Genetic Modification of T Lymphocytes for Adoptive Immunotherapy

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doi:10.1016/j.ymthe.2004.04.014

Genetic Modification of T Lymphocytes for Adoptive Immunotherapy Claudia Rossig1,* and Malcolm K. Brenner2 1

Department of Pediatric Hematology and Oncology, University Children’s Hospital Muenster, 48129 Muenster, Germany 2 Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX 77030, USA *To whom correspondence and reprint requests should be addressed. E-mail: [email protected].

Adoptive transfer of T lymphocytes is a promising therapy for malignancies—particularly of the hemopoietic system—and for otherwise intractable viral diseases. Efforts to broaden the approach have been limited by the physiology of the T cells themselves and by a range of immune evasion mechanisms developed by tumor cells. In this review we show how genetic modification of T cells is being used preclinically and in patients to overcome these limitations, by incorporation of novel receptors, resistance mechanisms, and control genes. We also discuss how the increasing safety and effectiveness of gene transfer technologies will lead to an increase in the use of gene-modified T cells for the treatment of a wider range of disorders.

INTRODUCTION That gene transfer could be used to improve the effectiveness of T lymphocytes was apparent from the beginning of clinical studies in the field. T cells were the very first targets for genetic modification in human gene transfer experiments. Rosenberg’s group marked tumor-infiltrating lymphocytes ex vivo with a Moloney retroviral vector encoding neomycin phosphotransferase before reinfusing them and attempting to demonstrate selective accumulation at tumor sites. Shortly thereafter, Blaese and Anderson led a group that infused corrected T cells into two children with severe combined immunodeficiency due to ADA deficiency. While neither study was completely successful in terms of outcome, both showed the feasibility of ex vivo gene transfer into human cells and set the stage for many of the studies that followed. More recently, a second wave of interest in adoptive T cell therapies has developed, based on their success in the prevention and treatment of viral infections such as EBV and cytomegalovirus (CMV) and on their apparent ability to eradicate hematologic and perhaps solid malignancies [1 – 6]. There has been a corresponding increase in studies directed toward enhancing the antineoplastic and antiviral properties of the T cells. In this article we will review how gene transfer may be used to produce the desired improvements focusing on vectors and genes that have had clinical application.

TRANSDUCTION OF HUMAN T CELLS: VECTOR SYSTEMS Currently available viral and nonviral vector systems lack a pattern of biodistribution that would favor T cell trans-

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duction in vivo—as occurs, for example, with adenovectors and the liver or liposomal vectors and the lung. This lack of favorable biodistribution cannot yet be compensated for by the introduction of specific T-cell-targeting ligands into vectors. Hence, all T cell gene transfer studies conducted to date have used ex vivo transduction followed by adoptive transfer of gene-modified cells. This approach is inherently less attractive for commercial development than direct in vivo gene transfer and has probably restricted interest in developing clinical applications using these cells. On the other hand, ex vivo transduction may be more readily controlled, characterized, and standardized than in vivo efforts and may ultimately produce a better defined final product (the transduced cell). Because T cells are highly proliferative, almost all clinical studies to date have used integrating vectors to ensure that all the progeny of the modified cells contain equal doses of the transgenes. Certain proposed applications may require only transient expression—for example of immunostimulatory cytokines—and in these settings it may be possible to substitute nonintegrating vector systems engineered for greater efficiency. Possibilities now being studied include fiber-modified adenovectors, liposomal plasmid complexes, or flowthrough electroporation techniques. For the moment, however, the great majority of T cell gene transfer studies reported so far have used Moloney murine leukemia virus (MoMLV)based vector systems [7 – 10]. The Moloney-based vector system has many technical limitations (and safety concerns—see Optimizing the Safety of Infused T Cells). As these vectors infect only dividing cells, retroviral gene transfer requires specific or nonspecific T cell activation. Such activation may con-

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tribute to reduced immunocompetence in genetically modified T cells [11 – 13]. The use of CD3 and interleukin 2 (IL-2) for the generation of large numbers of activated T cells was associated with a reduction of T cell receptor diversity and increased susceptibility to apoptosis [13,14]. Based on the observation that concurrent costimulation during T cell expansion allows T cell function and T cell receptor (TCR) repertoire diversity to be retained, current protocols include antibody coligation of the CD28 receptor [15,16]. Recently, in vitro T cell activation has been further improved by activation of the costimulatory molecule CD137 (4-1BB) by artificial antigen-presenting cells (APC) [17]. Furthermore, ex vivo T cell expansion in the presence of IL-15, a cytokine with antiapoptotic and proliferative properties on T cells, was shown to promote in vivo T cell survival and function [18]. Even when the T cells have been successfully transduced, the limitations of MoMLV vectors are substantial. Methylation of the viral long terminal repeat may produce transcriptional silencing, so that transgene expression declines over time [19]. It may be possible to overcome these problems too, by using alternative murine retroviral vectors, such as murine stem cell virus, which are less sensitive to silencing [20], or by introducing cisacting DNA elements that stabilize expression [20,21]. Despite the above limitations, the development of pseudotyped retroviral vectors for human T cells [22], the introduction of recombinant fibronectin to help colocalize vector and target cells [23], and the implementation of regimens to optimize T cell growth and proliferation mean that 80 – 90% of target T cells can now be transduced, almost a 2 log improvement over early efforts. Ultimately, MoMLV will likely be superseded by other vector systems. Lentiviral vectors currently attract the most attention, since they can transduce and perhaps integrate into nondividing cells (avoiding the need for T cell activation prior to transduction) and may also express higher transgene levels for a longer period. The first clinical trials using T cells modified with these vectors have now begun in patients with human immunodeficiency virus (HIV) (see Enhancing the Performance of T Cells—Transfer of viral resistance genes) and their safety and efficiency will be followed with great interest.

USES OF TRANSDUCED T CELLS FOR IMMUNOTHERAPY Gene transfer may be used to enhance any component of T cell function after adoptive transfer, for example: (1) to increase the reactivity of T cells to weak or poorly presented antigens, such as those expressed by tumor cells, by modifying the T cell receptor; (2) to favor survival of T cells even in a hostile tumor environment, by providing them with endogenous

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growth factors or by deleting receptors to inhibitory cytokines or death molecules; (3) to mark T cells so that they can be tracked in vivo by imaging techniques or by PCR analysis of blood and tissue, to determine their fate and performance in vivo; or (4) to provide a regulatory mechanism whereby unwanted proliferation or activity of modified T cells can be controlled. (1) Increasing Reactivity of T Cells to Weak or Poorly Presented Antigens Specific cytotoxic T cells (CTL) are potent mediators of the physiological immune defense against allogeneic or virus-infected cells and have been attributed an important role in controlling tumor growth. The most successful attempts at transferring T cell immunity have been reported from clinical trials with virus-specific T cells. Antigen-specific T cells have been shown to eradicate specifically and efficiently Epstein – Barr virus (EBV)infected target cells in patients with posttransplant lymphoproliferative disease [3,4,6,24] and reconstitute cellular immunity to CMV after allogeneic marrow transplantation [25,26]. A further example is the potent antitumor effect associated with allogeneic stem cell transplantation and subsequent donor lymphocyte transfusions. Recognition of minor histocompatibility antigens by allospecific T cells within the transferred donor T cell population results in T cell activation, leading to elimination of residual tumor cells. Infusion of donor lymphocytes induced durable remissions in 60% of patients with chronic myelogenous leukemia and 20 to 40% of patients with acute myelogenous leukemia relapsing after allogeneic transplant [27]. Unfortunately, attempts to extend this strategy to non-viral malignancies in the syngeneic setting have been largely unsuccessful. Most cancers are poorly immunogenic and can evade major histocompatibility complex (MHC)-restricted T-cell-mediated immune recognition [5]. Furthermore, in contrast to viral antigens, most tumor-associated proteins lack specificity since they are coexpressed on normal cells or at certain developmental stages. Due to self-tolerance, presentation of such antigens results in a peripheral T cell repertoire that is devoid of high-avidity antigen-specific CTL [28]. Therefore, obtaining useful quantities of functional antitumor T cells from the individual patient’s repertoire is difficult and involves a lengthy process of in vitro T cell selection, characterization, and expansion. In vivo, these activated cells have limited persistence after adoptive transfer [29]. Genetic engineering of T cells. Whereas many viral antigens are highly immunogenic and thus induce potent antiviral T cell responses, most tumor antigens only weakly stimulate the immune response. Hence, efforts

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to raise CTL directed to these antigens may founder due to an inability to expand selectively the scanty precursor cells that are specific for the desired target. While overexpression of weak antigens in ‘‘professional’’ APC may be one way of using gene transfer to overcome this problem, an alternative approach is to engineer T cells genetically with desired specificities, extending the range of antigens for adoptive T cell immunotherapy. The most obvious way of retargeting is to transfer selected TCR genes. It was first shown in transgenic mice that the antigen specificity of T cells is determined solely by the a and h chains of the TCR. Transferring both TCR a and h chains from one T cell to another was sufficient to endow the recipient T cell with the specificity of the donor T cell [30]. This strategy was then used to generate tumor-specific T cells. Human peripheral blood T cells transduced with cDNA encoding the full-length a and h TCR chains derived from MHC-restricted tumor antigenspecific T cell clones could be efficiently redirected to solid tumor antigens (MART-1 [31,32], MAGE-3 [33], MDM2 [34]) or infectious targets (HIV-1 [35], EBV [36]). In immunodeficient mice bearing syngeneic tumors expressing an influenza nucleoprotein (NP) peptide, adoptively transferred T cells transduced to express NPspecific TCR a and h chains were activated by antigen, homed to effector sites, and promoted tumor rejection [37]. Importantly, the redirected T cells showed marked clonal expansion after in vivo antigen exposure. One risk of this strategy is the formation of hybrid TCR between endogenous and transduced a and h T chains. T cells with unintended specificity may be generated, including autoreactive T cells. A more substantive limitation of TCR-derived recognition domains, however, is that interaction with antigen is MHCrestricted. The requirement for HLA matching precludes the use of universal receptors for the treatment of patients with disparate MHC phenotypes. Furthermore, tumors may escape from recognition by the modified T cells by downregulating surface MHC expression or by deficient antigen processing and presentation. As a complete and long-lasting antitumor immune response involves the concerted action of both CD4+ and CD8+ tumor-specific T cells, receptor genes for both MHC

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class I and class II presented peptides would have to be expressed in T cells. Moreover, nonprotein tumorassociated antigens, such as glycolipid and carbohydrate molecules, are not generally targeted by ah T cell receptors. This limits the use of TCR gene-modified T cells to cancers for which tumor-associated protein antigens have been identified. Finally, it is difficult to isolate tumor-specific CTL clones from which TCR a and h chains can be prepared. In an attempt to overcome this problem, human HLA-A2-transgenic mice immunized with tumor peptides were used to generate murine TCR recognizing antigen in the context of human HLA-A2 [34]. Human T cells transduced with chimeric receptors joining the TCR variable domains from these T cells to human TCR constant regions were shown to be capable of lysing a wide range of human HLA-A2+ tumor cell lines, and this approach is being further developed. A more popular strategy for creating tumor-specific T cells is based on the observation that proteins belonging to the ~ receptor family are capable of mediating signals that suffice to induce immune effector functions [38 – 41]. Ligation of an extracellular domain fused to the ~ chain of the TCR results in tyrosine phosphorylation of immune-receptor activation motifs present in the cytoplasmic domain of the ~ chain, initiating T cell signaling to the nucleus and recruitment of effector function [42]. Chimeric T cell receptors (chRec) thus combine antigen recognition and signal transduction in a single molecule. The antigen specificity of the artificial receptor relies on the binding characteristics of its extracellular domain, which usually consists of the variable domains of a monoclonal antibody, linked together as a single chain Fv (scFv) molecule (Fig. 1). Specific killing of tumor cells by chRec-modified T cells, as well as target-induced release of stimulatory cytokines, has been shown in vitro [42]. Furthermore, chRec-modified T cells have shown in vivo homing and antitumor activity in model systems [43 – 45], including elimination of established Burkitt’s lymphoma following adoptive transfer in a xenogeneic SCID mouse model [18]. Immunotherapy with chRec-modified T cells has advantages over strategies relying on native T cell spec-

FIG. 1. Chimeric receptor design is based on structural similarities between the recognition domains of an immunoglobulin molecule and the T cell receptor. Antibodies recognize their targets through hypervariable regions located within the variable (V) domains of their heavy and light chains. T cell recognition is mediated by regions within the a and h chains of the T cell receptor that share structural homology with the antibody V domains. Antibody V domains can be linked directly to T cell receptor signaling domains.

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ificity. ChRec interaction with antigen, defined by its scFv component, is independent of MHC restriction and antigen processing. Thus, major mechanisms of tumor escape from immune recognition are efficiently bypassed. Furthermore, patients with tumors that express a defined surface antigen can be treated with the same chRec construct, irrespective of their HLA phenotype. By grafting T cells with antibody-derived recognition domains, the spectrum of T cell targets is extended to a variety of surface proteins as well as nonprotein structures. An additional advantage is the relative ease with which tumor-specific T cells can be generated. As genetic modification of T cells does not require prior selection for native antigen specificity, this strategy allows generation of large numbers of tumor-specific T cells within weeks. Finally, the clinical success of monoclonal antibodies directed to tumor antigens has encouraged investigators to make use of these same specificities in chimeric receptors, which can then conscript the efferent activities of the T cell by which they are expressed. Chimeric receptors for solid tumors and hematologic malignancies. Chimeric receptors have been developed against a wide range of tumor antigens associated with solid tumors and hematological malignancies (Table 1). While most are derived from antibody molecules, receptors or their ligands have also been used as antigen recognition domains. Examples are neuregulin ligand, which binds to erb-B oncoreceptor family members overexpressed on human adenocarcinoma cells

TABLE 1: Chimeric T cell receptor specificities currently under investigation for cancer therapy Specificity Solid tumors Neu/HER2 Folate-binding protein (FBP) CEA EGP40 TAG-72 G250 PSMA GD3 GD2 KDR Epithelial glycoprotein-2 (EGP2) Hematological malignancies CD30 CD33 CD19

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Tumor target

Reference

Breast cancer Ovarian cancer

[43,82,131,132] [44,133,134]

Colorectal cancer Colorectal cancer Adenocarcinomas Renal cell carcinoma Prostate cancer Melanoma Neuroblastoma Tumor neovasculature Multiple malignancies

[59,135,136] [137] [65,138] [139 – 141] [81] [142] [77,143] [144] [145,146]

Lymphomas Myeloid leukemia B cell malignancies

[147,148] [78] [18,149]

[46], and vascular endothelial growth factor, which engages a receptor critically involved in tumor angiogenesis [47]. In these studies, the extracellular domains of the a and h chains each were fused to the TCR ~ chain, or single-chain TCR in which the a and h chain variable domains were connected by a linker were generated [48,49]. As nonphysiological a and h chain pairings are not expected to occur, this strategy may help to avoid the risk of generating autoreactive T cells associated with the transfer of unmodified a and h chain genes. Chimeric receptors for infectious and autoimmune diseases. Beyond specificities for tumor-associated antigens, receptors have been generated against molecules involved in autoimmune [50 – 52] and infectious diseases [53 – 56]. In a recent study of autoimmune encephalomyelitis, autoreactive T cells were targeted by genemodified T cells expressing a heterodimeric receptor that genetically links an autoantigenic peptide, its restricting MHC, and the TCR ~ chain [50]. In infectious disease, chimeric receptors have been designed to redirect T cells to HIV Env protein gp120 on infected cells via the extracellular domains of CD4 [53 – 56]. In 1995, investigators initiated an adoptive immunotherapy trial targeting HIV with CD4-specific T cells. Although chRectransduced T cells were functionally active in vitro and efficiently lysed CD4+ T cells infected with HIV [55], transfusions of ex vivo expanded CD4 ~-modified syngeneic CD8+ T cells in HIV-infected twin pairs failed to induce objective clinical responses [57]. The observed lack of antiretroviral activity was associated with a rapid decline in gene-marked cells in the blood following the cell transfusions. Limitations of chimeric receptor-expressing T cells. Failure of chimeric T cells to persist and function in vivo has been reported from other clinical studies and raised the question of the in vivo function of chRec-modified T cells. The observed discrepancy between highly efficient in vitro cytolysis and target-specific cytokine secretion and the failure of chRec-transduced T cells to induce therapeutic responses in vivo is likely a consequence of the biology of the system. (1) The short life span of adoptively transferred genemodified T cells can be due to the host’s immune response to foreign gene products, including coexpressed immunogenic marker genes. The immunogenicity of recombinant receptors can be reduced by using human antibody fragments as recognition domains [58,59]. Currently available murine hybridoma antibodies can be humanized by replacing murine framework regions [60], and fully human recombinant single-chain antibodies can be generated by phage display technology [61]. However, even

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in the absence of detectable immune rejection of transferred T cells, their in vivo persistence was impaired. (2) In murine models of viral infection, specific T helper cells have been shown to be of critical importance for the maintenance of CTL function [62,63]. Thus, cotransfusion of antigen-specific CD4+ T cells may be necessary for long-term maintenance of adoptively transferred chimeric T cells. Importantly, not only CD8+ CTL but also CD4+ T cells can be activated by ligand-bound chimeric receptors [38]. Beyond their role as amplifiers of the CD8+-mediated cytolytic immune response, CD4+ cells can lyse tumor targets by Fas-independent mechanisms [64,65] and contribute directly to an enhanced immune response. Coadministration of both CD4+ and CD8+ chRecmodified T cells to HIV-infected patients resulted in the continued presence of detectable chRec-modified cells in vivo for at least 1 year. Unfortunately, no significant therapeutic effects were observed, and antiviral activity rapidly declined over time [57]. Based on the experience that immunotherapy in general is most effective in subjects with low disease burden [2], a subsequent phase II randomized trial was performed in HIV-infected subjects with undetectable plasma viremia after antiretroviral therapy. Adoptive transfer of CD4 ~-gene-modified T cells still showed no significant antiviral effect, despite prolonged and stable persistence of gene-modified T cells in peripheral blood [66]. Effective and persistent control of infection or tumor growth by specific T cells requires that the transferred T cells continue to proliferate in vivo, repopulate the host, and enter the memory compartment, so that they both establish and maintain long-term tumor-specific immune responses. The continued detection of the transferred cells in vivo alone is obviously not predictive of their potential to fulfill these tasks. (3) Artificial T cell receptors may have poor signaling capacity. Physiologically, TCR engagement by agonist peptide – MHC complexes on APC results in the initiation of a signaling cascade that leads to transcriptional activation of genes encoding proteins involved in functional activation responses (Fig. 2A). T cell activation studies in transgenic mice have shown that, in contrast to native receptor triggering via agonist peptide – MHC binding, stimulation of the chimeric receptor is inadequate to induce a proliferative response in primary T cells [67,68]. The quality of the activation signals transduced through the chRec is thus inferior to that obtained after native TCR engagement. chRec differ from the native T cell receptor in several important aspects. Compared to the TCR ~ complex, a restricted subset of T cell receptor signaling domains is present within chRec, potentially limiting the activation response induced

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by receptor engagement (Fig. 2B). Moreover, TCR activation is a dynamic process that involves formation of an immunological synapse [69] by recruitment of engaged receptors and coreceptors into kinase-rich microdomains (‘‘lipid rafts’’). The role of coreceptor recruitment and lipid raft formation in chRec-mediated signal transduction has not yet been defined. (4) In addition to TCR-mediated signaling, appropriate costimulation is a critical determinant for efficient T cell activation. Interactions between costimulatory molecules on APC and their counterreceptors on T lymphocytes play a key role in the induction and maintenance of cell-mediated immune responses and the prevention of activation-induced anergy [70]. Both in vitro and in vivo studies of T cells stimulated by antigen through their conventional receptors have indicated that in the absence of continuing costimulation, there is a progressive decrease in proliferation and loss of cytotoxic T cell activity. As tumor cells are generally deficient in expression of costimulatory molecules, chRec engagement of genemodified T cells by tumor cells is accompanied by inappropriate costimulation, resulting in anergy or cell death (Fig. 3). Improving chimeric receptor function. Based on the observation that costimulatory signaling is crucial for the antitumor activity of adoptively transferred T cells [18,18,71,72], further attempts at enhancing the in vivo performance of engineered T cells have focused on providing adequate costimulation. The best characterized costimulatory signal is the one delivered through the CD28 receptor on T cells after engagement of one of its ligands, B7-1 or B7-2 [70,73], expressed on APC. The first attempt to exploit the phenomenon was to express B7 in tumor cells [74]. This strategy proved to be clinically ineffective, likely due to expression in only a small fraction of tumor cells and to the inability of B7 stimulation alone to induce immunity against nonimmunogenic tumors [75]. Recent efforts have thus focused on the introduction of costimulatory signals into tumor-reactive T cells. Ligation of an extracellular scFv antibody fragment fused to the signal transduction domain of CD28 can result in effective CD28 signaling in primary T cells [76]. Double transfectants simultaneously expressing scFv-CD28 and scFv-CD3~ chimeras result in effective primary and second signaling upon encounters with antigen [77]. In an attempt to deliver primary and costimulatory signals through a single receptor, chimeras were constructed that combined the signaling domains of both the TCR ~ chain and the CD28 receptor, again linked to an extracellular antitumor antibody domain (Fig. 4). Ligand engagement by the combined receptor (CD28 – CD3~) significantly enhanced the levels of IL-2 secretion in Jurkat cells compared to the CD3~ receptor [78] and

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FIG. 2. (A) Signal transduction by the TCR links antigen recognition with functional responses. T cell receptor engagement by agonist peptide – MHC complexes on APC initiates a signaling cascade, which results in recruitment and activation of various proteins with enzymatic activity. Coordinated initiation of downstream biochemical pathways results in activation of transcription factors that stimulate the expression of genes involved in T cell responses. (B) Proximal signaling events induced by agonist peptide stimulation of the TCR include T cell receptor clustering, phosphorylation of TCR-associated proteins, and recruitment and activation of protein tyrosine kinases. The molecular mechanisms of T cell activation via chimeric receptors have not been identified in detail. Limited proximal signaling may contribute to the poor activation response observed after chRec triggering.

induced IL-2 secretion as well as specific proliferation of human peripheral blood T cells even in the absence of professional APC [58,79 – 81]. ChRec providing both CD3~- and CD28-mediated signals were further shown to exhibit greater in vivo activity against lung metastases in a mouse model [82]. Thus, CD28 signaling can restore or

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enhance T cell proliferation in response to chRec target antigen and may enhance the in vivo activity of genemodified T cells. In a recent study [83], chimeric receptors containing not just CD28 but also signaling domains from additional costimulator molecules (ICOS, CD134, or CD137) in

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FIG. 3. T cell activation relies on recognition of specific antigen in the presence of adhesion (LFA-1, LFA-2), coreceptor (CD4, CD8), and costimulatory receptor crosslinking by their respective ligands on APC. Originally, costimulation was thought to involve only CD28 receptor interaction with its ligands on the APC. It is now known that costimulation is more complex and involves additional signals from various molecules, including further members of the immunoglobulin superfamily (ICOS) or of the tumor-necrosis factor receptor superfamily, such as OX-40 (CD134) and 4-1BB (CDw137). Tumor cells are poor APC that lack expression of most costimulatory ligands. Thus, chRec engagement by tumor antigen is not accompanied by adequate second signaling.

series with the TCR ~ region were expressed in T cells and compared to a receptor providing TCR ~ signaling alone. Each of the costimulatory receptors alone enhanced the level of antigen-induced release of TH1 cytokines and enabled resting primary T cells to survive and proliferate in response to antigen in the absence of any exogenous factors. Optimal activation of gene-modified T cells by tumor antigen in vivo may require a combination of various costimulatory signals, which may be achieved by in-series fusion of several signaling regions. The design of such combined receptors should take into account the different costimulatory requirements of individual T cell subsets. Mutating selected motifs within chRec signaling domains may be another means of optimizing receptor function. Recently, a critical dileucine motif was identi-

FIG. 4. Second-generation antitumor chimeric receptors provide costimulation or coreceptor signaling by in-series ligation of signaling domains with the TCR ~ chain.

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fied in murine CD28 that limited expression and function of chRec-modified T cells [84]. Specific inactivation of this motif resulted in enhanced chRec-mediated effector functions. To improve the signaling characteristics of chRec and bypass TCR-mediated proximal signaling events, which are often impaired in T cells from tumor patients [85 – 88], receptors in which the TCR ~ chain was linked directly to intracellular kinases were constructed [89,90] (Fig. 4). Engagement of a receptor combining TCR ~ and the src family kinase lck promoted formation of a qualitatively superior signal-transducing complex, reflected by enhancement of early events in TCR signal transduction and in a greater quantity of IL-2 release [79]. None of these new receptors has yet been clinically validated. An alternative approach to overcoming the limitations associated with deficient T cell activation by chRec is the genetic engineering of an effector T cell that possesses native specificity for a strong agonist antigen, such as viral or alloantigens. Stimulation of such dual-specific T cells with antigen recognized by the native receptor will provide a powerful proliferation and activation stimulus, while at the same time chRec expression will redirect the activated cells to tumor cells. The first dual-specific antitumor T cells were generated using CTL specific for EBV, which are capable of massive expansion in vivo and are associated with persistent and life-long control of viremia [91]. Following genetic modification with antitumor chRec genes, EBV-specific T cells were shown to be expanded and maintained long term in the presence of EBV-infected B cells. They recognized EBV-infected targets through their conventional T cell receptor and tumor targets through their chimeric receptors, and they efficiently lysed both [92]. Due to the high prevalence of EBV infection in the population and continued presence of EBV antigen in vivo, periodic restimulation of the

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transferred cells may occur in vivo, permitting long-lasting tumor control. ChRec-transduced dual-specific T cells have recently been tested in vivo in murine tumor models. Ovarian tumor xenografted mice were treated with alloreactive T cells transduced with tumor-specific chRec. The adoptively transferred T cells expanded in vivo in response to allogeneic stimulation and protected the mice against tumor challenge [45]. In a NOD/SCID mouse model, mice bearing human CD19+ tumor xenografts and treated with influenza-specific T cells expressing a CD19-specific chimeric receptor achieved significant tumor responses when rechallenged with influenza antigen-expressing APC [93]. The therapeutic value of chRec-transduced, dual-specific T cells awaits clinical investigation. Nonspecific enhancement of T cell targeting. While modified chRec may help improve the desired specificity and functionality of T cells, it is also important to note that these specific modifications may also be coupled with nonspecific means of genetically enhancing T cell function. For example, if T cells are modified to express an apoptosis-inducing ligand, they can serve as mediators of apoptotic cell death independent of their TCR specificity. Membrane-bound tumor necrosis factor-related apoptosis inducing ligand (TRAIL) was shown to trigger the caspase-dependent death receptor pathway in malignant but not in normal cells [94]. Jurkat cells inducibly expressing TRAIL induced apoptosis in human target tumor cells in a paracrine fashion and inhibited tumor growth of human Burkitt lymphoma xenografts in mice [95]. This approach can be coupled with other ‘‘performance-enhancing’’ strategies as we next describe. (2) Enhancing the Performance of T Cells Transfer of stimulatory cytokines. The first genetic strategy designed to enhance T cell survival and function in vivo was gene transfer of stimulatory cytokines [96]. Because of their homing capacity, tumor-specific T cells can be used as vehicles for cytokine delivery into the T cellinhibitory microenvironment created by many tumors. Hence, T cells transduced with a gene encoding IL-2 were shown to support their own long-term proliferation [97,98]. Disappointingly, in clinical trials neither IL-2[99] nor TNF-a- [100] transduced tumor-infiltrating lymphocytes (TIL) had improved activity compared with nontransduced TIL. This failure was attributed to low levels of transgene expression following Moloney retrovirus transduction and to potentially proapoptotic effects of IL-2. In a recent preclinical study, EBV-specific CTL transduced to produce IL-12 showed a proliferative advantage toward unmodified CTL in the presence of inhibitory supernatants from Hodgkin tumor cells [101]. T-cellmediated delivery of IL-12 or other stimulatory cytokines may thus create a favorable environment for the direct antitumor effects of adoptively transferred T cells, while

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avoiding the systemic cytotoxicity of recombinant cytokines. Ultimately, gene transfer may not be the best way of expressing biologically active molecules in and around T cells, and nongenetic alternatives, including exogenous delivery of fusion proteins that anchor to the cell surface [102], may prove to be simpler, easier to standardize, and potentially safer. These approaches are already being used to generate cancer ‘‘vaccines’’ and could in principle be extended to T lymphocytes. Transfer of protective genes. Protection from the immune-inhibitory tumor environment may also be accomplished by endowing T cells with the ability to resist inhibitory cytokines. One of the best-defined examples of active tumor immune evasion is secretion of transforming growth factor h (TGF-h), a ubiquitous cytokine with multiple immunosuppressive properties, by many solid and hematological malignancies [103]. Abrogation of TGF-h signaling was shown to be crucial for CD8+ T cell effector function in transgenic mice [104]. To prevent TGF-h-mediated suppression of antitumor T cell responses, Bollard et al. transduced ex vivo-expanded human EBV-specific CTL from patients with Hodgkin lymphoma with a dominant-negative TGF-h receptor, resulting in functional resistance to the inhibitory effects of TGF-h and a strong survival advantage over nontransduced T cells [105]. Transfer of inhibitory genes. Most interest to date has focused on using stimulatory cytokines to improve target cell destruction or on methods to evade inhibitory signals from tumor cells. However, the setting of autoimmune disease is one in which T cells with enhanced inhibitory/ regulatory function may be desirable, since cytokines are known to play a central role in the initiation as well as the maintenance of autoimmune disease [106]. T cell clones and hybridomas, as well as antigen-specific primary CD4+ T cells, have been used to deliver modulatory cytokines, such as IL-4 [107] and IL-12 [108] in experimental mouse models of autoimmune disease. Irrespective of TCR specificity, T-cell-mediated adoptive gene therapy resulted in T cell trafficking to the site of inflammation and local delivery of the regulatory cytokines and provided long-term disease suppression. Retention of the cells at the site, however, required interaction of the TCR with site-specific antigens [109]. Nonetheless, unlike immune control of malignant tumor growth, even transient local expression of a regulatory cytokine may be sufficient to shift the local immune response permanently toward a more benign outcome. Transfer of viral resistance genes. For most viral diseases, gene transfer is combined with T cell immunotherapy to enhance antiviral T cell effector mechanisms—that is to help the T cell recognize and control infected target cells. A different tack has been taken for HIV. Since this virus

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targets T cells themselves, in this disease the primary purpose of gene transfer is to boost T cell defenses against the virus itself, by protecting the T cell from infection or—once infected—limit viral replication. Genes to block viral entry. HIV requires both CD4 and a coreceptor on the cell surface for productive infection. More than a dozen chemokine receptors function as HIV coreceptors in vitro, but the two most critical for the replication of T-cell-tropic and macrophage-tropic isolates in vivo are CXCR4 and CCR5, respectively. The latter is an attractive target because individuals who are homozygous null (the so-called D32 mutation) are phenotypically normal but relatively resistant to HIV infection [105]. Chemokine genes that contain a KDEL sequence have been developed so the resultant proteins do not leave the cytoplasm but sequester their receptors in the rough endoplasmic reticulum [106]. This ‘‘intrakine’’ strategy increased the resistance of cells to HIV infection (both M- and T-tropic strains) while preserving cell function, although levels of protection are far from complete. Genes to block viral transcription. HIV encodes a transcriptional transactivator (Tat) that acts at the level of RNA elongation in conjunction with cellular factors. A stem-loop RNA element, TAR, is recognized by Tat as well as by cyclin T1. This complex recruits the cyclindependent kinase CDK9 to hyperphosphorylate the carboxy-terminal domain of RNA polymerase II and increase enzyme activity. Anti-Tat single-chain variable fragment antibodies (intrabodies) interfere with Tat function intracellularly, while anti-TAR ribozymes cleave TAR, and polymeric TAR decoys compete with TAR for Tat binding [107]. Transdominant mutants of Tat have also been developed [108], which sequester cellular cofactors, including cyclin T1 and CDK9, but in general these mutants produce only limited inhibition of HIV replication and remain to be evaluated in clinical trials. Viral RNA transport and assembly [109]. The HIV protein Rev directs the export of intron-containing viral messenger RNAs from the nucleus to the cytoplasm and interacts with the Rev-response element (RRE), an RNA stem-loop structure found within the tat – rev intron. In the presence of both Rev and the RRE, competition between the host’s splicing apparatus and Rev allows export of intron-containing viral mRNAs. Anti-Rev intrabodies, similar to those against Tat, have been tested, as have RRE decoys. Dominant negative Rev mutants such as Rev M10 bind to the RRE and thus interfere with wildtype Rev RNA export. Escape mutants have rarely, if ever, been observed and Rev M10 has now been introduced into clinical trials. Alternatively, inhibitors of envelope protein synthesis, including env antisense molecules,

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have been used to block viral assembly and an antisense construct in a lentiviral vector is now in trials (see below). Combination therapy. Two or more anti-HIV genes have been used in combination, analogous to highly active anti-retroviral therapy. For example, multimeric TAR decoys bearing a transdominant or antisense Gag sequence synergistically inhibited HIV replication, while combination of an anti-Rev intrabody with a RRE ribozyme blocked HIV replication in established T cell lines for prolonged periods of time. Although untested in vivo, these types of strategy have been the most effective in preclinical models. Clinical trials of HIV gene therapy and their ultimate value [110 – 113]. Clinical studies have utilized T cells modified to resist HIV infection or occasionally to kill infected target cells. All have shown safety and some have shown evidence of diminishing HIV reservoirs and a trend toward fewer patients with recurrent viremia. Most recently studies using lentiviral vectors and antisense env have been initiated by ViIRxSYS and collaborators at the University of Pennsylvania; in these studies isolated CD4+ T cells are transduced ex vivo and after approximately 10 days of culture are reinfused in a dose-escalation study. The investigators will measure persistence, function, and resistance. This study is currently in progress and its safety and outcome will have major implications for future use of these vectors in T cell immunotherapy in general (Minutes of the Recombinant DNA Advisory Committee, September 6, 2001, and March 2, 2004, p. 13). Given the multiplicity of effective small-molecule antiretroviral agents approved or in study it is questionable whether gene therapy will ever be able to make a major contribution to HIV treatment. It has been suggested that the greatest importance of HIV gene therapy is that it is providing a platform from which lentiviral vectors can legitimately be evaluated. It seems more likely that gene transfer to T cells will find its place in HIV therapy, albeit to supplement rather than supplant recombinant HIV vaccines and conventional small-molecule therapeutics. (3) Gene Marking of T Cells Gene marking strategies allow investigators to monitor the migration of T cells and their survival and function in vivo. Long-term monitoring of the persistence and trafficking of virus-specific CTL can be achieved by quantitative PCR amplification of sequences within the retroviral vector itself or in the gene of interest [6,110]. Analysis of peripheral blood samples from 33 patients following infusion with EBV-specific CTL for relapsed Hodgkin disease or posttransplant lymphoproliferative disease resulted in detection of a retroviral marker sequence for up to 85 months, with the level of detection ranging from < 0.01 to 4% [111]. No adverse events

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attributable to the marking component were observed. A nongenetic alternative to the monitoring of T cells with known antigen specificity is their detection with fluorescence-marked MHC tetramers [112], but these cannot be considered unique markers for infused cells. New imaging techniques that permit the direct visualization of antigen-specific and gene-marked T cells in vivo have recently been developed. Based on active accumulation of a radiolabeled reporter probe, cells expressing the reporter gene can be visualized by scintigraphy or by positron emission tomography [113]. Bioluminescence imaging of luciferase reporter gene expression was recently used to follow the migration of T cells in vivo in real time in mouse models of tumors or autoimmunity [108,109,114]. In another study, human EBVspecific T cells transduced with the herpes simplex virus thymidine kinase (HSV-tk) gene and labeled with the radiotracers 131iodine – FIAU and 124iodine – FIAU, both substrates for HSV-tk but not mammalian thymidine kinase, were noninvasively tracked to autologous EBVtransformed B cell tumors implanted in SCID mice [115]. Beyond their value for evaluating immunotherapy, reporter genes may allow visualization of T cell activation in vivo [116] and thus contribute to our understanding of T cell biology. (4) Optimizing the Safety of Infused T Cells While these improvements may overcome some of the limitations of Moloney retroviral vectors, recent clinical experiences with X-linked SCID have further called into question the safety of retroviral-mediated gene transfer. Two cases of acute T cell leukemia were reported in patients who had undergone retrovirus-mediated gene therapy of CD34+ selected stem cells for X-linked SCID T cell leukemia [117]. A comparable side effect was previously reported in a murine model of retroviral gene transfer into hematopoietic stem cells [118]. These findings have been extensively reviewed elsewhere and will not be further discussed here. However, it is important to note that no serious adverse effects have been associated to date with the adoptive transfer of gene-modified mature T cells. In 26 patients posttransplant and 7 patients with Hodgkin disease who had received gene-marked EBV – CTL, treatment was well tolerated and, importantly, no patient developed any new malignancies. Integration site analysis performed on peripheral blood of these patients has demonstrated multiple different integration sites with no predilection for oncogene regions [111]. In contrast to hematopoietic stem cells that will pass through multiple rounds of proliferation and differentiation, mature T cells have a limited menu of differentiation and may thus have a lower risk of malignant transformation following proviral integration. Nevertheless, extensive investigations still have to clarify the actual malignant potency of retroviral T cell gene therapy. As with other potentially mutagenic agents, the clinical use of gene-modified T cells will have to

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be carefully considered according to a clinical risk – benefit evaluation. Lentiviral vector systems have theoretical advantages beyond their higher transgene expression levels (see Enhancing the Performance of T Cells), since they do not require cell division for stable integration of the transgene and have not been associated with malignant transformation in T cells. Other integrating vector systems, synthetic or viral, may also prove to be of value in the future [119]. Transgenes That Add Safety To enhance the safety of adoptive T cell immunotherapy, genetic strategies have been developed that enable the specific elimination of the transferred T cells and thereby control undesired T cell activity, including retrovirusinduced lymphoproliferation. The most common strategy relies on the inclusion of a suicide gene, which should permit elimination of genetically modified T cells upon treatment with a drug. Modification of donor lymphocytes with a suicide gene was first evaluated as a means of preventing graft-versus-host disease (GVHD) in patients treated with donor lymphocyte transfusions following allogeneic stem cell transplantation. Suicide genes originally coded for enzymes that are capable of activating an inert substance into highly cytotoxic metabolites. The most extensively studied system is the HSV-tk suicide gene. Expression in T cells of HSV-tk confers sensitivity to the prodrug ganciclovir, providing an effective means to delete the transferred cells [120]. Ganciclovir treatment of patients starting to develop symptoms of GVHD was effectively used to eliminate the modified T cells [12,121,122]. Although proof of principle was established in the first clinical studies, not all patients appeared to benefit from T cell suicide gene therapy, and several suffered from GVHD despite ganciclovir treatment. An evident problem was the expression of a truncated form of HSV-tk by a small percentage of donor T cells that had lost sensitivity to ganciclovir. This problem has now been overcome by introducing mutations that preserve the sensitivity of HSV-tk protein splice variants to ganciclovir [123]. Nonetheless, a selection step using cotransfected antibiotic resistance genes is still required because of the low efficiency of murine retroviral vectors for T lymphocytes. Instead of including drug resistance genes in the vector, it is possible to select transduced cells based on expression of a transgenic cell surface molecule that binds to specific antibodies. Coexpression of a truncated low-affinity human nerve growth factor receptor gene [9] or CD34 splice variant gene [124] with suicide genes permits enrichment of transduced cells to high purity using magnetic or fluorescence-based cell sorting technology, as well as monitoring of the persistence of transduced cells in vivo by flow cytometry [120]. Another approach is to transduce T cells with the CD20 molecule and then use positive selection with CD20 MAb followed, if necessary,

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with in vivo lysis using the CD20 antibody Rituximab. Hence, this approach using CD20 provides both a selection marker and a suicide gene [125]. One drawback may be additional depletion of circulating B cells by Rituximab treatment. The gene products of suicide and coexpressed resistance genes are highly immunogenic and may induce immunemediated rejection of the transduced cells. In one study, the persistence of adoptively transferred autologous CD8+ HIV-specific CTL clones modified to express the hygromycin phosphotransferase (Hy) gene and the herpesvirus thymidine kinase gene as a fusion gene was limited by the induction of a potent CD8+ class I MHC-restricted CTL response specific for epitopes derived from the Hy-tk protein [126]. Less immunogenic suicide and selection marker genes, preferably of human origin, may reduce the immunological inactivation of genetically modified donor lymphocytes. Human-derived prodrug-activating systems include the human folylpolyglutamate synthetase/methotrexate [127], the deoxycytidine/cytosine arabinoside [128], or the carboxylesterase/irinotecan [129] systems. These systems do not activate nontoxic prodrugs but are based on enhancement of already potent chemotherapeutic agents. The administration of methotrexate to treat severe GVHD may not only kill transduced donor lymphocytes but may also have additional inhibitory activity on nontransduced but activated T cells. Finally, endogenous proapoptotic molecules have been proposed as nonimmunogenic suicide genes. A chimeric protein that contains the FK506-binding protein FKBP12 linked to the intracellular domain of human Fas [130] was recently introduced. Addition of the dimerizing prodrug induces Fas crosslinking with subsequent triggering of an apoptotic death signal. SUMMARY Genetic engineering of T lymphocytes should help deliver on the promise of immunotherapies for cancer, infection, and autoimmune disease. Improvements in transduction, selection, and expansion techniques and the development of new viral vectors incapable of insertional mutagenesis will reduce the risks and further enhance the integration of T cell and gene therapies. Nonetheless, successful application of the proposed modifications to the clinical setting still requires many iterative studies to allow investigators to optimize the individual components of the approach. RECEIVED FOR PUBLICATION MARCH 16, 2004; ACCEPTED APRIL 26, 2004.

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