Generation of Graft-Versus-Leukemia (GVL)-Effector T Cells through Targeting of Leukemia-Associated HY Antigens Via TCR Transfer.
Authors: Ivanov Roman , Hol Samantha , Aarts Tineke , Hagenbeek Anton , Ebeling Saskia 
Published: October 2019

Abstract
In this study we present two novel leukemia-associated antigens and their specific targeting through TCR-transfer. First, two CD8+ CTL clones were characterized that specifically recognized normal and malignant male B and T cell lymphoblasts, but not fibroblasts, resting lymphocytes or monocytes. One of clones recognized the HLA-B*5201-restricted TIRYPDPVI epitope of the RPS4Y protein, while the other recognized the HLA-B*5201-restricted MQQMRHKEV epitope of the UTY protein. Western blot analysis demonstrated the overexpression of RPS4 proteins in normal and malignant lymphoblasts. However, for UTY, real time RT-PCR failed to reveal differential expression of UTY mRNA in lymphoblasts, indicating that posttranscriptional regulation of UTY protein expression and/or improved processing and presentation of this epitope in lymphoblasts are likely causes of its differential recognition. These findings open up the possibility to specifically eliminate leukemic cells by targeting either RPS4Y or UTY. We evaluated the feasibility of transfer of the RPS4Y- and UTY-specific T cell receptors (TCRRPS4Y and TCRUTY, respectively) to naïve T cells to specifically eliminate RPS4Y- and UTY-expressing lymphoblasts. For each clone, the TCR α- and β-chains were cloned into separate pMX retroviral vectors. T cell transduction with either TCRRPS4Y or TCRUTY and subsequent purification resulted in 57–75% of cells expressing both α- and β-chain. TCRRPS4Y/TCRUTY-transduced T cells efficiently lysed B*5201-positive EBV-LCL (100%/92%), PHA blasts (72%/72%), and B*5201-transduced Raji (71%/56%), YT (21%/53%) and RPMI 8226 (28%/47%) cell lines, in the absence of significant recognition of B*5201-transduced fibroblasts. Killing of B*5201-positive primary leukemias could not be checked due to the paucity of B*5201-positive samples. Interestingly, despite the 1.4–1.7 fold lower expression of the exogenous TCRs on transduced T cells, both TCR-transduced populations had the same requirements for the minimal antigenic peptide concentration as the respective clones. This suggests that the limited peptide availability and not the TCR-affinity determines the selective pattern of recognition of these HY antigens. In addition, the amount of IFN-γ produced by TCRUTY-transduced T cells was significantly higher (5455±1286 pg/ml) than that of the original clone (721±134 pg/ml) (p=0.003). Similarly, IFN-γ production by the original RPS4Y-specific clone and TCRRPS4Y-transduced T cells was 391±35 vs. 1498±431 pg/ml (p=0.01). TCR-transduced T cells also displayed a significant increase in stimulation-induced production of IL-2 and TNF-α in comparison to T cell clones, produced less IL-10 and demonstrated great proliferative potential. This suggests that while preserving the fine specificity and differential pattern of recognition of the original clone, TCR-transduced T cells may have much more pronounced anti-tumor effect in vivo. This is likely due to their ‘freshness’ compared to the original clones, which had been in culture for more than 6 months. In conclusion, this is the first demonstration of the possibility to preserve the specific pattern of a T cell response to a differentially recognized antigen after TCR-transfer and to augment the amplitude of this response concomitantly. Our experiments support the feasibility of the selective targeting of male B and T cell lymphoblasts by T cells transduced with UTY- and RPS4Y-specific TCRs. This approach is currently being investigated in vivo in RAG2−/− γc−/− mice bearing male EBV-LCL.