T lymphocyte engineering ex vivo for cancer and infectious disease

Transcription

1 Review General T lymphocyte engineering ex vivo for cancer and infectious disease 1. Introduction 2. T lymphocyte subsets 3. Ex vivo culture methods for T-cell therapy 4. Clinical-scale culture of co-stimulated T lymphocytes for cell therapy 5. Clinical trials of co-stimulated T lymphocytes 6. Expert opinion Bruce L Levine The University of Pennsylvania School of Medicine, Hospital of the University of Pennsylvania, Department of Pathology and Laboratory Medicine, Philadelphia, PA , USA Background : Basic research contributions towards the molecular and cellular understanding of immune mediated control of cancer and infectious diseases have created opportunities to develop new forms of T-cell-based vaccination for cancer and chronic infections like HIV. In the past two decades, there has been a dramatic increase in the number of cell therapy clinical trials around the world aimed at enhancing antitumor immunity, restoring immune function to infectious diseases and augmenting vaccine efficacy. Objective : To provide a review of new and emerging methods of T lymphocyte engineering, gene transfer to T cells and clinical trials. Methods : A review of recent clinical trials, along with a brief historical perspective, with a focus on challenges and recent advances in the field and requirements for successful T-cell therapies. Conclusion : Advances in the technological approaches and methods for ex vivo manipulation have led to T lymphocytes endowed with enhanced potency and unique functions, with promise as the new generation of infused therapeutics. Keywords: adoptive immunotherapy, clinical trials, gene therapy, T lymphocytes, vaccines Expert Opin. Biol. Ther. (2008) 8(4): Introduction Cell therapy laboratories have developed from their roots in blood banking and blood and bone marrow transplantation into what can now be described as cellular engineering laboratories, where cells can be isolated, enriched, transduced, activated, expanded and manipulated in ways that change or enhance their function, prior to reinfusion. Together with evidence for the role of the immune response in controlling cancer and infectious disease, technological advances have facilitated a paradigm shift from the use of cells and tissues only for homologous function to the engineering of cells for non-homologous or improved function. An ideal cell therapy would have the following properties: i) demonstrated potency against tumor or infectious organism; ii) efficient engraftment, enabling a high effector to target ratio; iii) long-term persistence and memory; and iv) being easily obtained and efficiently manufactured. Adoptive T-cell therapies that would meet the above criteria depend on the ability to optimally select, genetically engineer or induce cells with targeted antigen specificity while preserving their effector function and engraftment and homing abilities. 2. T lymphocyte subsets 2.1 Activation, differentiation and memory Naive CD4 + and CD8 + T cells undergo unique developmental programs after antigen activation, resulting in the generation of effector memory and long-lived central memory T cells, TEM cells and TCM cells, respectively. Naive T cells, because of their higher threshold for activation and their inability to react to most / Informa UK Ltd ISSN

2 T lymphocyte engineering ex vivo for cancer and infectious disease tumors and pathogens in rapid fashion, are not thought to be useful for adoptive transfer unless first primed in vivo and/or activated and expanded in vitro. Models for the development and maintenance of T-cell memory include those based on linear differentiation [1], signal strength [2], and memory stem cells [3]. Some studies indicate that naive T cells first differentiate into effector T cells, a proportion of which then differentiate into TCM cells [4]. Other studies suggest that parallel differentiation occurs, with naive T cells directly differentiating into TCM and effector cells simultaneously through asymmetric division [5]. The recent demonstration of asymmetric division of T lymphocytes following antigenic stimulus [6] lends further credence to the parallel differentiation model. However, it may still be true that some TCM are derived from TEM that do not undergo apoptosis when antigen is no longer abundant. Understanding the mechanism of TEM and TCM generation is important so that culture systems can be devised to optimally derive populations of TEM and TCM cells. TCM cells retain the capacity for marked clonal expansion [7]. It has also been shown that naive T cells have a longer chromosomal telomere length compared with memory T cells and that telomere length correlates with lymphocyte replicative history and residual replicative potential [8]. With regard to memory T cells, the demonstration that TCM and TEM cell subsets have discrete trafficking and functional properties [9] has important implications for adoptive T-cell therapy. For example, CCR7 and CD62L, both important for homing to lymph nodes, are not expressed or are expressed at lower levels in TEM cells. In retrospect, previous clinical trials have primarily tested adoptively transferred populations of CD8 + TEM cells [10]. This was due to the available tissue culture technologies at the time, which resulted in rapid differentiation of T cells to late-stage effector cells which are CD28-negative, express CD57 and have poor replicative capacity [11,12]. In vitro, TEM cells are superior to TCM cells at tumor cytotoxicity. However, in vivo, TCM cells exhibit superior therapeutic effects when compared with TEM cells on a per cell basis [10,13]. Therefore, in principle, adoptive T-cell transfer strategies to generate long-lived populations of TCM cells capable of immune surveillance as well as tumor eradication would be an attractive approach. 2.2 CD8 + and CD4 + T lymphocytes for adoptive transfer Many studies show that the generation and/or maintenance of CD8 + T-cell memory requires CD4 + T-cell help [14,15] and that immunity specific for tumors lacking expression of MHC class II molecules is enhanced with CD4 + T-cell help [16]. It may be counterintuitive that CD4 + T cells enhance antitumor effects in hosts bearing tumors that lack MHC class II. However, adoptively transferred CD4 + T cells have the potential to augment tumor immunity by several mechanisms that might enhance the survival and function of CD8 + T cells, including the secretion of cytokines and the expression of CD40L [17]. Besides their intimate involvement in priming tumor-specific cytotoxic T lymphocytes (CTLs), CD4 + cells participate in additional effector functions. Clinical adoptive transfer studies also show that the persistence of adoptively transferred cytotoxic CD8 + effector T cells is enhanced with the concomitant administration of IL-2 [18] or CD4 + T cells [19]. Recent studies in patients with myeloma show that the adoptive transfer of mixed populations of pathogen-specific CD4 + and CD8 + T cells promoted the establishment of immunity with a robust central memory component [20]. However, it is not yet known if this approach enhances the establishment of immunity to self antigens in cancer patients. At present, one of the most important issues facing the field is the complexity of CD4 + -lineage T cells. Until recently, CD4 + T helper (T H ) cells were separated into two different subsets named T H 1 and T H 2 cells, based on the pattern of cytokines that they produce when stimulated. Additionally, several types of CD4 + regulatory/suppressor T cells (Tregs) have now been described in humans [21], and it is probably important to remove Tregs from adoptively transferred T-cell populations because they suppress antitumor immunity [22]. Tregs are CD25 + CD127 - [23] and could be removed by flow cytometry sorting, though at present the technical hurdles, as described in section 3.1 below, for tetramer sorting would need to be overcome. An alternative approach that has been tested in a clinical trial is to remove Tregs from an apheresis product via anti-cd25 antibody conjugated to magnetic beads [24]. Since Tregs are often enriched in tumor-infiltrating lymphocytes and the peripheral blood in cancer patients [25], it is possible that the outcomes of previous adoptive T cell therapy clinical trials were compromised because the adoptively transferred T-cell populations inadvertently contained Tregs. 3. Ex vivo culture methods for T-cell therapy 3.1 Source of T cells and initial processing The only forms of adoptive cellular therapy routinely employed in the practice of medicine are allogeneic bone marrow and peripheral blood stem cell transplantation. In this setting, donor leukocyte infusions mediate various potent antitumor effects [26]. The adoptive transfer of activated donor (allogeneic) T cells shows promise of augmenting this effect [27,28]. Ex vivo culture approaches to altering the ratio of effector T cells to Tregs have the potential of decreasing the risk of graft-versus-host disease (GVHD) while preserving antitumor effects [29]. Engineered adoptive T-cell therapy approaches to date have used peripheral blood, tumors, malignant effusions and draining lymph nodes as the anatomical sources of input T cells for adoptive transfer [30-33]. Given that the bone marrow from patients with breast cancer, pancreatic cancer and myeloma has recently been shown to be enriched in tumor-reactive 476 Expert Opin. Biol. Ther. (2008) 8(4)

3 Levine Starting T cell repertoire Polyclonal stimulation Time Time TILs or PBMCs Antigen-specific stimulation 7 10 d Antigen and APC Ctclic stimulation with CD3-and CD28-specific antibodies Antigen and APC 6w Functional development Treg cell depletion Genetic modification T cell selection and/or expansion in the host Figure 1. Polyclonal and antigen-specific T-cell propagation. Left: Antigen-specific T-cell culture, particularly without prior selection or enrichment, requires several rounds of stimulation with antigen-pulsed antigen presenting cells or feeder cells. Right: Polyclonal stimulation via CD3/CD28 stimulation maintains the T-cell receptor repertoire of the starting population. PBMC: Peripheral blood mononuclear cell; TILs: Tumour-infiltrating lymphocyte; Treg: Regulatory T cell. CD8+ T cells [34-37], it is important to determine whether improved antitumor effects are observed when bone marrow resident T cells are used for adoptive transfer. Preclinical data demonstrating that activated marrowinfiltrating lymphocytes show enhanced tumor-specific activity compared with peripheral blood lymphocytes has led to a clinical trial testing this approach [37]. With leukapheresis, mononuclear cells or more may be obtained as starting material with small modifications of established protocols [38,39]. With the other sources of T cells, such as bone marrow and tumors, the number of cells obtained as starting material is usually much less and may vary widely among patients and with disease status, which indicates the critical importance of validation studies prior to the initiation of clinical trials. Two basic approaches are being tested for clinical adoptive T-cell therapy (Figure 1). The first approach is to isolate and activate antigen-specific T cells from peripheral blood, bone marrow, or tumor specimens in vitro and then to use repetitive stimulation to clonally expand the antigenspecific T cells in vitro by various approaches. In the second approach, polyclonal ex vivo activation of the T cells is performed based on three assumptions: first, that antigenspecific T cells are present in the patient; second, that antigen-specific T cells are primed in the patient; and third, that the in vivo function of the antigen-specific T cells in the patient is impaired. In this second approach, the cells are activated in vitro by various means that preserve the polyclonal repertoire and are then reinfused into the patient with the expectation that they will respond directly to antigens presented by antigen-presenting cells (APCs) or on virally infected cells. The first approach guarantees antigen specificity but is costly in materials, labor and time needed for the expansion. The second approach is technically more rapid, feasible and in general less expensive. In practical terms, the second approach has been efficient and robust enough to support randomized clinical trials [20], and therefore, only this approach has the potential for regulatory approval. The rationale for the second approach has been substantially strengthened by the realization that many patients are already primed to their tumors [40,41]. A combination approach, whereby patients are first vaccinated and then primed T cells are collected for polyclonal expansion may also be taken [20,42,43]. As vaccination approaches with defined tumor or infectious disease antigens may cover only a proportion of patients or may induce a narrow range of antigenic T-cell specificities, vaccination with a broad range of antigens, antigen matched to the patient or tumor lysate, may generate more antigen-specific T cells that could be expanded in vitro for adoptive transfer. In a research setting, MHC tetramers may be used to sort for antigen-specific T cells by flow cytometry. This approach faces several hurdles prior to testing in clinical trials. First, clinical grade tetramers must be generated and tested at Expert Opin. Biol. Ther. (2008) 8(4) 477

4 T lymphocyte engineering ex vivo for cancer and infectious disease significant cost. HLA-A2 tetramers would cover less than 50% of most patient populations; thus additional tetramers would need to be manufactured to cover many more patients. Each tetramer would isolate T cells specific for only one peptide and would thus complicate generation of a multimeric cellular therapy. Additionally, current flow cytometry sorter rates are such that sort times of several hours are usually required to isolate enough antigen-specific T cells sufficient for clinical scale expansion. Clinical flow sorters pose challenges for sterility of the sorted cells and containment due to the generation of aerosols, although new designs and technological advances may address these concerns. An alternate approach for the isolation of antigenspecific T cells that has been evaluated in clinical trials is the magnetic capture of IFN- γ secreting cells [44]. Polyclonal cells isolated from peripheral blood, bone marrow or tumors are first stimulated with antigen in vitro. T cells secreting interferon- γ are then isolated with clinical-grade commercially available antibody-conjugated magnetic particles. The advantages of this method are that the antigen need not be defined or limited to a particular peptide or protein and that T cells of multiple antigenic specificities can be generated and isolated. With either of the above positive selection approaches, Tregs could be effectively removed. For donor lymphocyte infusions, alloreactive cells may be depleted in vitro using the cytokine capture system and host cells as stimulators, and thus reducing the risk of GVHD. 3.2 Presenting antigen to T cells ex vivo The most appropriate methods of ex vivo T-cell culture mimic the physiological processes whereby dendritic cells (DCs) generate a constellation of antigen-specific and costimulatory signals in the T cells. However, although useful for therapeutic vaccination, due to practical considerations such as substantial manufacturing costs and the logistics of maintaining independent culture systems for each type of cell, DCs are not useful as APCs for large-scale adoptive T-cell therapy trials. In addition, for ex vivo expansion of autologous T cells, it is desirable to have APCs with extensive replicative potential to facilitate both the scaling up of the process and multiple rounds of T-cell stimulation. The rapid expansion method developed by Riddell and co-workers uses irradiated allogeneic peripheral blood mononuclear cells as APCs (also known as feeder cells) to expand CTLs for adoptive transfer [18]. The main limitation of this approach is in clinical scale-up, because conforming to FDA-mandated requirements for the validation and qualification of allogeneic feeder cells can be tedious and expensive. Schultze and co-workers have shown that CD40-stimulated B cells, which have an extensive replicative potential, are an efficient means of propagating antigen-specific T cells [45]. Therefore, although currently available tissue culture approaches have provided proof of concept for adoptive T-cell therapy, a current priority is to develop alternative approaches, such as artificial antigen-presenting cells, that can support the randomized large-scale trials required for FDA approval. 3.3 Cytotoxic T lymphocyte therapy At present, there is a plethora of suitable CTL targets for many tumors and viruses. In melanoma, CTLs derived from peripheral blood lymphocytes were used to treat patients with refractory, metastatic melanoma, and 8 out of 20 patients had minor, mixed or stable antitumor immune responses [18]. Furthermore, the infusion of autologous Melanoma-associated Antigen Recognized by T cells-1 (MART-1)-specific CD8 + T cells into a patient with metastatic melanoma resulted in T-cell infiltration into both the skin and tumor tissue [46]. These results were confirmed in a recent study in which engraftment of the CTLs, as measured by an elevated frequency of circulating T cells able to bind tetramers loaded with MART-1 peptides, was detectable up to 2 weeks after T-cell transfer in all patients, with a maximal frequency of 2% of the total CD8 + T cells [47]. Despite this high level of engraftment in all patients, only 3 out of 11 patients had clinical antitumor responses, and a selective loss of MART-1 expression was observed. Therefore, perhaps the most worrisome issue revealed with CTL transfers is the emergence of antigen escape variants. It is possible that this problem can be addressed by cell isolation and culture methods that facilitate a broad tumor-antigen-specific T-cell repertoire. Immunotherapy of human viral infection using adoptively transferred antigen-specific T cells has been studied in the setting of cytomegalovirus (CMV), Epstein Barr virus (EBV) and HIV infection. Riddell et al. have explored this strategy using T-cell clones with HLA restricted antigenic specificity for CMV [48,49]. Recovery of CMV-specific CTL activity was observed and adoptively transferred CTL persisted in vivo for up to 12 weeks. In a similar study, Brenner, Heslop and coworkers administered donor-derived EBVspecific CD8 + and CD4 + T cells, genetically marked with the neomycin resistance gene, to six recipients of T-celldepleted allogeneic bone marrow allografts that persisted for as long as 18 months [50,51]. Autologous EBV-specific T cells can also be generated [52-54], however, culture of a sufficient number of EBV-specific cells may require months, and this amount of time may not be practical and may result in a population of cells that is near replicative senescence. While the generation of EBV-specific cell lines could be attempted for each patient with the goal of infusing the cells if posttransplant lymphoproliferative disorder (PTLD) develops, if the incidence of PTLD is < 10% this would require a substantial investment in time and resources for each treated patient. A novel combination strategy to generate CTL specific for multiple viruses was reported recently [55]. In this study, a chimeric adenovirus-cmv vector was used to modify EBV-expressing B cells lines to serve as APC. Multi-virus-specific T cells were then generated and infused into 11 patients where they could be detected in vivo. In the 478 Expert Opin. Biol. Ther. (2008) 8(4)

5 Levine setting of HIV, the infusion of HIV-specific CTL s has been less encouraging. While infused HIV-specific CTL have been shown to be present in peripheral blood and to traffic to lymph nodes following infusion, persistence was limited to only days or weeks [56,57], and no significant clinical benefit was noted. This may have been due to the severe defects in the CD4 helper response resulting from HIV infection. 3.4 Tumor-infiltrating lymphocyte therapy Adoptive transfer therapy with tumor-infiltrating lymphocytes (TILs) requires the isolation of T cells from fresh patient biopsy specimens and the progressive selection of tumor-specific T cells ex vivo using high levels of IL-2. The adoptive transfer of these cells, with perhaps one exception [30], has been almost uniformly disappointing [58-60]. However, recent studies suggest that prior host conditioning with chemotherapy increases the response to adoptive immunotherapy with TILs [31,61]. An objective response rate of 50% was confirmed in a subsequent report from the same group [61]. Importantly, the TILs showed prolonged engraftment compared with TILs transfused to patients without prior treatment with these chemotherapeutics, and the levels of engraftment correlated with the clinical responses. However, the ability to successfully generate TILs for therapy could in itself be predictive of a more favorable clinical outcome [40,62]. In the absence of a randomized clinical trial it is not possible to determine how much lymphoablative chemotherapy, highdose IL-2 administration and TIL therapy contributed to the promising results of these recent trials. This indicates the importance of the measurement of cell yield, expression of homing receptors, potency and immune assessment assays in developing improved methods of cell processing. Technical issues with producing tumor-specific T cells currently present a formidable barrier to conducting randomized clinical trials using TILs. Only 30 40% of biopsy specimens yield satisfactory T-cell populations, and the process is labor- and time-intensive, requiring about 6 weeks to produce the T cells for infusion [63]. Therefore, randomized trials based on rigorous intent-to-treat analysis design (in which all data from all patients are included in the data analysis and any patients who are discontinued or otherwise non-evaluable are considered to be treatment failures) cannot be performed using currently available tissue culture technologies, and the trials reported to date have been performed based on an ad hoc, as-treated analysis plan. Should technical limitations of current tissue culture approaches be overcome, TILs harvested from other commonly encountered epithelial cancers, in addition to melanoma, could be expanded to clinically relevant numbers. 3.5 Genetic engineering of T-cell function In patients with congenital and acquired immunodeficiency, genetically modified T cells have been shown to persist for years following adoptive transfer [51,64-66], which indicates that the general approach is feasible. Persistence with conditions such as adenosine deaminase deficiency, where there is a strong selective advantage for the corrected genetically modified cells, have shown the most durable persistence. A potential safety concern when infusing individuals with engineered T cells is one that arose with genetically engineered hematopoietic stem cells (HSCs) for severe combined immunodeficiency in four patients in one study [67], and recently one patient [68] in a second similar study [69], when viral insertional mutagenesis was shown to cause cellular transformation [70]. Although there is little clinical experience with engineered T cells for cancer therapy, it is notable that clinical trials to date using cells engineered to express suicide molecules have indicated that the approach is safe. It may be that terminally differentiated cells, such as T lymphocytes pose less of a risk for transformation than hematopoietic stem cells. Unlike HSCs, currently available retroviral vectors provide high-level expression of transgenes in T cells in vitro. The advent of lentiviral vectors has greatly increased the efficiency of human T-cell engineering, and a recent pilot study with lentivirus-engineered T cells that expressed an antisense HIV vector showed promise in patients infected with HIV [71]. As mentioned above, insertional mutagenesis is a safety concern with any integrating viral vector. It is reassuring that the natural history of HIV does not include an increased incidence of T-cell leukemia; this provides empirical data that lentiviral vectors might be safer in this respect than oncoretroviral vectors. Furthermore, side-by-side tests in preclinical models indicate that lentiviral vectors are less prone to insertional mutagenesis [72]. Nevertheless, longterm observational studies with large patient safety data sets are required to determine the ultimate safety of this approach. A novel targeted gene correction technique employs chimeric zinc-finger proteins coupled to endonucleases [73]. With these zinc-finger nucleases, genes can be corrected or disabled in T cells in a specific fashion without the need for integrating vectors, an approach that could be applied to a number of monogenic congenital and acquired diseases such as HIV [74]. Ex vivo gene delivery to T cells, has advantages over in vivo delivery of gene vectors in terms of efficiency and persistence, and may be a safer approach that reduces the risk of off-target genetic modification and adverse events T cells engineered to express tumor-antigen-specific receptors One genetic engineering approach has been to endow T cells with novel receptors by introduction of T bodies, chimeric receptors that have an antibody-based external receptor linked to cytosolic domains that encode signal transduction modules of the T-cell receptor [75]. These constructs can function to retarget T cells in an MHC-unrestricted manner to attack the tumor or virally Expert Opin. Biol. Ther. (2008) 8(4) 479

6 T lymphocyte engineering ex vivo for cancer and infectious disease infected cell while retaining MHC-restricted specificity of their endogenous TCR. An additional benefit is that a population of antigen-specific T cells can be generated without extensive T-cell expansion and differentiation to a TEM population. One trial that tested T cells expressing a T body receptor specific for a folate-binding protein present on ovarian carcinoma cells indicated that the approach was safe but poor expression and persistence of the transgene encoding the T body receptor were observed in vivo [76]. Similarly, a pilot test in children with neuroblastoma treated with autologous T cells retargeted for a tumor-associated adhesion molecule indicated that the approach was safe but limited by poor persistence of the T cells [77]. Lamers and colleagues recently tested T cells expressing a T body receptor specific for carbonic anhydrase IX (CAIX), an antigen present on the surface of clear-cell renal cell carcinoma [78]. They observed an unexpected serious hepatic toxicity in several patients within a week of T-cell infusion that seemed to be due to CAIX expression in the biliary tract. If confirmed, this would indicate that engineered T cells can traffic to and exert effector function at sites of antigen expression in vivo. However, this study indicates that the targets of chimeric antigen receptors must be carefully chosen to avoid unwanted autoimmune and other adverse effects, or that additional safety features, such as suicide switches (see below), should be incorporated. In several of the patients in the studies described above, the engineered cells persisted for several days to weeks before elimination by host immune responses [76-78], indicating that a technical challenge for this approach is to prevent a host immune response from eliminating the adoptively transferred cells. Optimizing the ligand-binding domain and incorporating costimulatory signaling domains [79,80], may ensure better long-term survival of the T cells so that the proper costimulatory signals can be delivered upon target recognition [79,81]. T cells are also being transduced to express natural TCR specific for MART-1 in a study where lymphodepleted patients with melanoma were given a single infusion of engineered T cells followed by IL-2 [82]. A concern with transgenic TCR has been that additional, novel receptor specificities might be generated by pairing of the transgenes with the endogenous TCR chains [83]. A general limitation of inserting TCR as opposed to antibody/tcr chimeric receptors for humans is that each TCR is specific for a given peptide-mhc complex, such that each TCR vector would only be useful for patients that shared both MHC alleles and tumor antigens. Engineering enhanced specificity of T cells through receptor modification has recently been reviewed in further detail [84,85] Engineering enhanced T-cell survival or death A limitation of adoptive transfer of expanded CTLs discussed above is that they have short-term persistence in the host in the absence of antigen-specific Th cells and/or exogenous cytokine infusions. In some settings, where passenger lymphocytes or HSC will eventually reconstitute the lymphoid compartment, this may be acceptable. In chronic infections or residual disease where immediate and sustained immune augmentation is required, other strategies are being pursued. Greenberg and co-workers have transduced human CTLs with chimeric GM-CSF IL-2 receptors that deliver an IL-2 signal when they bind GM-CSF [86]. Stimulation of the CTLs with antigen caused GM-CSF secretion and resulted in an autocrine growth loop such that the CTL clones proliferated in the absence of exogenous cytokines. This type of genetic modification has the potential to increase the circulating half-life of the CTLs and, by extension, the efficacy of these ex vivo-expanded cells. A related strategy to rejuvenate T-cell function is to engineer T cells to ectopically express CD28 [87] or the catalytic subunit of telomerase [88]. Severe and potentially lethal GVHD represents a frequent complication of allogeneic immunotherapy and donor lymphocyte infusion (DLI). The promising results with DLI have created increased interest in developing T cells with an inducible suicide phenotype. Expression of herpes simplex virus thymidine kinase (HSV-TK) in T cells provides a means of ablating transduced T cells in vivo by the administration of acyclovir or ganciclovir. Using this strategy, Bordignon and colleagues infused allogeneic donor lymphocytes engineered to express HSV-TK into patients with refractory hematologic malignancies who had suffered complications following allogeneic bone marrow transplants and donor lymphocytes [89]. The HSV-TK lymphocytes survived for up to a year, and complete or partial tumor remission in five of eight patients was achieved. Tumor regression coincided with onset of GVHD, and in most cases, GVHD was abrogated when ganciclovir was administered. A recent Phase II clinical trial has confirmed and extended these results [90]. It is possible that the first form of adoptive therapy with engineered T cells to enter clinical practice will be the use of allogeneic T cells with a conditional suicide switch, as a Phase III clinical trial is planned to test this approach in the setting of haploidentical HSC transplantation. The principal concern with the HSV-TK approach has been that it would generate potent HSV-TK specific immune responses as others have found that humans efficiently reject cells engineered to express HSV-TK or similar constructs [91,92]. Therefore, HSV-TK might confer immunogenicity to the transfused cells, leading to their impaired survival and the inability to retreat a patient with a DLI of cells engineered to express HSV-TK should the tumor recur. In patients who are immunosuppressed, this may not be the case. Development of vectors that encode less immunogenic suicide proteins will be required to extend this approach to immunocompetent hosts. Recently, investigators have developed suicide systems comprised of fusion proteins containing a human FAS or caspase death domain and a modified FK506-binding protein (FKBP) [93,94]. T cells expressing these modified 480 Expert Opin. Biol. Ther. (2008) 8(4)

7 Levine Figure 2. First generation artificial antigen presenting cells. Magnetic beads 4.5 µm in diameter are coupled to monoclonal antibodies directed against CD3 and CD28 on T lymphocytes. Beads are added to T lymphocytes at a 3:1 ratio enabling efficient activation and expansion [96]. chimeric proteins are induced to undergo apoptosis when exposed to a drug that dimerizes the modified FKBP. These approaches have the advantage that the suicide switches are expected to be non-immunogenic because they are based on endogenous proteins. 3.6 Tuning up T lymphocytes with artificial antigen presenting cells In recent years, a greater understanding has emerged of the receptor signaling pathways for T-cell activation, particularly the recognition that both a primary specificity signal via the T-cell receptor (TCR) (Signal 1) and a costimulatory/regulatory signal via the CD28 receptor (Signal 2) are simultaneously required for the generation of full T-cell effector function and a long-lasting immune response [81]. In cancer or infectious disease, antigen-specific T cells may have been deleted or tolerized due to suboptimal T-cell activation. With this knowledge, we have developed efficient and reproducible methods of mimicking the signal provided to T cells by dendritic cells but without delivering a negative costimulatory signal. With consistent lots of artificial antigen presenting cells (aapc), appropriate signals can reproducibly be delivered to T cells to improve on the function, activation/expansion and length of T-cell survival in vivo. These aapc methods allow for T cells to be grown rapidly ex vivo to clinical scale for therapeutic applications. The technology enables direct T-cell activation, instead of indirect activation via vaccines, which can be modulated by the nature of cell dose as necessary to achieve a clinical response [95,96]. The first generation of off-the-shelf aapc was developed by covalently linking clinical grade anti-human CD3 and anti-cd28 monoclonal antibodies to magnetic beads, which serves to crosslink the endogenous CD3 and CD28 receptors on the T cell ( Figure 2 ). This bead-based aapc enables the most efficient growth of human polyclonal naive and memory CD4 + T cells reported [96]. In terms of cell function, the expanded cells retain a highly diverse TCR repertoire and, by varying the culture conditions, can be induced to secrete cytokines characteristic of Th1 or Th2 cells [27]. One important advantage of this bead-based system is that it does not crossreact with CTL antigen 4 (CTLA4) and therefore provides unopposed CD28 stimulation for more efficient expansion of T cells. Another, unanticipated discovery was that crosslinking of CD3 and CD28 with bead-immobilized antibody renders CD4 + T lymphocytes highly resistant to HIV infection. This is due to the downregulation of the chemokine receptor CCR5, a necessary co-receptor for the internalization of HIV, and the induction of high levels of β -chemokines, the natural ligands for CCR5 [97,98], and allows for the efficient culture of CD4 + T cells from HIV-infected study subjects. Ex vivo expansion may also indirectly enhance T-cell activity by removing T cells from a tumor-induced immunosuppressive milieu [99,100]. Other key features are that exogenous growth factors or accessory cells are not needed to enable T-cell stimulation and expansion, as with previous methods. 3.7 Cell-based artificial antigen presenting cells Recently, aapc lines derived from the chronic myelogenous leukemia line K562 have been described [ ]. K562 cells do not express MHC) or T costimulatory ligands, and these cells may represent DC precursors that retain many other attributes that make DCs such effective APCs, such as cytokine production, adhesion molecule expression and macropinocytosis. These cells have been transduced with a library of lentiviral vectors that allows for the customized expression of stimulatory and costimulatory molecules that can be used to activate and expand different subset of T cells, and can be further modified to amplify antigen specific T cells in culture. These aapcs offer the advantage of expression of molecules additional to CD3 and CD28 on their surface. The K562 aapcs have been transduced with vector to express the antibody Fc-binding receptor and the costimulatory molecule 4-1BB. The expression of CD64, the high affinity Fc receptor, on K562 aapc s allows the flexibility of loading antibodies directed against T-cell surface receptors. CD3 and CD28 antibodies are added to the cells and are bound by the Fc receptor to yield a cell that expresses anti-cd3 mab, anti-cd28 mab and 4-1BB. These cell-based aapc s have proved to be more efficient at Expert Opin. Biol. Ther. (2008) 8(4) 481

8 T lymphocyte engineering ex vivo for cancer and infectious disease LV-aAPC T cell Figure 3. Second-generation artificial antigen presenting cells optimized for CD8 T lymphocytes and antigen-specific T lymphocyte activation. K562 cell lentivirally transduced to express CD64 and CD137L and loaded with anti-cd3 and anti-cd28 antibodies stimulates a CD8 + T lymphocyte [103]. activating and expanding T cells, especially CD8 +, CD28 - TEM, and antigen-specific T cells ( Figure 3 ), than the magnetic bead-based aapc [103]. In addition, the cells are capable of stimulating CD4 cells efficiently. Thus, K562 cells may represent ideal scaffolds to which the desired MHC molecules, costimulatory ligands, and cytokines can be introduced in order to establish a DC-like aapc that has the advantages of DCs (e.g., high levels of MHC expression, a wide array of costimulatory ligands and the ability to engage in cytokine crosstalk with the T cell) without any of the disadvantages (i.e., the need to derive natural DCs from either G-CSF mobilized CD34 + cells or monocytes using cytokines that are not currently available as good manufacturing practice reagents, patient-specific expansion, limited lifespan and limited replicative capacity). Moreover, these cells have been injected into humans as part of a tumor vaccine [104], signifying that these cells can be used in a good manufacturing practice manner. Additionally, this lab and its colaborators have now developed either bead- or cell-based aapcs optimized for Th2 cells [27,105], and for T regulatory cells [106]. 4. Clinical-scale culture of co-stimulated T lymphocytes for cell therapy 2 µm One challenge in translating research findings to clinical scale is technical, there may be differences in cell yield and function due to changes in nutrient and metabolite exchange, gas transfer, shear stress, or other conditions at large scale that are not apparent at small scales. A second challenge is the availability of clinical-grade reagents, materials and equipment to enable cellular engineering at the clinical scale that mimics as closely as possible the methods developed at the research scale. The use of either the bead-based or cell-based aapc s described above serves as an efficient GMP-compatible method for robust production of engineered T lymphocytes. In the case of bead-based aapc, a cohort of clinical trials (described in the next section) has demonstrated safety and feasibility of co-stimulated T lymphocytes, and the potential for efficacy in malignancies and HIV infection. Independent of which of the aapcs is used, the manufacturing procedure remains similar, starting with an apheresis product and depleting monocytes either by adherence to magnetic beads or via an Elutra counterflow centrifugal elutriator. If a CD8 + or CD4 + T-cell product is desired, the depletion of CD4 + or CD8 + T cells can be accomplished via large-scale magnetic cell selection in a closed and sterile system. T cells can then be cultured in a nutrient media and stimulated to divide and grow via the addition of either antibody-coated magnetic beads or the irradiated and antibody preloaded K562 aapc s described above. With either method, gene transduction with either retroviral or lentiviral vectors is very efficient [65,71,107]. T cells may be cultured either in static gas-permeable bags [108] or in a dynamic wave action bioreactor at high concentrations. Measurement of nutrients and metabolites in the cell culture media can provide invaluable assistance in optimizing conditions beyond what can be gleaned from simply counting and phenotyping cells. After completion of cell culture, antibody coated beads are removed by passing the cells over a magnet using a closed system of bags and tubing [108]. Prior to infusion, engineered T cells must be tested to ensure that they meet release criteria such as T-cell phenotype, cell viability, pyrogenicity, sterility and demonstration of the removal of residuals such as beads used in manufacturing to address regulatory requirements regarding purity, potency and sterility [109,110]. 5. Clinical trials of co-stimulated T lymphocytes To date, several hundred infusions of co-stimulated beadexpanded T cells have been safely administered to treat several types of malignancies with both autologous and allogeneic T cells and HIV with autologous T cells. In patients with high risk lymphoma, expanded co-stimulated T cells were infused on day 14 post CD34-selected hematopoietic stem cell transplantation (HCT), and immune reconstitution was assessed following post-transplant T-cell reconstitution. Of the 16 subjects, 5 had an unexpected lymphocytosis following T-cell infusion and the frequency of interferon- γ secreting cells also increased markedly in some patients. This trial indicated for the first time that it is possible to accelerate immune reconstitution in patients with advanced lymphoma who are given high dose therapy and autologous stem cell transplantation. In a subsequent trial 482 Expert Opin. Biol. Ther. (2008) 8(4)

9 Levine for chronic myelogenous leukemia, a second objective was to determine the frequency of hematologic, cytogenetic and molecular remissions from the treatment approach of autologous transplants followed by T-cell infusions. Of the four subjects who proceeded through the trial regimen, all had rapid recovery of lymphocyte counts following T-cell infusion and had complete cytogenetic remissions early after transplantation, three also became PCR negative for the bcr/abl fusion mrna [111]. A randomized Phase I/II study in subjects with advanced myeloma was designed to examine the relative benefits of pre- and post-transplant vaccine immunizations in combination with adoptive T-cell transfer. Post-stem-cell-transplant lymphocyte reconstitution and the pneumococcal vaccine (Prevnar) response were evaluated in 42 subjects. As in the lymphoma trial, the infusion of activated autologous T cells by day 14 post-transplant resulted in the induction of what appeared to be homeostatic T-cell proliferation in the first few weeks following transplantation. In addition, only those subjects that received antigen-experienced T cells showed robust antibody responses. This in vivo prime-and-boost with ex vivo -expanded and co-stimulated T cells would be a hybrid of the two approaches shown in Figure 1 and may prove to be a useful way to generate and/or enhance protective antitumor immunity [20,42] and in infectious diseases where effective vaccines are not yet available. A follow-on trial is now open in which the potency of a putative myeloma-specific peptide vaccine is being tested to evaluate the induction of T-cell-mediated graft versus myeloma effect. In an allogeneic setting, activated donor leukocyte infusions (adli) were administered in a trial to treat relapsed advanced hematologic malignancies after allogeneic bone marrow transplantation and standard DLI [28]. Of the 17 subjects evaluable for response, 8 achieved a complete remission with 6 still alive in complete remission a median of 17 months after adli. This trial suggests that adoptive transfer of activated allogeneic T cells is associated with durable complete remission in a subset of subjects without excessive GVHD or other toxicity. In general, these trials demonstrate that infusion of activated and expanded T cells, in combination with other therapies such as stem cell transplantation, chemotherapy and alkylating agent therapy (i.e., melphalan-containing regimens) has been associated with complete and partial responses in the treated subjects. In follicular lymphoma, regulatory-t-cell-depleted and co-stimulated T-cell infusion results in significant CD4 + and CD8 + numerical and functional lymphocyte recovery after cyclophosphamide-fludarabine chemotherapy [24,112]. In HIV, adoptive transfer of co-stimulated autologous CD4 + T cells resulted in a dose-dependent increase in CD4 counts and in the CD4:CD8 ratio following infusions. Sustained increases in CD4 + T-cell numbers and decreases in the percentage of CD4 + CCR5 + cells in patients were also found, suggesting augmentation of natural immunity to HIV infection [113]. More recently, co-stimulated T cells genetically modified using vectors that express proteins or antisense RNAs that target specific HIV genes have been generated and the safety and feasibility of this gene transduction and expansion method has beenassessed in the first clinical trial of a lentiviral vector [71,114]. In HIV+ study subjects who had failed at least two prior combinationantiviral drug regimens, following T-cell infusion, viral loads were stable or decreased in all five subjects. One subject has had a prolonged 2-log decrease in viral load for at least 2 years. CD4 counts remained stable in all patients and circulating genetically modified cells were detected in all patients for at least 6 months. Sustained lentiviral gene transfer was demonstrated in all subjects, and has persisted for more than 2 years in three of the patients. Promising results in heavily pretreated patients, from these early trials for various cancers and HIV, have led to a second series of randomized trials to address the efficacy of engineered T-cell therapies. 6. Expert opinion Cell therapies created through ex vivo manipulation may entail the isolation and/or propagation of T lymphocytes with a greater specificity for tumor antigens or pathogens along with enhanced function, or engineered with novel functions. Considerations of the best method to produce a large population of functional antigen-specific T cells ex vivo have centered on the source of T cells followed by repetitive stimulations in extended ex vivo culture. This requirement may be surmountable by redirection of T-cell specificity through gene transfer of chimeric receptor or engineered TCR constructs. Alternatively, the ex vivo manipulation may serve to activate, reeducate or endow T cells with enhanced or novel functions in a way that was not possible in vivo, perhaps due to masking of antigens or disease-induced immunosuppression. Central to the success of future clinical trials of engineered T lymphocytes is the determination of whether one approach or a combination of approaches should be employed and how to engineer and manufacture the respective T lymphocytes for human testing in a variety of disease settings. It may be that a combination approach, where patients are first vaccinated with peptides, proteins, DNA or peptide pulsed DCs, followed by collection of the primed T cells, expansion and reinfusion may be the best approach in settings of immune deficiency or tumor tolerance [20,42,43]. Box 1 lists some points to consider in engineering effective T lymphocyte therapies in the light of our current understanding of T lymphocyte biology and homeostasis. Unfortunately, until recently many trials were carried out before the complexity of T-cell biology and costimulation was understood. As a result, cells were propagated under what are now understood to be suboptimal conditions that impair the essential functions and long-term engraftment of T cells. Similarly, in vitro Expert Opin. Biol. Ther. (2008) 8(4) 483

10 T lymphocyte engineering ex vivo for cancer and infectious disease Box 1. Points to consider in ex vivo engineering of an effective T lymphocyte therapy. The infused T-cell population should retain properties that permit persistence and homing to tumor or lymph node Engraftment and persistence of adoptively transferred CTLs may well depend on adequate CD4 T-cell help or exogenous cytokine support Transduced gene product of transferred cells should not be immunogenic T-cell manufacturing process should avoid induction of replicative senescence Effector to target ratio: host tumor burden should not exceed killing capacity of adoptively transferred cells Tumor- or HIV-antigen-specific T cells may have been deleted or tolerized in the donor by previous chemotherapy or by the tumor itself Lack of costimulation may induce anergy or apoptosis of adoptively transferred cells activity observed in preclinical experiments may not translate to in vivo activity due to a lack of costimulation in the local tumor environment or any of several other reasons listed in Box 1. There is now a more detailed understanding of the regulation of T-cell activation and signaling, the process of immunosenescence, and a greater appreciation of the heterogeneity of T-cell memory, effector and regulatory/suppressor (Tregs) cell subsets. The implications of these findings need to be incorporated into the translation of therapeutic approaches from animal models to clinical settings. In addition, the ideal ex vivo culture process in a research setting must be adapted to clinical scale and clinically compatible reagents to achieve regulatory compliance and yet still be robust enough to support large scale trials of potent cells [109]. With improved methods of T lymphocyte engineering, a remaining barrier is widespread access and application of new technologies consistent with optimum and durable T-cell function. Frequently, reagents or materials that serve the research market are either not available in clinical grades or are prohibitively expensive. There are limited options for equipment for large-scale cell isolation, washing and concentration with sterile fluid paths. Should cell therapies be viewed as a traditional vaccine manufactured in central plants or processing facilities or more like surgery or stem cell transplantation? What federal, philanthropic and industry-funded mechanisms will ensure the clinical development, manufacturing and conduct of definitive clinical trials? These major challenges facing the field at present must be met to expand upon the promise of early successful trials. Demonstrated clinical benefit in randomized clinical trials will then justify the regulatory approval for licensure of customized T lymphocyte therapies. Acknowledgements The author would like to acknowledge helpful discussions and assistance from C June, J Riley, R Carroll, A Chew, G Binder and D Powell. Declaration of interest BL Levine is a consultant for GE Healthcare. 484 Expert Opin. Biol. Ther. (2008) 8(4)

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15 Levine Dropulic B, Carter B, editors. Hoboken, NJ, John Wiley & Sons; Rapoport AP, Levine BL, Badros A, et al. Molecular remission of CML after autotransplantation followed by adoptive transfer of costimulated autologous T cells. Bone Marrow Transplant 2004 ;33(1): Andreadis C, Levine BL, Nasta SD, et al. Adaptive immunotherapy with autologous CD25-depleted and CD3/CD28-costimulated T-cells enhances lymphocyte recovery after cyclophosphamide fludarabine chemotherapy in patients with low-grade follicular lymphoma. Blood 2006 ;108(11): Levine BL, Bernstein WB, Aronson NE, et al. Adoptive transfer of costimulated CD4(+) T cells induces expansion of peripheral T cells and decreased CCR5 expression in HIV infection. Nat Med 2002 ;8(1): Kohn DB. Lentiviral vectors ready for prime-time. Nat Biotechnol 2007 ;25(1):65-6 Affiliation Bruce L Levine PhD The University of Pennsylvania School of Medicine, Hospital of the University of Pennsylvania, Department of Pathology and Laboratory Medicine, M6.40 Maloney, 3400 Spruce Street, Philadelphia, PA , USA Tel: ; Fax: ; LevineBL@mail.med.upenn.edu Expert Opin. Biol. Ther. (2008) 8(4) 489

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