Peter Kurre, MD
Dr. Kurre indicated no relevant conflicts of interest.
Provasi E, Genovese P, Lombardo A, et al. Editing T cell specificity towards leukemia by zinc finger nucleases and lentiviral gene transfer. Nat Med. 2012;18:807-815.
Case reports of an inverse relationship between incidence of disease recurrence and extent of graft-versus-host disease (GVHD) after hematopoietic stem cell transplantation (SCT) along with subsequent confirmatory retrospective studies in larger patient cohorts suggested that T lymphocytes have potent anti-tumor activity. This concept was strongly supported by the demonstration that donor lymphocyte infusions could produce disease remission in patients who had relapsed after allogeneic SCT. Frustratingly, the inability to segregate anti-tumor effects from anti-host reactivity (GVHD) in the allogeneic setting and the formidable technical challenges of in vitro manipulation of T cells in the autologous situation have hampered clinical development of effective cancer immunotherapy. Efforts to direct anti-tumor activity by cloning and transferring antigen-specific T-cell receptors (TCRs) revealed two additional problems: inadequate expression of the tumor-specific TCR and mispairing of components of the tumor-specific TCR complex with those of the endogenous TCR. The compromised target specificity resulted in both low avidity and auto-reactivity.
Chiara Bonini and colleagues from Vita Salute San Raffaele University in Milan appear to have overcome these obstacles, and their report marks another milestone in the quest to implement effective immunotherapy through adoptive T-cell transfer. The authors used an elegant two-stage approach in creating tumor-specific T cells. In the first stage, the endogenous TCR α and β chains were disrupted using specifically designed zinc-finger nucleases (ZFNs). The idea behind ZFNs is to target a specific DNA site for disruption. The zinc-finger portion of the molecule is the recognition component that binds to the targeted sequence, positioning the endonuclease to cleave the adjacent DNA. In the current experiments, the zinc-finger DNA-binding domain targeted the catalytic portion of the endonuclease to a specified sequence in the endogenous TCR coding region. Nuclease-induced, genomic double-strand breaks are typically repaired byan error-prone process (called non-homologous end joining) that almost inevitably disrupts the reading frame. Thus, T cells treated with the specific ZFNs fail to express their endogenous TCR. Absence of expression of the CD3-TCR complex allowed enrichment of the TCR-negative population through immunoselection, and because central-memory T cells were targeted in these experiments, in vitro expansion did not require TCR engagement but could be accomplished through cytokine supplementation with interleukin (IL)-7 and IL-15. Part two of the process of creating tumor-specific T cells involved the transfer of a TCR specific for the target antigen into the edited T cells. In this case, a lentiviral vector was used to transfer a TCR specific for Wilms tumor 1 (WT1) antigen, an inherently leukemogenic transcription factor commonly expressed in acute myeloid leukemia (AML) and already known to be a T-lymphocyte target in some patients.1 Complete editing with genomic elimination of endogenous TCR chain expression and transgenic replacement by the WT1-specific TCR required sequential rounds of culture, transduction, and sorting, but avoided TCR mispairing while providing functional T cells. In the end, the edited T cells showed strong antigen-specific avidity without in vitro off-target reactivity, a normal immunophenotype and unaltered in vitro expansion characteristics. Finally, the authors demonstrated appropriate WT1-specific lytic activity against AML patient samples in vitro, and xenogeneic transplantation studies provided evidence that fully TCR-edited cells can eliminate WT1+ AML grafts in vivo in the absence of broad host-directed reactivity.
Success in murine xenotransplantation does not predict how edited T cells will perform in patient trials, but the studies of Provasi and colleagues demonstrate the way forward for clinical use. Several aspects of their protocol lend themselves to broad application. As recently demonstrated,2 the same ZFN-based TCR-editing strategy can be paired with the introduction of a chimeric antigen receptor. Similarly, cytokine-driven rather than TCR-driven expansion of central-memory T cells may prove to be a generally effective method for ex vivo enrichment, and WT1 targeting serves as a paradigm for tailoring the technique to the desired malignancy-specific antigen. The strategy described by Provasi et al. produces functional T cells with redirected specificity and constitutes an important proof of concept on the way to generating generic, off-the-shelf T-cell therapy. The improved avidity, reduced off-target reactivity, and TCR-independent in vitro expansion represent key accomplishments. While the strategy is technically feasible and potentially effective, the considerable complexity of the human immune system requires careful translation of this exciting new technique into use in the clinic.
Adoptive, cancer-directed T-cell immunotherapy using genome engineering and gene transfer represents perhaps the ultimate progression of cellular therapy in the quest to eradicate tumors.
1. Gao L, Bellantuono I, Elsässer A, et al. Selective elimination of leukemic CD34+ progenitor cells by cytotoxic T lymphocytes specific for WT1. Blood. 2000;95:2198-2203.
2. Torikai H, Reik A, Liu PQ, et al. A foundation for “universal” T-cell based immunotherapy: T-cells engineered to express a CD19-specific chimeric-antigen-receptor and eliminate expression of endogenous TCR. Blood. 2012. [Epub ahead of print]
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