Transfer of Human T-Cell Receptors (TCR) Containing Murine Chimeric Constant Beta-Gamma-Chain Sequences Reduces the Risk of Mixed Heterodimers and Shows Enhanced in Vitro-Accumulation of TCR-Tranduced Effector Cells.

Adoptive transfer of T-cell receptor (TCR)-transduced T cells may represent an attractive and promising novel approach to specifically treat malignant diseases and has been previously successfully applied in the clinic. This approach promises the availability of sufficient numbers of effector cells with defined specificity for any tumor-associated antigen as also TCR from T cells with specificity for tumor-associated self antigens usually deleted in the autologous host may be isolated from an allorestricted or xenorestricted environment. We have previously identified the HLA-A2-allorestricted T-cell clone (SK22) with specificity for a peptide derived from Formin-like protein 1 (FMNL1) restrictedly expressed in hematopoietic tissue and overexpressed in diverse leukemias and other malignant tissue. SK22 demonstrated specific cytotoxicity against FMNL1-overexpressing cells as EBV-transformed B cells, lymphoma cell lines and native malignant cells derived from patients with chronic lymphocytic leukemia whereas healthy tissue was mainly spared. The TCR of this T cell clone may therefore represent a suitable tool for the treatment of diverse malignant diseases using TCR-transduced T cells. However, there are different concerns which need to be addressed to further improve this therapeutic approach. First, the formation of heterodimers between endogenous TCR chains and transduced TCR chains derived from receptors with low interchain affinity may abrogate specific TCR function and harbours a particular risk for unknown specificities. Although a number of TCR chain modifications has been previously applied to solve this problem further improvements are necessary. Second, longterm survival of TCR-transduced T cells has been demonstrated to be critical for the effectivity of this approach and novel approaches are needed.

We have isolated the TCR-chain genes of the FMNL1-specific T cell clone SK22 and cloned them into the retroviral vector pMP71. Transduction of unmodified TCR-chain genes of SK22 in CD8α-transfected TCR-deficient Jurkat76 cells resulted in multimer-positive cells indicating that correct TCR-chain genes have been isolated. However, peripheral blood mononuclear cells (PBMC) transduced with these native TCR chains did neither show TCR expression nor specific T-cell function suggesting that TCR SK22 represents a weak TCR with low interchain affinity. Expression and function of this TCR could be significantly improved by current optimization strategies as codon-optimization and murinization of constant chains. Effector cells transduced with these optimized TCR chain genes showed reactivity against transformed cells of different origin whereas non-transformed HLA-A2 positive target cells as lung fibroblasts, embryonic cardiomyocytes, CD4- and CD8-positive T cells as well as activated PBMC were not recognized. However, substantial mispairing persisted despite of murinization of constant chain sequences. Using human TCR chain genes containing murinized chimeric constant βγ-chains previously reported to exert improved signaling in murine T cells and cell lines, we created a hybrid TCR with high functional efficiency after transfer in human effector cells. Moreover, usage of murinized chimeric constant βγ-chains of SK22 clearly reduced the formation of heterodimers in human PBMC. In addition, we observed enhanced in vitro-accumulation of CD8- and CD4-positive cells expressing the transgenic receptor when optimized murinized chimeric constant βγ-chains in comparison to optimized murinized constant β-chains without γ-chain sequences were used. These results could be confirmed after transfer of two alternative TCR with specificities for HER2/neu and GP100 containing murinized chimeric constant βγ-chains.

These data show that transfer of the optimized TCR SK22 may be an attractive therapeutic tool for the treatment of malignancies of hematologic and other origin. Moreover, the transfer of TCR chain genes containing optimized murinized chimeric constant βγ-chains may have a significant impact on the improvement of safety and efficiency of this therapeutic approach.

No relevant conflicts of interest to declare.