) of TCR-gene transduced autologous T cells has become a potent therapeutic tool. We hypothesized that a) the use of allogeneic T cells as TCR vehicles after allogeneic HCT results in a reduced risk of graft versus host disease (GVHD) due to competitive downregulation of the alloreactive receptor, b) cell division of TCR-transduced T-cells by continuous allostimulation leads to prolonged in vivo persistence, and as a consequence to c) long lasting specific graft versus leukemia effects (GVL).

Studies were performed using the OT-1 anti-ovalbumin (OVA) reactive TCR introduced in B10.A donor T cells for AT after MHC-mismatched HCT (B10.A mice (H-2^a) into C57BL/6 (H-2^b) mice). For direct quantification of an alloreactive endogenous receptor 2C mouse-derived T cells (endogenous TCR directed against H-2^d) and anti-Ld antibodies were used in some experiments. After HCT, leukemia-bearing (C1498-OVA) mice (syngeneic HCT-recipients served as reference) received AT and were monitored for GVHD, GVL, and T cell persistence. Transferred T cells of surviving mice were retrieved for TCR-spectrotyping analysis and assessed for remaining alloreactivity.

1) Up to 1×10⁹ TCR-transduced CD8+ T cells were generated in vitro within 7 days using a retroviral vector system linking the genes for the α- and β-chain via a 2A sequence. 40% of CD8+ T cells coexpressed the respective TCR chains Vα2 and Vβ5. 2) The introduced TCR mediated comparable specific cytotoxicity against C1498-OVA in vitro being functional on autologous and allogeneic T cells. 3) After AT transduced T cells rescued up to 60% of mice in a dose dependent manner. (p = 0.001-0.01 versus mock-transduced controls). Minimal GVL-effects only were mediated by high doses of mock-transduced allogeneic T cells 4) After TCR-transfer the alloreceptor of 2C-derived T cells was downregulated by up to 60%. Increasing TCR transduction rates negatively correlated with the expression of the endogenous alloreactive receptor. Alloreactivity of B10.A-derived T cells against C57BL/6 stimulators was reduced by 50% in vitro. This translated into decreased GVHD-scores upon AT after allogeneic HCT (mock-transduced CD8+ cells served as controls (p=0.0011). 5) The degree of long-term persistence after AT was comparable to the autologous setting and correlated with tumor protection upon leukemia challenge after HCT. 13 weeks after AT the endogenous TCR-repertoire of TCR-engineered allogeneic T cells narrowed significantly as compared to their autologous counterpart. 100% of long term persisting T cells expressed the introduced TCR.

AT with TCR-engineered T cells after allogeneic HCT can decrease the risk of GVHD and provide potent GVL effects.

No relevant conflicts of interest to declare.