The first approved human gene therapy clinical trials used T cell-directed gene transfer approaches, and these approaches have remained an active area of investigation for applications involving the correction of inborn errors of metabolism (Blaese et al.
; Aiuti et al.
), as a potential therapy of HIV-1 infection (Ranga et al.
; Morgan et al.
), and as a treatment for a variety of malignancies (Rossig and Brenner, 2004
). The largest number of protocols using genetically engineered T cells has been for applications involving novel cancer treatments. The transgenes used in these applications were designed to provide direct therapeutic benefit (such as cytokines or TCR) or were indirectly used to monitor cell fate and/or as a potential safety switch (e.g., using HSV-Tk). A number of clinical trials using T cells engineered with antitumor-reactive TCRs or chimeric antigen receptors have been undertaken (Kershaw et al.
; Lamers et al.
; Morgan et al.
; Park et al.
; Till et al.
). We demonstrated the successful use of TCR-based T cell gene therapy for the treatment of melanoma using gammaretroviral vectors (Morgan et al.
). Although the response rate reported in this trial was lower than that observed for naturally occurring TILs (12% vs. >50%), the two responses observed were durable (>20 months) and additional patients have responded to the same TCR gene therapy (R.A. Morgan, unpublished observations).
The extensive analysis of promoter function in lentiviral vector-transduced T cells, performed herein, was undertaken in an attempt to develop an effective lentivirus-based TCR expression vector. Lentiviral vectors have a number of potential advantages in comparison with gammaretroviral vectors, including the ability to transduce minimally stimulated PBLs, and a potentially safer integration site preference (Cavalieri et al.
; Montini et al.
). Our previous observations using gammaretroviral vectors demonstrated that both internal promoters and IRES elements mediated coordinated TCR α and β chain expression leading to functional TCR chain pairing and biological activity (Morgan et al.
; Hughes et al.
). Although it was previously reported that the use of IRES elements in bicistronic lentiviral vectors led to biased expression of the transgenes (Yu et al.
; Amendola et al.
; Chinnasamy et al.
; Osti et al.
), the complete lack of functional TCR gene expression in IRES vectors 7 and 8 () was not expected. It was also reported that lentiviral vectors containing two independent internal promoters transferred high-level expression of multiple transgenes in human hematopoietic stem progenitor cells (Yu et al.
). Using highly active T cell promoters, we tried serial combinations of dual promoters in eight different lentiviral vectors, but consistently failed to achieve a high percentage of TCR expression in transduced PBLs. Expression of the first gene (the α chain) was observed by CD3 mobilization studies in SUP-T1 cells, yet these cells expressed significantly less of the second gene (the β chain), as functional pairing was not observed by tetramer staining (). Naldini and coworkers developed lentiviral vectors coordinately expressing dual genes driven by synthetic bidirectional promoters (Amendola et al.
). Although they observed coordinated gene expression in various cells and tissues, these synthetic promoters exhibited lower activities in activated PBLs (10%) and naive PBLs (5%). Why internal promoter and IRES vector designs were functional in gammaretroviral vectors but not in these lentiviral vectors is unknown.
The failure of internal promoters or IRES to yield significant TCR gene expression led us to the construction of lentiviral vectors expressing antitumor antigen TCR by the use of 2A peptides, which were previously reported to yield functional TCR expression in gammaretroviral vectors (Szymczak et al.
). The main advantage of using the 2A ribosomal skip peptides in the construction of bicistronic vectors is the potential for coexpression of both genes at equal levels. Our data from two different constructs (vectors 11 and 12) demonstrated both functional α and β chain pairing and biologically activity, using this approach (). These results were not specific for the anti-p53 TCR, as we observed similar results with the antimelanoma gp100 TCRs ().
There has been one report on the clinical use of lentiviral vector-transduced T cells in humans. In this report, June and coworkers used lentiviral vectors in the setting of HIV-1 infection, in which CD4+
T cells were engineered with a vector containing an antisense gene to the HIV-1 envelope protein (Levine et al.
). High levels of ex vivo
transduction were reported along with short-term persistence of the transferred T cells, with no adverse advents attributed to the lentiviral vector gene transfer system. On the basis of this initial report on the safety of this vector platform and the potential biological advantages in using lentiviral vectors to engineer human T cells, it is likely that lentiviral vectors expressing antitumor-reactive TCRs or similar genes may have immediate application in adoptive immunotherapy for cancer.