Sturm und Drang over Suicidal Lymphocytes (2002)

The idea that T cells can be controlled in vivo via the selective activation of a suicide mechanism is an important paradigm for gene therapy. In the context of allogeneic bone marrow transplantation, this strategy may help resolve a longstanding dilemma in the treatment of leukemia. The infusion of donor T cells confers substantial anti-leukemic benefits, but exposes the recipient to the risk of graft versus host disease (GVHD), which can be lethal. The depletion of T cells from the graft averts GVHD but increases the risk of leukemia relapse. The infusion of T cells endowed with a recall mechanism opens the prospect of preserving anti-leukemic responses and preventing GVHD. This approach can be applied to any type of donor leukocyte infusion, such as the infusion of donor-derived T cell lines generated against viral or tumor antigens. The ability to selectively ablate T cells could also be useful in the autologous context, if the infused T cells were to engage self-antigens and inflict autoimmune damages. The concept of genetically controlling T cells originated over a decade ago, but its feasibility and utility still remain to be completely demonstrated. Several complications have emerged in the course of preclinical and clinical investigation, challenging the proposed methodologies and strategies. A recent report in Science [1Li Z. Murine leukemia induced by retroviral gene marking.Science. 2002; 296: 497Crossref PubMed Scopus (571) Google Scholar] raises concerns about the use of a cell surface marker commonly used to purify transduced cell populations. It is enlightening to deconstruct the complexity that underlies what appeared at first to be a simple concept. We focus here on the issues of vector design and T cell transduction. Vector design. An effective suicide strategy demands that the vector insures reliable expression of the suicide gene. This in turn requires the availability of a marker to select or purify the transduced T cells. This marker must be faithfully co-expressed with the suicide gene. Three approaches have been investigated so far: fusion genes, dual-promoter vectors, and bicistronic vectors [2Lupton S.D. Brunton L.L. Kalberg V.A. Overell R.W. Dominant positive and negative selection using a hygromycin phosphotransferase-thymidine kinase fusion gene.Mol. Cell. Biol. 1991; 11: 3374-3378Crossref PubMed Scopus (166) Google Scholar, 3Thomis D.C. A Fas-based suicide switch in human T cells for the treatment of graft-versus-host disease.Blood. 2001; 97: 1249-1257Crossref PubMed Scopus (168) Google Scholar, 4Lyons M. An improved retroviral vector encoding the herpes simplex virus thymidine kinase gene increases anti-tumor efficacy in vivo.Cancer Gene Ther. 1995; 2: 273-280PubMed Google Scholar, 5Verzeletti S. Herpes simplex virus thymidine kinase gene transfer for controlled graft-versus-host disease and graft-versus-leukemia: clinical follow-up and improved new vectors.Hum. Gene Ther. 1998; 9: 2243-2251Crossref PubMed Scopus (174) Google Scholar, 6Gallardo H.F. Tan C. Sadelain M. The internal ribosomal entry site of the encephalomyocarditis virus enables reliable co-expression of two trans-genes in human primary T lymphocytes.Gene Ther. 1997; 4: 1115-1119Crossref PubMed Scopus (53) Google Scholar]. The fusion gene strategy (Figs. 1A and 1B) guarantees co-expression of a selectable marker and the suicide gene. Its drawbacks are the obligatory T cell culture needed to enforce drug resistance [7Sauce D. Retrovirus-mediated gene transfer in primary T lymphocytes impairs their anti-Epstein-barr virus potential through both culture-dependant and selection process-dependant mechanisms.Blood. 2002; 99: 1165-1173Crossref PubMed Scopus (105) Google Scholar] and the possible immunogenicity of the fusion product [8Berger C. Non-myeloablative immunosuppressive regimen prolongs in vivo persistence of gene-modified autologous T cells in a non-human primate model.J. Virol. 2001; 75: 799-808Crossref PubMed Scopus (51) Google Scholar]. Dual-promoter vectors (Figs. 1C and 1D) allow for the selection of a promoter with optimal transcriptional characteristics for each vector-encoded gene, but run the risk of transcriptional interference [9Emerman M. Temin H.M. Quantitative analysis of gene suppression in integrated retrovirus vectors.Mol. Cell. Biol. 1986; 3: 792-800Crossref Scopus (118) Google Scholar]. It is therefore important to quantify, for any new vector, the fraction of T cells that express the marker gene but fail to adequately express the suicide gene. Bicistronic vectors (Fig. 1E) avoid the latter problem by relying on a single promoter to coexpress two genes. The internal ribosomal entry site of encephalomyocarditis virus has proven to be reliable in human primary T cells [6Gallardo H.F. Tan C. Sadelain M. The internal ribosomal entry site of the encephalomyocarditis virus enables reliable co-expression of two trans-genes in human primary T lymphocytes.Gene Ther. 1997; 4: 1115-1119Crossref PubMed Scopus (53) Google Scholar], but the level of expression of the 3′ gene, which can vary markedly from one vector to another, has to be sufficient. All of these strategies depend on the transcriptional control elements incorporated into the vector, which also warrant careful analysis. Different vectors vary in terms of their transcriptional requirements and propensity to silencing [10Pollok K.E. Costimulation of transduced T lymphocytes via TCR-CD3 complex and CD28 leads to increased transcription of integrated retrovirus.Hum. Gene Ther. 1999; 10: 2221-2236Crossref PubMed Scopus (54) Google Scholar, 11Agarwal M. Scaffold attachment region-mediated enhancement of retroviral vector expression in primary T cells.J. Virol. 1998; 72: 3720-3728PubMed Google Scholar]. Ultimately, the vectors need to be tested in vivo over relevant periods of time. Vector components. The herpes simplex virus-1 thymidine kinase (HSVTK) gene, the product of which confers sensitivity to the prodrug ganciclovir, remains the prototypic suicide gene [12Cohen J.L. Boyer O. Thomas-Vaslin V. Klatzmann D. Suicide genemediated modulation of graft-versus-host disease.Leuk. Lymph. 1999; 34: 473-480Crossref PubMed Scopus (23) Google Scholar]. The cytotoxic activity of HSVTK is biologically restricted to dividing cells. It remains to be established whether this restriction might pose a problem in specific clinical settings (by precluding the elimination of nondividing T cells). To address this hypothetical limitation, suicide genes that are active in nondividing cells, such as molecules encompassing the cytoplasmic domain of Fas (Fig. 1B), are under development [3Thomis D.C. A Fas-based suicide switch in human T cells for the treatment of graft-versus-host disease.Blood. 2001; 97: 1249-1257Crossref PubMed Scopus (168) Google Scholar]. Another possible limitation to the use of HSVTK is its potential immunogenicity, the clinical consequences of which remain to be fully delineated. It was recently reported that the HSVTK gene used in certain vectors could undergo RNA splicing and that the spliced transcript encoded an inactive enzyme [13Garin M.I. Molecular mechanism for ganciclovir resistance in human T lymphocytes transduced with retroviral vectors carrying the herpes simplex virus thymidine kinase gene.Blood. 2001; 97: 122-129Crossref PubMed Scopus (98) Google Scholar]. It is noteworthy, however, that there are different HSVTK gene sequences (GenBank), which may not be equally subject to RNA editing. The genomic integrity of variants that undergo splicing can be restored by silent mutations that disrupt splice recognition signals [14Chalmers D. Elimination of the truncated message from the herpes simplex virus thymidine kinase suicide gene.Mol. Ther. 2001; 4: 146-148Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar]. The enzymatic activity of HSVTK variants, in particular regarding the phosphorylation of ganciclovir, will also need to be evaluated. Another key issue in the suicide gene strategy is the selection of an appropriate marker for purifying transduced T cells and monitoring vector expression. An ideal marker is a cell surface molecule that is human, non-polymorphic, and absent from resting or activated T cells. The transduced molecule should be expressed at levels that permit efficient cell separation by panning or bead technologies using a specific monoclonal antibody. Fulvio Mavilio and colleagues proposed the use of a modified form of human p75, also known as the low affinity nerve growth factor receptor (LNGFR), which has a truncated cytoplasmic domain (ΔLNGFR) [15Mavilio F. Peripheral Blood Lymphocytes as targeted cells of retroviral vector-mediated gene transfer.Blood. 1994; 83: 1988-1997PubMed Google Scholar]. ΔLNGFR has subsequently been adopted by several other investigators [16Kornblau S.M. Preemptive control of graft-versus-host disease in a murine allogeneic transplant model using retrovirally transduced murine suicidal lymphocytes.Cancer Res. 2001; 61: 3355-3360PubMed Google Scholar, 17Orchard P.J. Development of a successful clinical scale procedure for activation, transduction and selection of human T cells: factors affecting cellular function.Blood. 2001; 98: 422aCrossref PubMed Scopus (131) Google Scholar, 18Burt R.K. Anti-tumor effectiveness of herpes simplex thymidine kinase gene transduced donor lymphocyte infusions for relapsed hematologic malignancies following allogeneic hematopoietic stem cell transplantation.Blood. 2001; 98: 698aGoogle Scholar, 19Berger C. Evaluation of a novel suicide gene for T cell therapy.Blood. 2001; 98: 694aGoogle Scholar]. Christopher Baum and his colleagues now describe murine bone marrow chimeras with acute leukemia following bone marrow transduction with ΔLNGFR [1Li Z. Murine leukemia induced by retroviral gene marking.Science. 2002; 296: 497Crossref PubMed Scopus (571) Google Scholar]. In this study, 6 of 10 mice engrafted with bone marrow pooled from five primary recipients developed acute myeloid leukemia (AML), an uncommon tumor in mice. The clonality of the tumors and the transcriptional activation of the Evil oncogene by the integrated retroviral vector point to insertional mutagenesis as a likely cause of the disease. The association between retroviral activation of Evil and myeloid leukemia has been previously reported [20Morishita K. Retroviral activation of a novel gene encoding a zinc finger protein in IL-3-dependent myeloid leukemia cell lines.Cell. 1988; 54: 831-840Abstract Full Text PDF PubMed Scopus (355) Google Scholar]. Noting that overexpression of Evil alone in murine bone marrow cells is not leukemogenic, and that the tumors obtained in their chimeras proliferated in response to NGF, Zhixiong Li et al. suggest that ΔLNGFR may have contributed to the transformation [1Li Z. Murine leukemia induced by retroviral gene marking.Science. 2002; 296: 497Crossref PubMed Scopus (571) Google Scholar]. The biological function of p75/LNGFR is complex, and includes pro-apoptotic and anti-apoptotic effects depending on its association with TrkA and TrkB [21Chao M.V. Bothwell M. Neurotrophins: to cleave or not to cleave.Neuron. 2002; 33: 9-12Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar]. The elimination of the cytoplasmic domain of p75/LNGFR does not preclude formation of high-affinity NGF receptors [22Hantzopoulos P.A. Suri C. Glass D.J. Goldfarb M.P. Yancopoulos G.D. The low affinity NGR receptor, p75, can collaborate with each of the Trks to potentiate functional responses to the neurotrophins.Neuron. 1994; 13: 187Abstract Full Text PDF PubMed Scopus (261) Google Scholar, 23Esposito D. The cytoplasmic and transmembrane domains of the p75 and Trk A receptors regulate high affinity binding to nerve growth factor.J. Biol. Chem. 2001; 276: 32687-32695Crossref PubMed Scopus (193) Google Scholar]. To abrogate the ability of ΔLNGFR to interact functionally with the Trks, at least one additional mutation in the extracellular domain of p75/LNGFR is required [22Hantzopoulos P.A. Suri C. Glass D.J. Goldfarb M.P. Yancopoulos G.D. The low affinity NGR receptor, p75, can collaborate with each of the Trks to potentiate functional responses to the neurotrophins.Neuron. 1994; 13: 187Abstract Full Text PDF PubMed Scopus (261) Google Scholar, 23Esposito D. The cytoplasmic and transmembrane domains of the p75 and Trk A receptors regulate high affinity binding to nerve growth factor.J. Biol. Chem. 2001; 276: 32687-32695Crossref PubMed Scopus (193) Google Scholar], and possibly another in the transmembrane region [23Esposito D. The cytoplasmic and transmembrane domains of the p75 and Trk A receptors regulate high affinity binding to nerve growth factor.J. Biol. Chem. 2001; 276: 32687-32695Crossref PubMed Scopus (193) Google Scholar]. It is therefore possible that ΔLNGFR exhibits anti-apoptotic effects, as shown in fibroblasts [22Hantzopoulos P.A. Suri C. Glass D.J. Goldfarb M.P. Yancopoulos G.D. The low affinity NGR receptor, p75, can collaborate with each of the Trks to potentiate functional responses to the neurotrophins.Neuron. 1994; 13: 187Abstract Full Text PDF PubMed Scopus (261) Google Scholar]. Additional studies are required to address this point. Interestingly, an in-frame deletion in TrkA has been previously associated with human AML [24Reuther G.W. Lambert Q.T. Caligiu M.A. Der C.J. Identification and characterization of an activating TrkA deletion mutation in acute myeloid leukemia.Mol. Cell. Biol. 2000; 20: 8655-8666Crossref PubMed Scopus (85) Google Scholar], suggesting a possible link between leukemogenesis and neurotrophin receptors. These putative transforming mechanisms may not apply to T cells, but it should be kept in mind that T lymphocytes can be transformed by retroviruses [25Uchiyama T. Human T cell leukemia virus type I (HTLV-1) and human diseases.Annu. Rev. Immunol. 1997; 15: 15-37Crossref PubMed Scopus (350) Google Scholar]. Collectively, these observations serve as a reminder to carefully scrutinize “inactive” markers and cautiously assess other markers such as CD8, CD34, or Thy1. The use of oncoretroviral vectors dictates that the T cells be cycling at the time of infection. The T cells can be activated by mitogens, such as PHA or anti-CD3 monoclonal antibody, or by antigen presenting cells (APC) that engage specific T-cell receptors. Both ways permit efficient gene transfer, but the latter provides the additional benefit of favoring the transduction of the antigen-specific T cells that are activated by the APC at the time of retroviral infection [26Koehne G. Rapid selection of antigen-specific T lymphocytes by retroviral transduction.Blood. 2001; 4: 1115-1119Google Scholar]. The technique used to activate T cells is not the fundamental concern. The main issue is to transduce and expand potent T cells that retain their therapeutic properties in vivo. Successful therapy requires an appropriate dose of T cells that are poised to traffic to appropriate target sites without succumbing to activation-induced cell death or anergy. Resting and activated T cells differ greatly in these respects. Mitogen-activated allogeneic T cells exhibit a reduced GVHD-inducing potential in comparison with resting lymphocytes [27Contassot E. In vivo alloreactive potential of ex vivo-expanded primary T lymphocytes.Transplantation. 1998; 65: 1365-1370Crossref PubMed Scopus (53) Google Scholar, 28Weijtens M. van Spronsen A. Hagenbeek A. Braakman E. Martens A. Reduced graft-versus-host disease-inducing capacity of T cells after activation, culturing, and magnetic cell sorting selection in an allogeneic bone marrow transplantation model in rats.Hum. Gene Ther. 2002; 13: 187-198Crossref PubMed Scopus (32) Google Scholar]. This may explain why the incidence of GVHD was less than anticipated in earlier clinical trials [29Ciceri F. Long-term follow-up in 30 patients receiving HSV-Tk transduced donor lymphocytes after allo-BMT.Blood. 1999; 94: 668aGoogle Scholar, 30Champlin R. Phase I/II study of thymidine kinase (TK)-transduced donor lymphocyte infusions (DLI) in patients with hematologic malignancies.Blood. 1999; 94: 324aGoogle Scholar], thus hampering the evaluation of the suicide gene strategy. In addition to increasing the antigen-specific cytotoxic T-cell frequency, T-cell activation by APCs such as dendritic cells, Epstein-Barr virus-transformed B cells, or artificial APCs may be important to preserve and/or enhance T-cell potency and thus decrease T-cell doses. More studies are needed to address these questions, which are likely to be investigated—and debated—for quite some time. We thank Moses Chao (Skirball Institute, New York University) for helpful discussion and Jakob Dupont (Memorial Sloan Kettering Cancer Center) for reviewing the manuscript.