Immunotherapy of primary leukaemia by WT1-TCR engineered patient’s autologous T cells
Year: 2008
Session type: Poster / e-Poster / Silent Theatre session
University College London, Royal Free Hospital, London, UK
Abstract
Previously we have shown that a Wilm’s Tumour Antigen-1 specific TCR (WT1-TCR), isolated from an allogeneic CTL line, was functionally active in vitro and in vivo. When this WT1-TCR was retrovirally transduced into normal donor T cells, these TCR transduced T cells were able to recognise their specific targets and kill human leukaemia cell lines in vitro, and eliminate a human leukaemia cell line in a NOD/SCID mouse model.
Now we are developing this TCR for a clinical trial application. Both TCR alpha and beta chains were inserted into one single vector (WT1-TCR-MP71). To minimise the potential oncogenic activity of the woodchuck hepatitis virus derived post-transcriptional regulatory element (PRE) within this retroviral vector, we generated a WT1-TCR retroviral vector without the PRE element (WT1-TCR-∆-PRE). Transduction and expression experiments demonstrated that this WT1-TCR-∆-PRE vector gave similar level of TCR expression and transduction efficiency in peripheral blood T cells when compared with a PRE-containing vector.
To improve the TCR expression, we have generated a codon optimized WT1-TCR, and linked the genes for the TCRα and β chains with a viral 2A sequence. Furthermore, to reduce the misparing between introduced and endogenous TCR chains, we introduced an extra interchain disulphide bond between the TCR α and β chains (WT1-ss-TCR). To further enhance the preferential pairing between the introduced TCR α and β chains, we have replaced the human TCR constant domains with murine sequences and also maintained the extra interchain disulphide bond between the TCR α and β chains (WT1-hybrid-TCR).
Detailed experiments showed that these newly modified WT1-TCRs can be efficiently expressed in human T cells and functionally active as measured by tetramer staining and intracellular cytokine staining after antigen specific stimulation. Recently, we used CD34+ leukemia progenitor cells isolated from a CML patient with lymphoid blast crisis for in vivo experiment in the NOD/SCID mice, followed by adoptive immunotherapy with WT1-TCR or control TCR transduced patient’s autologous T cells.
Preliminary results showed that the WT1-CTL treated mice had much better survival rate than the control CTL treated mice. FACS analysis demonstrated that control CTL treated mice showed engraftment of human leukemia cells, while mice treated with WT1-TCR engineered T cells had no detectable leukemia cells. These data have provided a solid base for a phase I clinical trial application.
Previously we have shown that a Wilm’s Tumour Antigen-1 specific TCR (WT1-TCR), isolated from an allogeneic CTL line, was functionally active in vitro and in vivo. When this WT1-TCR was retrovirally transduced into normal donor T cells, these TCR transduced T cells were able to recognise their specific targets and kill human leukaemia cell lines in vitro, and eliminate a human leukaemia cell line in a NOD/SCID mouse model.
Now we are developing this TCR for a clinical trial application. Both TCR alpha and beta chains were inserted into one single vector (WT1-TCR-MP71). To minimise the potential oncogenic activity of the woodchuck hepatitis virus derived post-transcriptional regulatory element (PRE) within this retroviral vector, we generated a WT1-TCR retroviral vector without the PRE element (WT1-TCR-∆-PRE). Transduction and expression experiments demonstrated that this WT1-TCR-∆-PRE vector gave similar level of TCR expression and transduction efficiency in peripheral blood T cells when compared with a PRE-containing vector.
To improve the TCR expression, we have generated a codon optimized WT1-TCR, and linked the genes for the TCRα and β chains with a viral 2A sequence. Furthermore, to reduce the misparing between introduced and endogenous TCR chains, we introduced an extra interchain disulphide bond between the TCR α and β chains (WT1-ss-TCR). To further enhance the preferential pairing between the introduced TCR α and β chains, we have replaced the human TCR constant domains with murine sequences and also maintained the extra interchain disulphide bond between the TCR α and β chains (WT1-hybrid-TCR).
Detailed experiments showed that these newly modified WT1-TCRs can be efficiently expressed in human T cells and functionally active as measured by tetramer staining and intracellular cytokine staining after antigen specific stimulation. Recently, we used CD34+ leukemia progenitor cells isolated from a CML patient with lymphoid blast crisis for in vivo experiment in the NOD/SCID mice, followed by adoptive immunotherapy with WT1-TCR or control TCR transduced patient’s autologous T cells.
Preliminary results showed that the WT1-CTL treated mice had much better survival rate than the control CTL treated mice. FACS analysis demonstrated that control CTL treated mice showed engraftment of human leukemia cells, while mice treated with WT1-TCR engineered T cells had no detectable leukemia cells. These data have provided a solid base for a phase I clinical trial application.
