Augmented Expression of WT1-Specific TCR and Inhibition of Mispaired-TCR Formation In TCR-Gene Modified T-Cells Can Concomitantly Be Achieved Using a Novel Retroviral Vector with Silencers for Endogenous TCRs

Adoptive engineered T-cell therapy using WT1-specific T-cell receptor gene-transfer is currently considered as a challenging strategy for cancer treatment. However, the formation of shuffled TCR between endogenous and introduced TCR α/β chains still remains a major issue to be solved, not only for the improvement of introduced TCR expression, but also for the prophylaxis of “GVHD-like syndrome” due to newly generated self-reactive T-cells bearing shuffled TCRs. Taking above, we have newly developed an novel WT1-specific TCR retroviral vector with built-in silencers to inhibit endogenous TCRs. In this study, we evaluated the feasibility of this novel WT1-targeting and silencers built-in vector for the clinical application.

WT1_235-243-specific and HLA-A*2402-restricted TCR α/β genes were cloned into this novel retroviral vector with built-in shRNA for endogenous TCRs. (siWT1-TCR) (1) The inhibitory effect on endogenous TCR in gene-modified T-cells was examined using the pre-established CTL clone. (2) Compared with the conventional WT1-TCR vector (without silencers), the functional avidity of siWT1-TCR-gene introduced T-cell was examined, using ⁵¹Cr-release assay, CD107a assay and intracellular IFN-γ assay, in vitro. (3) The in vivo anti-leukemia effect of siWT1-TCR introduced CD8⁺ T-cells was examined using NSG mice. (4) WT1-specific Th1 helper function of siWT1-TCR introduced CD4⁺ T-cell was examined. (5) The synergistic effect of WT1 peptide stimulation on siWT1-TCR introduced CD8⁺ T-cell was examined. (6) The on-target adverse effect of siWT1-TCR introduced CD8⁺ T-cells against autologous hematopoietic progenitor cells were examined using HLA-A*2402⁺ human cord blood CD34⁺ and TCR-gene modified autologous CD8⁺ T-cells, both in vitro and in vivo.

Compared with conventional WT1-TCR vector, siWT1-TCR vector remarkably increased the expression of functional WT1-specific TCR accompanied with the inhibition of endogenous TCR synthesis on gene-modified T-cells. CD8⁺ and CD4⁺ T cells engineered with siWT1-TCR gene transfer exerted WT1-specific and leukemia-specific cytotoxicity, and target-responsive Th1 cytokine production, in an HLA-A*2402-restricted fashion, respectively. Mainly because of remarkably increased WT1-specific TCR expression, the anti-leukemia effect exerted by siWT1-TCR introduced CD8⁺ T-cells was significantly up-regulated, compared with conventional WT1-TCR vector, which reflected the increased the target-responsive granular exocytosis. SiWT1-TCR introduced CD8⁺ T-cells also exerted in vivo anti-leukemia effect against inoculated leukemia cell lines in NSG mice, furthermore, such siWT1-TCR introduced CD8⁺ T-cells could kill autologous patients' leukemia cells, but not autologous hematopoietic progenitor cells, in vitro. Eventually, transplanted human hematopoietic progenitor cells which had been precultured with siWT1-TCR introduced autologous CD8⁺ T-cells into irradiated NSG mice demonstrated that those pre-treated CD34⁺ cells preserved abilities of engraftment, proliferation and differentiation. Additionally, it was demonstrated that repetitive WT1 peptide stimulations successfully expanded siWT1-TCR introduced CD8⁺ T-cells, in vitro, which suggests the synergistic effect of combined peptide vaccination in vivo.

Our novel WT1-targeting vector could provide not only the improved functional avidity of gene-modified T-cells, but also a promising option to solve the raised major concern in safety that is the potentially lethal GVHD-like syndrome due to newly generated T-cells bearing self-antigen reactive shuffled TCRs. Based on these pre-clinical observation, we are planning to conduct clinical trials against human hematological malignancies.

No relevant conflicts of interest to declare.