Co-expression of cytokine and suicide genes to enhance the activity and safety of tumor-specific cytotoxic T lymphocytes

Abstract

The antitumor effect of adoptively transferred tumor-specific cytotoxic T lymphocytes (CTLs) is impaired by the limited capacity of these cells to expand within the tumor microenvironment. Administration of interleukin 2 (IL-2) has been used to overcome this limitation, but the systemic toxicity and the expansion of unwanted cells, including regulatory T cells, limit the clinical value of this strategy. To discover whether transgenic expression of lymphokines by the CTLs themselves might overcome these limitations, we evaluated the effects of transgenic expression of IL-2 and IL-15 in our model of Epstein Barr Virus-specific CTLs (EBVCTLs). We found that transgenic expression of IL-2 or IL-15 increased the expansion of EBV-CTLs both in vitro and in vivo in a severe combined immunodeficiency disease (SCID) mouse model and enhanced antitumor activity. Although the proliferation of these cytokine genes transduced CTLs remained strictly antigen dependent, clinical application of this approach likely requires the inclusion of a suicide gene to deal with the potential development of T-cell mutants with autonomous growth. We found that the incorporation of an inducible caspase-9 suicide gene allowed efficient elimination of transgenic CTLs after exposure to a chemical inducer of dimerization, thereby increasing the safety and feasibility of the approach.

Development of optimal bicistronic lentiviral vectors facilitates high-level TCR gene expression and robust tumor cell recognition

“…Encouraged by the successful use of 2A peptides in g-retroviral vectors to express multiple genes in one construct, 24,25,27 we attempted to develop lentiviral vectors expressing antitumor antigen-specific TCRs using different 2A peptides. In Figure 1a, the amino acids from different 2A peptides are illustrated and the conservative domains highlighted.…”

“…An alternative to these approaches is the use of ribosomal skipping via 2A peptides. 23,24 Several viruses use 2A peptides to mediate protein cleavage, including foot-and-mouth disease virus (F2A), equine Rhinitis A virus, porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A). The 2A peptide consensus motif (DVEXNPGP) is extremely rare and is associated with cleavage-like activity between 2A and 2B genes through a ribosomal skip mechanism; the 2A peptide impairs normal peptide bond formation between 2A and 2B without affecting the translation of 2B.…”

“…2A peptides have been shown in g-retroviral vectors to initiate the production of up to four proteins both in vitro and in vivo. 24,25 When exogenous genes are linked using this approach, about 18 amino acids are left on the C terminus of the first gene and a single proline residue remains on the N terminus of the second gene. Although linking of the TCR-a and -b chains by 2A peptide has been reported, 25 the effect of the residual amino acids on the function of expressed TCR has not been fully explored.…”

Progress and prospects: graft-versus-host disease

“…This goal might be either achieved by expressing homeostatic cytokines, 76 or cognate receptors 77 into T lymphocytes, or by gene transfer and expansion of selected T-cell sub-populations with retroviral and lentiviral vectors. 78 We showed that a potent activation with cell-sized beads conjugated with antibodies directed to CD3 and CD28 recruits in cell cycle, thus facilitating retroviral transduction of naive and central memory (T CM ) lymphocytes.…”

Selective expansion and enhanced anti-tumor effect of antigen-specific CD4+ T cells by retrovirus-mediated IL-15 expression

“…As an important T cell growth factor, IL-15 has been demonstrated to prolong the persistence of adoptively transferred cytotoxic T cells (CTLs) in vivo (Quintarelli et al, 2007). The major effects of IL-15 on T cells include (1) enhanced cytotoxic ability and proliferation of CTLs in vivo (Klebanoff et al, 2004), (2) activation of the telomerase and elongation of telomeres (Li et al, 2005), (3) ability to rescue tumor-tolerant T cells for use in adoptive immunotherapy (Teague et al, 2006), and (4) inhibition of activation-induced cell death of T cells (Marks-Konczalik et al, 2000).…”