WT1-targeted immunotherapy of leukaemia

Blood Cells, Molecules, and Diseases 33 (2004) 288 – 290 www.elsevier.com/locate/ybcmd

WT1-targeted immunotherapy of leukaemia

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S. Xue, L. Gao, R. Gillmore, G. Bendle, A. Holler, A.M. Downs, A. Tsallios, F. Ramirez, Y. Ghani, D. Hart, S. Alcock, A. Tranter, H.J. Stauss*, E. Morris Tumour Immunology Section, Department of Immunology, Imperial College London, London W12-0NN, UK Submitted 6 August 2004 Available online 25 September 2004 (Communicated by M. Lichtman, M.D., 12 August 2004)

Abstract Since malignant cells are derived from normal cells, many tumour-associated antigens are also expressed in normal tissues. For examples, WT1 is expressed at elevated levels in most leukaemias, but it is also expressed at reduced levels in normal CD34+ haematopoietic stem cells and in progenitor cells of other tissues. Antigen expression in normal tissues is likely to trigger immunological tolerance and thus blunt T cell responses. This could explain the observation that WT1 vaccination in mice frequently fails to stimulate high avidity cytotoxic T cell responses. In order to circumvent tolerance, we have isolated from HLA-A2-negative donors high avidity CTL specific for HLA-A2presented peptide epitopes of WT1. These allorestricted CTL efficiently kill HLA-A2-positive leukaemia cells but not normal CD34+ haematopoietic stem cells. However, adoptive cellular therapy with allorestricted CTL could only be performed in leukaemia patients rendered tolerant to the infused CTL by prior allogeneic stem cell transplantation. In order to circumvent this limitation, we propose to exploit the TCR of allorestricted CTL as therapeutic tool. TCR gene transfer can be used to take advantage of the specificity of allorestricted CTL and transfer it to patient CTL, while avoiding the transfer of immunogenic alloantigens from the donor CTL to the patient. D 2004 Elsevier Inc. All rights reserved. Keywords: Immunotherapy; CTL; TCR; Cancer; Leukaemia; WT1

Introduction

Allogeneic T cell therapy The infusion of allogeneic T lymphocytes into patients undergoing allogeneic stem cell transplantation is a therapeutic strategy in humans with proven curative potential of malignancies. It is now well documented that donor lymphocytes can eliminate leukaemia cells [1] and that this graft-versus-leukaemia (GvL) effect is dependent upon $ This paper is based upon a presentation at a Focused Workshop on Haploidentical Stem Cell Transplantation sponsored by The Leukemia and Lymphoma Society held in Naples Italy from July 8–10, 2004. * Corresponding author. Tumour Immunology Section, Department of Immunology, Hammersmith Hospital, Imperial College London, London W12-0NN, UK. Fax: +44 208 383 2788. E-mail address: [email protected] (H.J. Stauss).

1079-9796/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bcmd.2004.08.018

histoincompatibility between donor and recipient [2]. The fact that allogeneic histocompatibility antigens represent the major target for donor lymphocytes is the basis for the most serious side effect of allogeneic T cell therapy, graft-versushost disease (GvHD). GvHD is primarily triggered by host antigen presenting cells (APC) presenting alloantigens to donor lymphocytes [3,4]. Occasionally, GvL can occur without GvHD, indicating that the two immune-mediated effects are not always linked. Several strategies have been developed to reduce the GvHD risk of donor lymphocytes. Host-specific tolerance in donor T cells can be achieved by costimulation blockade using CTLA-4-Ig [5] or disruption of the CD40/CD40L pathway [6,7] or the stimulation of donor T cells with host antigen-presenting cells in the presence of IL-10 [8,9]. These approaches have the potential to induce host-specific tolerance, or in the case of CD40L blockade and the IL-10 stimulation protocol, regulatory T

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cells that can suppress antihost immune responses by naRve donor T cells. It is also possible to deplete host-reactive donor T cells using antibodies against T cell activation markers such as CD25 [10] and CD69 [11]. Finally, dsuppressorT cells such as CD4/CD25 T cells [12,13], NKT cells [14] and veto cells [15] have been shown to decrease the risk of GvHD in animal models. It is likely that the described strategies will lead to GvHD control without general immune suppression.

Increasing the specificity of allogeneic T cells The inactivation of alloreactive donor T cells is expected to maintain donor T cells specific for leukaemia-associated antigens, although the frequency of leukaemia-specific donor T cells would be expected to be low. It is therefore essential to identify leukaemia-associated target antigens that can be effectively recognised by T cells. In the case of CML, the leukaemia-specific BRC/ABL fusion protein has been extensively studied. Frequently, peptides containing the BCR/ABL fusion sequence are not naturally presented at levels sufficient for CTL recognition, although recent studies suggest that fusion peptides can be presented by HLA-A3 molecules [16]. Haematopoietic-specific minor histocompatibility antigens have been explored as GvL targets in HLAmatched transplant settings [17–19]. These antigens would provide a target for CTL attack of patient haematopoietic cells, including leukaemic cells, but not of donor cells. Finally, proteinase 3, a myeloid-specific differentiation antigen expressed at high levels in CML, can function as CTL target in patients treated by allogeneic transplantation or IFN-a [20]. It has been observed that IFN-a-treated patients with hepatitis C also develop anti-proteinase-3 CTL (unpublished), indicating these responses are not limited to leukaemia patients.

Immunotherapy with allorestricted CTL Our laboratory has generated CTL specific for haematopoietic differentiation antigens and proteins involved in malignant transformation. The CTL targets were chosen based on known expression patterns and on their involvement in the initiation or maintenance of the malignant phenotype of transformed cells. The selected target proteins are usually expressed at elevated levels in tumour cells, but also at lower levels in normal tissues. In order to overcome possible tolerance of autologous CTL, we have developed the allorestricted CTL approach [21]. Allorestricted human CTL were raised against peptides presented by HLA-A0201 (A2) [22–25], which is the most frequent HLA class I allele in the Caucasian population. Allorestricted murine CTL were generated to explore their use for tumour immunotherapy in vivo [26]. Murine allorestricted CTL were targeted against MDM2, a transcription factor that is

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expressed at elevated levels is many tumours. In vitro, MDM2-specific CTL can selectively kill tumour cells while sparing normal cells, and in vivo they can delay tumour growth without causing damage to normal tissues. These experiments suggest that the risk of immune attack of normal tissues is relatively low, even when CTL are specific for an antigen, such as MDM2, that is expressed in most normal cells. However, preliminary data suggest that antigen expression in normal tissues is not ignored by MDM2-specific CTL. Rather than triggering immune attack and tissue destruction, it appears that antigen encounter in normal tissues can induce tolerance of MDM2-specific CTL and thus limit their in vivo efficacy in terms of tumour protection. Thus, prevention of tolerance induction of adoptively transferred CTL may be an important strategy to enhance antitumour efficacy. This may be achievable by combining adoptive therapy with repeated vaccinations, with the goal to compete with tolerogenic signals from normal tissues by stimulating and activating the CTL with immunogenic vaccine preparations. In the human system, we have generated allorestricted CTL specific for Wilms tumour antigen 1 (WT1). The WT1 protein is an attractive target for immunotherapy of leukaemia and solid cancers since elevated expression has been demonstrated in AML, CML, MDS and in breast and colon cancers. In the past, we have isolated from HLA-A2-negative donors high avidity CTL specific for a WT1-derived peptide presented by HLA-A2. In vitro analyses demonstrated that these CTL showed WT1-specific killing activity, lysed A2positive leukaemia cell lines and also killed fresh leukaemia cells isolated from patients. For example, the CTL killed leukaemic CD34+ progenitor/stem cells in CFU, LTC-IC and SCID/NOD engraftment experiments, while they did not affect the function of normal CD34+ cells in any of these functional assays. The TCR of allorestricted CTL can be used for therapy of leukaemia patients. In addition, it will be possible to use this TCR in breast cancer since approximately 80% of breast cancer cases express WT1 while normal breast tissue is WT1-negative. Retroviral gene transfer can equip patient T cells with WT1-specific TCRs that are not naturally present in the patient repertoire. The genetransduced patient T cells gain a novel specificity that allows them to effectively recognise and attack autologous tumour cells. It has been demonstrated that retroviral gene transfer can be used to direct human T cells against tumour-associated antigens that are not effectively recognised by autologous T cells [27]. In addition, transfer of TCR modified T cells into mice prevented the growth of tumour cells expressing the TCR-recognised antigen [28]. Allogeneic T cells provide a valuable source of TCRs with tumour specificity that may not be present in the repertoire of cancer patients. Isolation of such TCRs and transfer into patient T cells provides a strategy to produce effector T cells for antigen-specific immunotherapy of malignancies expressing the TCR-recognised target antigens.

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Acknowledgments This work was supported by the Leukaemia Research Fund, The Association of International Cancer Research, The Dinwoodie Trust and the Medical Research Council.

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