Gene transfer for activation of cmv specific t cells

Gene Transfer for Activation of CMV Specific T Cells Isabelle Andre´-Schmutz, Liliane Dal Cortivo, Yamina Hamel, and Marina Cavazzana-Calvo ABSTRACT: Cytomegalovirus (CMV) is responsible for significant morbidity and mortality in immunocompromised patients undergoing allogeneic hematopoietic stem cell transplantation. The limitations of antiviral drugs and a better understanding of the cellular immune response to CMV has lead to the development of alternative therapies that restore host cellular immunity to CMV. Infusion of donor T lymphocytes results in variable protection against CMV but a high incidence of graft-versus-host disease in the allogeneic setting. To prevent this complication and further improve anti-CMV immune response, several groups have developed new approaches, such as the introduction of a suicide gene to control alloreactivity against the host or the selective activation of CMV-specific T cells ABBREVIATIONS APC antigen presenting cells B-LCL B lymphoblastoid cell line CTL cytotoxic T lymphocytes CMV cytomegalovirus DC dendritic cells EBV Epstein Barr virus GCV ganciclovir

INTRODUCTION Reactivation of latent cytomegalovirus (CMV) is considered one of the most frequent viral complication in immunocompromised patients [1, 2]. When CMV disease (pneumoniae or enteritis) is established, treatment with antiviral drugs or intravenous immunoglobulins has been only partially effective and the mortality rate for patients developing CMV pneumonia is more than 50% From the INSERM U429 (I.A-S., M.C-C.) and Biotherapy Department (L.D.C., M.C-C.), Hopital Necker-Enfants Malades, Paris, France; Clinical Research Division, Fred Hutchinson Cancer Research Center and the University of Washington, Departments of Medicine and Immunology (Y.H.), Seattle, WA. Address reprint requests to: Isabelle Andre´-Schmutz, INSERM U429, Batiment Kirmisson, Hopital Necker-Enfants Malades, 149, rue de Se`vres. 75743 Paris Cedex, France; Tel: 33 1 44 49 57 47; Fax: 33 1 42 73 06 40; E-mail: [email protected]. Received August 12, 2003; revised January 15, 2004; accepted February 3, 2004. Human Immunology 65, 565⫺570 (2004) © American Society for Histocompatibility and Immunogenetics, 2004 Published by Elsevier Inc.

by antigen-presenting cells expressing CMV antigens introduced by gene transfer. Depending on the target cells and the strategy chosen, adenovirus, retrovirus or poxviruses derived vectors are used for gene transfer. The protocols as well as the preclinical and clinical results obtained in the field of anti-CMV immunotherapy using gene transfer are reported and discussed. Human Immunology 65, 565⫺570 (2004). © American Society for Histocompatibility and Immunogenetics, 2004. Published by Elsevier Inc. KEYWORDS: CTL; cytomegalovirus; gene transfer; suicide gene; dendritic cells

GVHD HSCT IE1 PBMC pp65 TK

graft versus host disease hematopoietic stem cell transplantation immediate-early protein 1 peripheral blood mononuclear cells phosphoprotein 65 thymidine kinase

[3]. The observations that CMV reactivation correlates with the absence of CMV-specific T cells [4] and that CD8⫹ T cells infused protected mice against lethal challenge mediated by CMV [5] led to the development of adoptive immunotherapy. The first trial of specific anti-CMV adoptive immunotherapy [6] was based on the activation of donor T cells by autologous fibroblasts infected with CMV, a method requiring 4 weeks to establish a fibroblast cell line derived from a skin biopsy, followed by at least 4 additional weeks to stimulate, isolate, and expand antiCMV CD8⫹ cytotoxic cell lines. Although this trial has demonstrated the feasibility and the efficiency (i.e., reconstitution of anti-CMV immunity and absence of CMV infection in treated patients) of this adoptive immunotherapy approach [6, 7], the use of a live virus to infect the fibroblasts and the fact that only CD8⫹ T cells 0198-8859/04/$–see front matter doi:10.1016/j.humimm.2004.02.015

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are activated by fibroblasts and may not be sufficient to provide a long-lasting resistance to CMV constituted a barrier to the extension of this protocol to other centers. Since then, great efforts have focused on the optimization of the procedure, in the aim to obtain within the shortest time T cells highly reactive against CMV, not alloreactive against the recipient, produced under Good Manufacturing Conditions (GMP). Beside the generation of virus-specific T-cell lines or clones from the donor, one alternative consists in the transfer of nonspecific immunocompetent donor T cells manipulated to control their antihost alloreactivity to prevent the development of GVHD. In this context, gene transfer can be used then either to insert CMV antigens in antigen-presenting cells (APC) to specifically activate anti-CMV specific T cells or a suicide gene in the T lymphocytes to control their alloreactivity. Gene Transfer in Effector T Cells: The Suicide-Gene Strategy To preserve the anti-infectious capacity of donor lymphocytes while preventing graft-versus-host disease (GVHD), the groups of Bordignon in 1995 and Tieberghien 6 years later have transferred using a retroviral vector the thymidine kinase (TK) suicide gene into donor lymphocytes to confer ganciclovir (GCV) sensitivity [8, 9]. TK converts nucleoside analogs such as GCV into monophosphate form, which is transformed into a potent inhibitor of DNA elongation, thereby causing cell death in dividing transduced T cells. The main application is the control of unwanted T-cell immune responses such as GVHD. In phase I clinical trials, the suicide-gene strategy allowed a quite efficient control of GVHD in adult patients with around 25% of adult patients developing acute GVHD of grade ⬎I [9, 10]. The infused cells were detected up to 60 months after infusion in some patients. Nevertheless, Tierberghien described three cases of EBVinduced lymphoproliferation out of 12 patients infused, an observation that raised some doubts on the antiinfectious capacities of TK-transduced T cells [9]. Indeed, the transformation of retrovirus RNA into DNA and integration in the cell genome requires cell division and thus polyclonal activation followed by 12 days of culture in the presence of interleukin-2 [8]. This procedure was shown to result in a global decrease of the immunocompetence of T cells. In a recent study by Marktel and colleagues, T cells were polyclonally activated during only 2 days, transduced with herpes simplex virus-TK for 3 days, sorted by magnetic selection, and expanded for another 14 days [11]. After the procedure, T cells were able to lyse Epstein-Barr virus (EBV)-transformed B cell lines as well

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as CMV-infected autologous fibroblasts as efficiently as expanded lymphocytes not transduced. HSV-TK strategy could thus be used in the treatment or prevention of CMV infection. Its advantage is linked to faster and easier manipulation method than antigenspecific T cells, as well as the fact that it may provide immunity against a wide range of other pathogens. Nevertheless, its use in the setting of hematopoietic stem cell transplantation (HSCT) may be excluded for those donors in which anti-CMV T cells are absent or present at very low frequency. Gene Transfer of Immunodominant Viral Antigens Into APC: Generalities Choice of APC. Different types of cells can be used as APC, including fibroblasts, autologous peripheral blood mononuclear cells (PBMC), EBV lymphoblastoid cell lines (B-LCL), and dendritic cells (DCs). Fibroblasts do not express costimulatory and accessory molecules, and thus cannot be used to stimulate a primary immune response. Furthermore, the absence of presentation of antigens by MHC class II molecules prevents the stimulation of CD4⫹ anti-CMV T cells, which seems to be required to sustain a prolonged and stable anti-CMV response in vivo. Finally, a minimum of 2 months are required to generate enough fibroblasts for immunotherapy from a skin biopsy. Autologous PBMC are a very convenient source of APCs because they can be used directly after blood sampling or cytapheresis. Nevertheless, their composition, which varies from one donor to the other, renders them less reliable both for gene transfer and presentation of antigens. For these reasons, B-LCL and DC, which constitutively express at different rates MHC class II, costimulatory and accessory molecules are preferred to generate antiviral specific responses. B-LCL proliferate well and are easily prepared from minimal amount of blood, but present the disadvantage of requiring 3– 4 weeks to get them in sufficient amounts. Among professional APC, DC can be prepared in large numbers from human monocytes in 2 to 7 days. Furthermore, they have the capacity of cross-presenting antigens that leads to the presentation of antigens both to CD4⫹ and CD8⫹ T cells [12, 13]. Choice of viral antigen. The choice of the viral antigen inserted is crucial because it will determine the efficiency of the CTL in vivo. The viral antigen has to be immunodominant (i.e., to be preferentially recognized by T cells among all viral antigens). In addition, it has to be (1) recognized in the context of several if not most human leukocyte antigen (HLA) alleles, so that it can be used for a maximum of patients; (2) recognized both by CD4⫹ and CD8⫹ T cells to sustain a long-lasting and

Peptide Configurations for T-Cell Stimulation

efficient cytotoxic activity; and (3) presented at sufficient levels not only by APC, but also by the in vivo infected target cells, so that cultured cytotoxic T lymphocytes (CTL) can recognize and kill them once administered to the patients. For CMV, the lower matrix 65kd phosphoprotein (pp65) has been shown to be the predominant viral antigen, targeted by 70 –90% of CMV-specific CTL [14]. pp65, which is the most abundant protein in viral particles can be presented by a wide variety of HLA alleles [14, 15]. However, in some individuals, IE1 may be the major target for CD8⫹ T cells, with 10 CTL epitope identified for different HLA alleles, mostly in exon 4 [15, 16]. Other CMV antigens such as pp150 and glycoprotein B (gB) seem to be recognized in about one third of individuals [15, 17, 18]. Choice of viral vector. The viral vector used to transduce the different APC has to present the following criteria: (1) it has to infect as efficiently as possible the APC, and thus present the corresponding receptors; (2) if retrovirus vectors are used, the cell has to cycle to allow integration and expression of the transgene; (3) the size of the transgene should not exceed the insertion capacity of the vector (around 7kb for retrovirus and adenovirus and 35kb for poxvirus); 4) its immunogenicity should not overlap the one of the transgene; and 5) their safety has to be controlled. Basically, mostly on the basis of the cell cycle status and viral receptors of the target APC, activated PBMC and B-LCL can be infected by retroviruses, whereas monocyte-derived DC are usually transduced by adenoviruses and poxviruses. Antigen Transfer in EBV-Transformed B-Cell Lines and Specific CTL Induction Sun and colleagues have generated pp65-specific CTL by stimulation of T cells with wild-type B-LCL expressing pp65 from a retroviral murine stem cell virus vector [19]. The disadvantage of this APC as compared with others was a quite low level of expression of the transgene. A recent study by Moosmann and colleagues has focused on the use of EBV itself to mediate antigen transfer to B cells [20]. They constructed an EBV plasmid containing less than half the EBV genome, lacking many lytic genes essential for viral replication, but still able to immortalize B cells and in which up to 80 kb of foreign DNA can be added [21]. Out of three donors for which peripheral lymphocytes were stimulated with B cells transformed with mini-EBV plasmid containing pp65, two showed induction of bispecific CTL directed against pp65 and EBV antigens [20]. The length of the procedure is clearly a disadvantage of this method since in addition to the 3–5 weeks required to prepare the

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mini-BLCL, 5– 8 weeks are necessary to obtain sufficient numbers of stimulating cells. Antigen Transfer in Autologous PBMC and Specific CTL Induction Although they do not represent fully competent APC, autologous PBMC have been used in some protocols of immunotherapy using poxviruses-mediated gene transfer. Poxviruses used for smallpox vaccination either in human (vaccinia virus) or fowl (Avipox viruses) present several characteristics that render them suitable for gene transfer: (1) they do not integrate in the cell genome, (2) they are easy to manipulate genetically, (3) they are capable of accommodating large genes, (4) they are able to infect a large number of mammal cells including PBMC and DC, and (5) they are not pathogenic for the individuals whatever their immune status. The only limitation to the use of poxviruses in gene transfer is the transitory expression of the transgene, usually 1–2 weeks, that is sufficient for antigen presentation, but not for gene correction. Avipox such as canarypox, contrary to vaccinia, are unable to replicate in mammal cells although they infect them with a high efficiency and do not induce the formation of neutralizing antibodies that could inhibit transgene expression [22, 23]. In the study of Gyulai and colleagues, autologous PBMC have been transfected with canarypox virus vectors encoding pp65, IE1 exon 4, gB, pp150, or pp28 to stimulate CTL induction from CMV-seropositive healthy donors [15]. CMV-specific CTL were obtained from all 26 donors against pp65 or IE1 antigens. They were able to lyse targets with an efficiency ranging from 40% using pp65 to 65% using IE1-exon 4-canarypox vectors. Other CMV proteins, pp150 and gB, induced a CTL response in about one third of the donors. Compared with other vectors, canarypox recombinants may enhance IE1-specific CTL induction [15]. This method has to be further tested to prove its efficiency in clinical trials. Antigen Transfer in DC and Specific CTL Induction Contrary to B-LCL, DC and fibroblasts present a high susceptibility to transduction by recombinant adenoviruses [24, 25]. Despite low levels of internalization, more than 90% of DC are infected at high doses of vectors (100 to 200 pfu/cell) [26]. Because almost everybody has been immunized with adenovirus, the risk of inducing an antiadenoviral cellular response when using adenovirus to transfer genes has to be evaluated. Hamel and colleagues [27] demonstrated that a cytotoxic-efficient CTL response against both the transgene and the adenovirus vectors could be induced when T cells were activated by APC transduced using first-generation adenoviral vectors.

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Multiple attenuated adenoviral vectors or adenoviral vectors lacking all viral genes have been developed to achieve long-term and a high level of gene expression [28]. When compared with E1- or E3-deleted first or second generation of adenovirus vectors, they were shown to induce a much higher level and longer lasting transgene expression [29]. These helper-dependent adenoviral vectors lack viral coding regions in the final infectious particles and were supposed to be less immunogenic. However, they induced a similar T-cell response as compared with E1/E3-deleted adenovirus vectors, suggesting that preformed components of the viral capsid are recognized [30]. However, polyclonal activation against the vector itself is of great interest in the setting of allogeneic HSCT in which patients are often multi-infected, with adenoviruses as well as CMV being two of the most frequent infections in pediatric patients. After two or three successive stimulation of donor lymphocytes, with pp65 adenovirus vectors transduced DC, T-cell lines were able to lyse pp65 expressing as well as CMV-infected targets with a very high efficiency [27, 31]. Keever-Taylor and colleagues compared the capacity of CTL induction of pp65-adeno monocytes, DC, and CMV-infected fibroblasts. Whereas no CTL could be generated using monocytes as APC, transduced DC allowed the generation of pp65-specific CTL from two donors. These CTL were as efficient if not more than those obtained with fibroblasts in killing pp65 expressing B-LCL and CMV-infected fibroblasts. Indeed 50 – 70% of target cells were lysed at a ratio of 12.5:1. Similar results were obtained by Hamel and colleagues after three rounds of stimulation with pp65 adeno DC. The study of T-cell receptor repertoire by immunoscope analysis showed that although restricted, the predominant TCRV␤ diversity of pp65-specific clones was conserved, demonstrating that the memory repertoire was preserved all along the procedure [14, 32]. These studies led to overall similar results as compared with those obtained with CMV-infected fibroblasts [27], although KeeverTaylor and colleagues found less efficient cytotoxicity, likely because of the priming of CTL to CMV antigens other than pp65 [31]. Nevertheless, as said previously, a clear advantage of DC as compared with fibroblasts is the stimulation of both CD4⫹ and CD8⫹ specific T cells, whereas fibroblasts only activate CD8⫹ T cells.

a recombinant vaccine canarypox candidate in seronegative donors included in a randomized placebo-controlled clinical trial. Subjects, who were not selected for specific HLA alleles, were immunized intramuscularly four times during a 6-month period. First CTL activity was detected as soon as month 3 and up to 26 months in all subjects tested. Final pp65 specific activity, detected at very low E:T ratio (6:1) and CTL frequency were similar in immunized seronegative and naturally seropositive subjects demonstrating the efficiency of this vaccination strategy. In view of these results, vaccination against CMV using pp65-expressing recombinant canarypox viruses might elicit a sufficient immunologic response to confer protection against CMV. Nevertheless, this strategy can be used only in patients presenting significant numbers of mature T cells.

In vivo Generation of CTL: The Vaccination Strategy If most studies have focused on the ex vivo generation of anti-CMV–specific CTL, in vivo generation of such a response becomes feasible by vaccination. One vaccination strategy is based on the direct injection of viral vectors encoding CMV antigens. In the study of Berencsi and colleagues [33], pp65-specific CTL were induced by

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Conclusion Between all the strategies described in this article, T cells transduced with a suicide gene still have to prove their efficiency in terms of prevention or treatment of CMV infection. In addition, the incomplete prevention of GVHD could hamper its use in pediatric patients submitted to partially incompatible HSCT. Vaccination, although apparently efficient, poses problems that will probably limit its use in immunocompromised patients. The most promising results come from CMV-reactive CTL generated ex vivo using APC transduced with viral immunodominant antigens. The adoptive transfer of virus-specific T-cell lines generated using CMV-infected fibroblast has demonstrated the efficiency and feasibility of this approach. Gene transfer of CMV antigens, avoiding the use of live CMV virions, is more suitable under GMP conditions for large-scale clinical trials. In addition, it allows the use of professional APC, such as DC that are very efficient in activating both CD4⫹ and CD8⫹ T cells. Although, pp65 recombinant vaccinia and adenovirus vectors give equivalent results in terms of CTL induction at least in preliminary results, adenovirus may be preferred for immunocompromised multi-infected patients, because they can induce CTL reactive against both CMV and adenoviruses. The generation of adenoviral vectors lacking all viral genes may improve further CMV-specific CTL induction.

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