Adoptive T-cell transfer in cancer immunotherapy

Transcription

1 Immunology and Cell Biology (2006) 84, doi: /j x Special Feature Adoptive T-cell transfer in cancer immunotherapy SIOK-KEEN TEY, CATHERINE M BOLLARD and HELEN E HESLOP Center for Cell and Gene Therapy, Baylor College of Medicine, The Methodist Hospital and Texas Children s Hospital, Houston, Texas, USA Summary Adoptive T-cell therapy has definite clinical benefit in relapsed leukaemia after allogeneic transplant and in Epstein Barr virus-associated post-transplant lymphoproliferative disease. However, the majority of tumour targets are weakly immunogenic self-antigens and success has been limited in part by inadequate persistence and expansion of transferred T cells and by tumour-evasion strategies. Adoptive immunotherapy presents the opportunity to activate, expand and genetically modify T cells outside the tolerising environment of the host and a number of strategies to optimize the cellular product, including gene modification and modulation of the host environment, in particular by lymphodepletion, have been developed. Key words: adoptive immunotherapy, bone marrow transplantation, gene therapy, neoplasm, T lymphocyte. Introduction The ability of adoptive cellular immunotherapy to restore immunity and eradicate tumours has been shown in numerous animal models. The first evidence of efficacy in humans was reported in the early 1990s, when several studies reported that patients with relapsed chronic myeloid leukaemia after allogeneic bone marrow transplantation (BMT) could be reinduced into complete and durable remission by the infusion of donor mononuclear cells from the original HLA-identical sibling marrow donor. 1,2 Around the same time, two studies showed that adoptive transfer of ex vivo-expanded antigen-specific T cells could reconstitute immunity against CMV 3 and Epstein Barr virus (EBV) 4,5 in immunosuppressed patients following BMT. The clinical benefit of the latter also extended to EBV-associated lymphoproliferative disease, thereby providing proof of the concept that antigen-specific T cells can eradicate established tumours. 6 These successes notwithstanding, the translation of preclinical studies and animal models into efficacious treatment for human malignancies has proved challenging. Advances in our understanding of T-cell antigen recognition, costimulation, trafficking, expansion, persistence and memory, and insights into tumour-evasion strategies and the tolerogenic host environment have provided clues for means to enhance the effectiveness of adoptive cellular therapy. Selecting and expanding antigenspecific T cells ex vivo can circumvent some of the regulatory and tolerising effects of the tumour environment that are seen in active vaccination. The host environment can also be manipulated before T-cell infusion to potentiate the antitumour response. In addition, T cells could be manipulated ex vivo before Correspondence: Dr Helen E Heslop, Center for Cell and Gene Therapy, Baylor College of Medicine, The Methodist Hospital and Texas Children s Hospital, 1102 Bates Street, Suite 1120, Houston, TX 77030, USA. hheslop@bcm.tmc.edu Received 12 February 2006; accepted 15 February adoptive transfer to enhance their numbers, specificity, antigen avidity and effector function. Target cancer antigens Cancer antigens that can be targeted in T-cell immunotherapy fall under several broad categories (Table 1). First, cancer-testis antigens are a group of tumour antigens that in health are restricted to germ cells of the testis, with occasional expression in ovary and trophoblast. 7 They can be aberrantly expressed in a broad range of tumours, particularly melanoma, sarcoma, lung cancer, breast cancer, prostate cancer and some other epithelial cancers. Examples of cancer-testis antigens include the family of MAGE genes (MAGE-1 to 10), BAGE, GAGE, SSX-1 to 9 and NY-ESO-1. 8 Second, antigens present in normal cells may be targeted if they are significantly overexpressed in malignant cells or if the normal cells involved are not functionally critical. Examples of overexpressed antigens include proteinase 3 and WT-1, which are overexpressed in leukaemia cells, and melanoma differentiation antigens, which include MART-1 and gp100, that are also present in normal melanocytes. 9,10 These antigens are frequently the target of melanoma-reactive T cells and autoimmunity in the form of vitiligo, or less frequently uveitis, can occur. 11,12 Third, cancer antigens that are unique to malignant cells as a result of somatic mutation during malignant transformation can be targeted. An example is the joining region of the BCR-ABL fusion protein in chronic myeloid leukaemia, which is a target for both CD4 1 and CD8 1 T cells. 13,14 Antigens from this group are probably the most ideal as they are specific for tumour cells and are likely to be crucial for malignant transformation. Fourth, antigens from oncogenic viruses, particularly EBV, can be targeted. 5,15 Finally, minor histocompatibility antigens can be targeted in the setting of allogeneic haematopoietic stem cell transplantation. These are naturally processed peptides from normal cellular proteins that may differ between donor and recipient and are usually targets for both graft-versus-tumour effect and graft-versus-host disease (GVHD). The lineage Ó 2006 The Authors Journal compilation Ó 2006 Australasian Society for Immunology Inc.

2 282 S-K Tey et al. Table 1 Target antigen in adoptive immunotherapy Category Example Tumour expression Reference Cancer-testis antigen NY-ESO-1 Melanoma, ovarian cancer, lung Zendman et al. 8 and Zhao et al. 67 cancer and breast cancer Overexpressed antigens Proteinase-3 Chronic myeloid leukaemia Molldrem et al. 9 WT-1 Acute leukaemia, renal cell carcinoma, Gao et al. 10 advanced breast cancer, ovarian cancer and melanoma Differentiation antigen MART-1, gp100 Melanoma Dudley et al. 1 and Yee et al. 74 Antigen arising from BCR-ABL fusion protein Chronic myeloid leukaemia ten Bosch et al. 13 and Yotnda et al. 14 somatic mutation Viral antigens EBNA3A (immunodominant) EBV-associated post-transplant Heslop et al. 5 and Rooney et al. 6 lymphoproliferative disease LMP1 and LMP2 (subdominant) EBV-associated Hodgkin s lymphoma and nasopharyngeal carcinoma Gottschalk et al. 56 and Bollard et al. 57 Minor histocompatibility antigen HA-1 and HA-2 (in allogeneic stem cell transplantation) Haematologic malignancies Marijt et al. 16 EBV, Epstein Barr virus; LMP, latent membrane protein. restriction of some minor histocompatibility antigens, particularly haematopoietic lineage restriction, provides a therapeutic window for graft-versus-leukaemia (GVL) effect without GVHD. 16 Adoptive T-cell therapy in the setting of allogeneic haematopoietic stem cell transplantation Allogeneic BMT is the most widely used and most potent form of adoptive cellular immunotherapy. Although the mechanism of leukaemia eradication is in part attributable to the ablative doses of chemotherapy and radiotherapy used in conditioning, the role played by the GVL effect has been described in animal models for many decades. The initial clinical evidence to support GVL came from observations in the 1970s that patients who developed GVHD had a lower incidence of leukaemia relapse compared with those who did not develop GVHD. 17 The critical role played by T cells was underscored by the finding that patients receiving T-depleted grafts, with or without GVHD, have significantly higher relapse rates than patients who received non-t-depleted grafts even if the latter did not develop GVHD. 18 Furthermore, patients with relapsed chronic myeloid leukaemia have high response rates to donor lymphocyte infusions from the original HLA-identical sibling marrow donor. 1,2,23,24 (Table 2). Donor leucocyte infusion could also eradicate EBV-associated post-transplant lymphoproliferative Table 2 Clinical studies of adoptive T-cell therapies Type of T cell Clinical application Reference Unmanipulated T cell Allodepleted T cell T cells non-specifically activated ex vivo Antigen-specific T cells Relapse or residual disease after allogeneic Drobyski et al. 1 and Kolb et al. 2,23,24 haematopoietic stem cell transplantation To reduce risk of relapse and infection after Amrolia et al., 27 Andre-Schmutz et al. 28 and allogeneic haematopoietic stem cell Solomon et al. 31 transplantation with reduced risk for GVHD Adjuvant therapy after resection of hepatocellular Takayama et al. 35 and Leemhuis et al. 40 carcinoma and salvage therapy for multiply relapsed lymphoma Accelerates lymphocyte recovery after autologous Laport et al. 38 and Rapoport et al. 39 haematopoietic stem cell transplantation; used in conjunction with vaccination to accelerate immune reconstitution To introduce suicide gene into T cells as a safety Bonini et al. 41 switch in allogeneic haematopoietic stem cell transplantation, used to treat relapse haematologic malignancy post-transplantation EBV-specific CTL to prevent and treat EBVassociated post-transplant lymphoproliferative and Haque et al. 53 Heslop et al., 5 Rooney et al., 6 Comoli et al. 52 disease EBV-specific CTL for treatment of relapsed or Straathof et al. 15 and Bollard et al. 55 refractory EBV-associated Hodgkin s Lymphoma and nasopharyngeal carcinoma LMP-1-specific or LMP-2-specific T cells for Gottschalk et al. 56 and Bollard et al. 57 Hodgkin s lymphoma Treatment of metastatic melanoma Dudley et al. 11 and Rosenberg and Dudley 78 Chimeric receptor-transduced T cells CD20 chimeric receptor transduced T cells for non-hodgkin s lymphoma Wang et al. 73 EBV, Epstein Barr virus; GVHD, graft-versus-host disease; LMP, latent membrane protein.

3 Adoptive T-cell therapy 283 Table 3 Strategies to increase effectiveness of adoptive immunotherapy Barrier Potential solution Example Reference Tolerance to self-antigens resulting in low number of T cells or T cells with low avidity Gene modification to redirect antigen specificity ab-tcr specific for HLA-2/MART-1 Chimeric T-cell receptor Inadequate T-cell expansion in vivo Dual-specific T cells EBV-specific CTL or allogeneic CTL redirected to recognize tumour antigens by gene modification Exogenous cytokine IL-2 in conjunction with infusion of CD8 1 tumour-specific CTL Lymphodepletion Fludarabine and cyclophosphamide Inadequate homing Inhibitory factors expressed in tumours Tumour evasion by downregulation of antigen presentation Combine with vaccination Gene modification to express chemokine receptor Gene modification to confer resistance Recognition of target antigens independent of HLA ab-tcr, ab-t-cell receptor; EBV, Epstein Barr virus. conditioning before T-cell infusion Antipneumococcal vaccination in conjunction with polyclonal activated T cells (not yet tested in tumour model) Clay et al. 66 Dotti and Heslop, 64 Sadelain et al. 65 and Wang et al. 73 Rossig et al. 71 and Kershaw et al. 72 Yee et al. 74 Dudley et al. 11 and Rosenberg and Dudley 78 Rapoport et al. 39 Chemokine receptor CXCR2 Kershaw et al. 81 Dominant negative TGF-b receptor T cells expressing IL-12 (Flexi IL-12) Chimeric T-cell receptors Polyclonal CD3 1 CD56 1 recognizing tumour cells via NKG2D Bollard et al. 82 Wagner et al. 83 Dotti and Heslop, 64 Sadelain et al. 65 and Wang et al. 73 Leemhuis et al. 40 disease (PTLD) following allogeneic BMT. 19 More recent studies have identified potential target antigens for the GVL response with an increase in T cells specific for proteinase 3 in patients with chronic myeloid leukaemia responding after BMT 9 and T cells specific for the haematopoiesis-restricted minor histocompatibility antigens 1 and 2 (HA-1 and HA-2) in patients responding to donor lymphocyte infusion. 16 In recent years, a graft-versus-tumour effect has also been reported in solid tumours, particularly metastatic renal cell carcinoma, where up to 57% of patients responded, including rare cases of durable complete responses. 20,21 The increased interest in GVL has led to the development of non-myeloablative conditioning regimens using immunosuppressive doses of chemotherapy with or without radiotherapy to allow donor engraftment and GVL effects without the toxicity of myeloablative chemoradiotherapy. 22 The response to donor lymphocyte infusion or non-myeloablative allogeneic stem cell transplantation is usually delayed, with median time to response of 4 to 6 months. 21,23 Such approaches are therefore not effective in patients with advanced, rapidly progressing malignancy. It should also be pointed out that the response to donor leucocyte infusion is highly dependent on the type and stage of leukaemia: chronic myeloid leukaemia has the highest response rate, acute myeloid leukaemia has only modest response rates and acute lymphoblastic leukaemia rarely responds. 23,24 The major drawback of donor lymphocyte infusion is the significant risk of GVHD, which is the most important source of treatment-related mortality. Given that the frequency of tumour or viral antigen-specific T cell is in most cases considerably lower than that of alloreactive T cells, it is necessary to expand antigen-specific T cells ex vivo not only to enhance antitumour activity but also to separate GVHD from a graft-versus-tumour effect. 25 An alternative method to separate graft-versus-host from graft-versustumour effect is to selectively deplete alloreactive donor T cells ex vivo. Selective depletion of alloreactive T cell in allogeneic haematopoietic stem cell transplantation Both the GVHD and the GVL effects of donor lymphocyte infusion are to some extent dose dependent. 26 The ability to selectively attenuate alloreactivity will allow the infusion of a higher total T-cell dose and accelerate immune reconstitution. Selective allodepletion is carried out by removing donor T cells that express activation markers following coculture with non-leukaemic recipient cells. Activation markers investigated include surface antigens, in particular CD25 27,28 and CD69, 29 and the increased sensitivity of activated T cells to photosensitising dyes. 30 A number of phase I/II studies using immunotoxin directed against CD25 have shown the feasibility of this approach with accelerated reconstitution of virusspecific and total T-cell numbers with low rates of severe GVHD. 27,28,31 Relapse rates remained high in these studies, which is likely a reflection of the high-risk nature of the patient population. Preclinical studies evaluating cytotoxic 32 and helper 33 T-cell precursor frequencies against leukaemic cells and the frequency of HLA-A2-PR1 tetramer directed at a candidate myeloid-associated tumour antigen, 34 proteinase 3, suggest that antitumour activity is retained in allodepleted T cells. T cells non-specifically activated ex vivo T cells can be expanded ex vivo through polyclonal activation using phytohaemagglutinin, OKT3 (anti-cd3 antibody) or a combination of OKT3 and anti-cd28 antibody and several groups have evaluated whether such expanded T cells might have antitumour activity (Table 2). Such expanded cells have also been mainly used in the autologous setting to treat relapse or to augment T-cell function against minimal residual disease. In a randomized controlled trial in patients undergoing curative liver resection for hepatocellular carcinoma, patients who received autologous expanded T cells had lower rates of recurrence. 35

4 284 S-K Tey et al. Over the past few years, several studies have correlated faster recovery of lymphocyte counts with improved outcome after autologous transplant for both non-hodgkin s lymphoma and Hodgkin s disease. 36,37 This has led to a phase I study evaluating the infusion of autologous CD3/CD28-activated cells after a CD34-selected stem cell graft. 38 Preliminary results suggest that this approach is associated with rapid recovery of lymphocyte counts. 38 The investigators subsequently combined this approach with a 7-valent pneumococcal conjugate vaccine in a phase 1/2 study of patients undergoing autologous stem cell transplantation for myeloma. 39 A four-way randomization assigned patients to receive T-cell infusions either on day 12 or on day 100 after transplant and either one pre-transplant and two post-transplant vaccinations or two post-transplant vaccinations only. Patients who received vaccine-primed T cells early after transplant had better antipneumococcal humoral response and better CD4 1 proliferative response to the vaccine carrier protein than other treatment groups. This study provides an insight into how vaccination and adoptive cellular therapy can be effectively combined in the setting of lymphopenia and may serve as a basis for the more challenging task of targeting tumour antigens. Another strategy uses IFN-g, IL-2 and OKT3 to generate large number of CD3 1 CD56 1 cytokine-induced killer T cells, which are a unique population of CTL with a characteristic CD3 1 CD56 1 phenotype. These cells can mediate non-mhcrestricted cytotoxicity through NKG2D. In a phase I trial of patients with relapsed lymphoma, there was no toxicity and transient response was observed in a minority of patients. 40 Polyclonal T-cell activation also provides a means for retrovirus transduction, which is efficient only in actively dividing cells. Allogeneic polyclonal T cell transduced with the HSV1- thymidine kinase (HSV-tk) suicide gene has been tested in clinical trials. Transduced cells could mediate GVL effect and reconstitute EBV immunity and could be eliminated by ganciclovir in the event of life-threatening GVHD. 41 The drawbacks of this system include the reduced immune function of transduced cells, 42 the immunogenicity of HSV-tk 43 and the occurrence of truncated HSV-tk due to cryptic splice donor and acceptor sites that render the cells resistant to ganciclovir. 44 Addition of CD28 costimulation to CD3 activation helps to preserve the immune competence of the T cells. 45,46 Alternative suicide genes based on the dimerization of Fas 47 or caspase 9 48 in the apoptotic pathway have been developed to circumvent the problem of immunogenicity of HSV-tk. Adoptive immunotherapy using antigen-specific T cells Antigen-specific T cells targeting viral antigen The use of ex vivo-expanded antigen-specific T cells was first reported by Riddell et al., who showed that CD8 1 T-cell clones specific for CMV given prophylactically postallogeneic transplantation could reconstitute CMV-specific CTL in vivo. 3 Our group evaluated the use of antigen-specific T cell in cancer immunotherapy using EBV-associated PTLD as a model (Table 2). PTLD is a life-threatening disease that arises in recipients of allogeneic haematopoietic stem cell transplants or solid organ grafts. PTLD, particularly those arising in the first year post-transplantation, are usually EBV driven and express all nine latent-cycle EBV antigens, including the immunodominant Epstein Barr Nuclear Antigens (EBNA3), thus making good targets for EBV-specific CTL. 49 To generate antigen-specific CTL, it is necessary to have a good source of APC to effectively present the antigen to T cells. EBV-infected lymphoblastoid cell lines (LCL) generated by infecting normal peripheral blood B cells with EBV function as excellent APC as they present EBV antigens efficiently and express high levels of costimulatory molecules. Provided the donor is immune to EBV, EBV-specific CTL can be generated and expanded ex vivo using LCL as stimulator cells. 4 This strategy generates polyclonal EBV-specific CTL containing both CD4 1 and CD8 1 cells, which are able to reconstitute EBV immunity and reduce high viral loads when transferred to haemopoietic stem cell transplant recipients at high risk of developing PTLD. 4,6 EBV-CTL also appeared to prevent progression to EBV lymphoma because none of the 60 patients who received prophylactic CTL developed this malignancy, compared with 11.5% of controls. 6 In addition, five of six patients who received CTL as treatment for overt lymphoma achieved complete remission. 6,50 In some patients, the ex vivoexpanded CTL were gene marked with neomycin-resistant gene to distinguish them from endogenous T cells. Using this technique, the gene-marked CTL were found to persist for up to 7 years and expand in response to EBV antigen in vivo and in vitro 5 and were present at sites of disease. 6 A similar strategy has been applied to PTLD arising after solid organ transplantation using either autologous 51,52 or allogeneic 53 EBV-specific CTL with reports of immune and clinical responses. Although other EBV-associated malignancies, particularly Hodgkin s lymphoma and nasopharyngeal carcinoma, are also potential targets for EBV-specific CTL immunotherapy, they are likely to be less responsive to such therapy. These tumours arise in immunocompetent hosts and express a more restricted pattern of weakly immunogenic EBV antigens expressing only EBNA1, latent membrane protein (LMP) 1 and LMP2 in a pattern known as type II latency. 54 Furthermore, at least in Hodgkin s lymphoma, the tumours are armed with a range of tumour-evasion strategies. Nevertheless, the subdominant EBV antigens may serve as targets for immunotherapy approaches and in phase I studies, we have evaluated the use of autologous EBV-specific CTL for patients with EBV-positive type II latency tumours. 15,55 Out of six patients with refractory nasopharyngeal carcinoma treated with autologous EBV-specific CTL, two achieved complete remission and one achieved partial remission. 15 In relapsed Hodgkin s lymphoma, 2 out of 11 patients with measurable disease at the time of EBV-specific CTL infusion achieved complete remission and one achieved partial remission. 55 Given that Hodgkin s lymphoma and nasopharyngeal lymphoma express mainly the subdominant LMP1 and LMP2 antigens, skewing the anti-ebv response towards these antigens by overexpressing them on dendritic cells could potentially increase their effectiveness and clinical trials are currently underway. 56,57 Targeting non-viral tumour antigens Developing immunotherapy strategies targeting non-viral tumour antigens is more challenging. Many of these tumour antigens are self-antigens and as a result of self-tolerance mechanisms, CTL against these antigens are of low frequency, have

5 Adoptive T-cell therapy 285 low T-cell receptor avidity or are anergic. In the allogeneic setting, preclinical studies have shown that it is possible to generate leukaemia-specific CTL using dendritic cells as APC 58 and a report has detailed a patient with relapsed chronic myeloid leukaemia who attained complete remission following infusion of leukaemia-specific CTL generated from the original allogeneic stem cell donor. 59 In the autologous setting, dendritic cells pulsed with tumour peptide epitopes have been used as APC. Using this approach, CTL specific for melan-a peptide, a melanocyte differentiation antigen, could be generated from the peripheral blood mononuclear cells of HLA-A2-positive patients and clinical responses were seen in a small minority of patients. 60,61 Another approach is to culture several independent lines of tumour-infiltrating lymphocytes (TIL), screen them for recognition of melanoma cells using IFN-g cytokine secretion assay and expand the selected lines with OKT3. The TIL lines generated usually contain both CD4 1 and CD8 1 T cells and have been shown to have specificity for MART-1 or gp100 epitopes. 11,62 However, clinical trials using TIL and high-dose IL-2 in nonlymphodepleted patients resulted in objective response in roughly a third of patients, with most responses being transient. 63 Retargeting T cells to tumour antigens by gene modification An alternative strategy to target weakly immunogenic tumour antigen is to alter the antigen specificity of T cells by gene modification. T cells can be transduced with genes encoding a different antigen receptor: either physiologic ab-t-cell receptor (ab-tcr) heterodimers or artificial chimeric T-cell receptors. 64,65 Several ab-tcr heterodimers specific for tumour antigens have been cloned, either from autologous CTL culture 66,67 or from a HLA-A*0201 transgenic mouse model, whereby high-avidity murine TCR can be cloned. 68,69 Although some inefficiency from heterologous pairing between endogenous and transduced TCR chains is expected, primary T cells transduced with cloned a and b TCR chains do mediate antitumour activity in vitro. 66,68,69 The main drawback of cloned TCR is HLA restriction, which limits its use to a subset of patients with a particular HLA type. Chimeric T-cell receptors recognize target antigens on tumour cell surface through an extracellular domain, which is most commonly derived from the variable domains of an immunoglobulin. The extracellular domain is linked via a spacer to the intracellular signalling domain, which triggers T-cell activation. The latter consists of either the f-chain of the TCR complex or the g-chain from FceRI. One or more costimulatory endodomains, such as CD28 and OX40, can be added to promote full T-cell activation. 64,65,70 Full activation of the transduced T cells can also be provided through engagement of the native TCR. This can be achieved by transducing antigen-specific CTL instead of primary T cells. For example, EBV-specific CTL transduced with a chimeric T-cell receptor specific for GD2 antigen expressed by neuroblastoma cells could expand in the presence of EBV-lymphoblastoid cell in vitro and could lyse both EBV-lymphoblastoid cells and neuroblastoma cells. 71 In an in vivo mouse model, alloreactive T cells transduced with a chimeric receptor recognizing folate-binding protein could expand and exert antitumour effect in response to immunization with allogeneic cells. 72 A number of target antigens for chimeric T-cell receptors have been studied in vitro and in mouse models and phase I clinical trials are in progress. 73 Because chimeric T-cell receptors recognize native antigens, recognition is not HLA-restricted, which circumvents the problem of tumour evasion through downregulation of HLA expression. Furthermore, unlike conventional TCR, chimeric T-cell receptors can be made to recognize carbohydrates and glycolipid antigens. Strategies to increase the effectiveness of adoptive cellular immunotherapy Although adoptively transferred T cells can fairly consistently mediate clinically significant therapeutic effects in specific settings, the majority of clinical trials have shown only modest and variable response rates. 5,6 Effective T-cell therapy requires the T cells to persist, expand, traffic and home to tumours and mediate effector function. Harnessing the antitumour effects of T cells will require strategies to overcome deficiencies intrinsic to the host immune system on the one hand and the evolution of tumour-evasion mechanisms on the other (Table 3). Problems intrinsic to the host include tolerance to self-antigen, the presence of network of regulatory T cells, T-cell anergy and impaired immune function from previous chemoradiotherapy. Tumour-evasion strategies include loss of tumour-antigen expression, downregulation of antigen presentation, downregulation of death receptors and secretion of immunosuppressive factors. Enhancing T-cell persistence and expansion Several approaches to enhance the in vivo T-cell persistence and expansion have been studied. Administration of IL-2 in conjunction with adoptive transfer of antigen-specific T cell can prolong the in vivo survival of infused T cells, particularly if the T-cell product lacks CD4 1 cells. 74 However, systemic administration of IL-2 is toxic, the clinical benefit is minimal 74 and in vivo IL-2 contributes to the growth of CD4 1 CD25 1 regulatory T cells. 75 Targeting IL-2 to the desired T-cell population could be achieved though transduction with gene encoding IL-2 76 or a chimeric GM-CSF/IL-2R receptor 77 but a concern would be the potential for transformation into lymphoproliferative disorder. More recently, lymphodepletion before adoptive transfer has emerged as a promising strategy. 11,78 This manoeuvre enhances the efficacy of adoptive therapy by facilitating homeostatic T- cell proliferation, possibly in part through reduced competition for cytokines and by removal of regulatory T cells. 79 In homeostatic proliferation, low-affinity antigens such as self-antigens may be sufficient to trigger proliferation and activation. 79 In a study of patients with metastatic melanoma, immunodepleting doses of fludarabine and cyclophosphamide were given before the infusion of autologous antigen-specific T cells with concomitant high-dose IL-2. 11,78 Approximately half of the treated patients showed clinical responses. 78 In patients with skewed expression of the beta chain variable region (Vb) of TCR within the infused T cells, the same Vb skewing was present in the peripheral blood at 1 week after cell transfer. 11 Marked Vb skewing was persistent at 4 months

6 286 S-K Tey et al. in two of the responding patients, one of who also displayed very high levels of circulating HLA-A2/MART-1 tetramerpositive cells. The lymphodepleted host environment therefore promotes the survival, persistence and expansion of infused T cells. Further studies involving a larger cohort of patients showed that persistence of infused cells at 1 month, as shown by Vb skewing, is significantly associated with disease response, thus validating the concept that in vivo T-cell number and expansion is important for success. 80 Promoting T-cell trafficking and homing to tumour sites The presence of transferred T cells in the peripheral blood alone is unlikely to be effective if these T cells do not localize to tumour sites. T-cell migration to tumour sites can be facilitated by transducing them with appropriate chemokine receptors. One potential candidate is the chemokine receptor CXCR2, which binds to CXCL1 expressed by tumour cells. 81 In vivo testing will be required to test the effect of transduced CXCR2 on T- cell migration, function and recirculation. Increasing resistance to inhibitory factors Tumours can evade immune surveillance through the secretion of inhibitory cytokines, either by the malignant cells themselves or by non-malignant tumour-infiltrating cells. One of the most commonly involved cytokines is TGF-b that inhibits T-cell proliferation and promotes tolerance. One means of overcoming this inhibition is to render adoptively transferred cells resistant to TGF-b. In a preclinical study, EBV-specific T cells transduced with a dominant negative TGF-bII receptor were shown to be insensitive to the inhibitory effects of TGF-b although their phenotype, function and growth characteristics were otherwise unchanged. 82 This strategy could therefore be useful in Hodgkin s lymphoma, which is known to secrete TGF-b and has a Th2 environment. Another strategy to overcome the Th2 environment in Hodgkin s lymphoma is to deliver IL-12 locally at the tumour site using gene-modified EBV-specific CTL. 83 EBVspecific CTL transduced with a retrovirus vector expressing the p40 and p35 subunits of IL-12 as a single molecule (Flexi-IL- 12) produce IL-12 following antigenic stimulation. This results in increased production of Th1 cytokines, reduction in the Th2 cytokines and resistance to the effects of TGF-b. 83 therapy. 85 Targeting tumour antigens that are critical to tumour transformation, in addition to using polyclonal CTL, may circumvent part of the problem. Practical considerations for adoptive cellular therapy Currently, cellular therapy remains a specialized area of therapeutics that is relatively expensive and resource intensive. Cell processing facilities are required to adhere to good manufacturing practice and other local regulatory requirements. Innovations that produce promising results in preclinical studies may sometimes prove too expensive or difficult to scale up. Furthermore, clinical-grade reagents may not always be available and most cellular products are tailormade for individual patients. The challenge of bringing benchtop research to the bedside has been recognized by national bodies. To this end, the National Institutes of Health in the USA recently designated specialized centres for product assistance to provide consulting, manufacturing and regulatory support to other research centres within the country. 86 Conclusions There is now a large body of evidence supporting the ability of adoptively transferred T cells to eradicate tumours in humans. However, consistent and definitive benefit is currently achieved only in specific settings such as EBV-associated malignancies, relapsed leukaemia after stem cell transplantation and in some cases of metastatic melanoma. Aside from donor lymphocyte infusion after stem cell transplantation, adoptive cellular therapy is largely conducted in the context of clinical trials. Ongoing basic, translational and clinical research in optimizing both the cellular product and host environment will continue to improve adoptive immunotherapy approaches. Acknowledgements This work was supported by the National Institutes of Health grants PO1 CA94237, the GCRC at Baylor College of Medicine (RR00188), a Specialized Center of Research Award from the Leukemia Lymphoma Society, a Doris Duke Distinguished Clinical Scientist Award to H. E. H. and a Young Investigator Scholarship from the Haematology Society of Australia and New Zealand to S.-K. T. Tumour evasion by loss of antigen expression or presentation By nature, antigen-targeted therapies, both cellular and humoral, are susceptible to tumour evasion by loss of antigen expression. In T-cell therapy, this can be caused either by the loss of antigen expression or by the downregulation of antigen processing pathways. This is a particular concern if T-cell lines of restricted specificity are infused but can occur even when polyclonal CTL lines were used. We have described a case of EBV-associated PTLD that was resistant to EBV-specific CTL through the deletion of the immunodominant viral EBNA3B antigen. 84 Loss of tumour antigens or HLA class I expression has also been reported in relapsed melanoma after initial successful vaccine References 1 Drobyski W, Keever C, Roth M et al. Salvage immunotherapy using donor leukocyte infusions as treatment for relapsed chronic myelogenous leukemia after allogeneic bone marrow transplantation: efficacy and toxicity of a defined T-cell dose. Blood 1993; 82: Kolb H, Mittermuller J, Clemm C et al. Donor leukocyte transfusions for treatment of recurrent chronic myelogenous leukemia in marrow transplant patients. Blood 1990; 76: Riddell SR, Watanabe KS, Goodrich JM, Li CR, Agha ME, Greenberg PD. Restoration of viral immunity in immunodeficient humans by the adoptive transfer of T cell clones. Science 1992; 257:

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