Dendritic cell vaccines in acute leukaemia

Best Practice & Research Clinical Haematology Vol. 21, No. 3, pp. 521–541, 2008 doi:10.1016/j.beha.2008.07.010 available online at http://www.sciencedirect.com

10 Dendritic cell vaccines in acute leukaemia Caroline Duncan *

MBBS, MRCP (Ed)

Clinical Research Fellow

Huw Roddie

MBChB, PhD

Consultant Haematologist Department of Haematology, Western General Hospital, Crewe Road South, Edinburgh EH4 2XU, UK

There is a need for novel treatment for acute leukaemia as relapse rates remain unacceptably high. Immunotherapy aims to stimulate the patient’s immune responses to recognize and destroy leukaemia cells whilst activating immune memory. The qualities of the most potent professional antigen-presenting cell, the dendritic cell (DC), can be used to stimulate leukaemia-specific cytotoxic T cells. DCs can be loaded with leukaemia antigens, or leukaemia blasts can be modified to express DC-like properties for use in vaccine therapy. This chapter will review the rationale for DC vaccine therapy, the preclinical and clinical trials to date, the barriers to successful DC vaccine therapies and the role of immune adjuncts to improve outcomes. Key words: acute leukaemia; dendritic cell; immunotherapy.

REQUIREMENT FOR NOVEL TREATMENT IN ACUTE LEUKAEMIA Acute myeloid leukaemia (AML) and acute lymphoblastic leukaemia (ALL) are heterogeneous diseases arising from clonal proliferation of neoplastic precursors in the bone marrow. A variety of prognostic factors have been identified that predict for outcome, most notably, the presence of defined cytogenetic abnormalities. Intensive combination chemotherapy treatment for acute leukaemia results in excellent remission rates. Interim analysis from the latest Medical Research Council (MRC) AML trial (AML 15) reveals that 85% of patients achieve complete remission1 (CR) as defined by <5% blasts in the bone marrow. Ninety-one percent of patients with ALL entered into the MRC UKALL XII trial achieved CR. Unfortunately, many patients relapse from a state of minimal residual disease (MRD), particularly patients in poor-risk categories. Even in the best prognostic categories, patients with AML have overall survival (OS) rates * Corresponding author. Tel.: þ44 0131 537 1182; Fax: þ44 0131 537 1172. E-mail address: [email protected] (C. Duncan). 1521-6926/$ - see front matter ª 2008 Elsevier Ltd. All rights reserved.

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of 50–55% (results from the MRC AML 10 trial2), and patients with ALL have 5-year OS of 37–53%.3 Allogeneic stem cell transplantation is associated with a lower risk of relapse compared with conventional chemotherapy, and results in improved disease-free survival rates in AML2 and ALL3 in some settings. However, significant toxicity and transplant-related mortality limit the efficacy of this procedure and may abrogate the potential benefits of a lower relapse rate. The graft-vs-leukaemia effect mediated by donor cytotoxic T lymphocytes (CTLs) that potentially recognize and destroy the residual malignant cells is thought to be the mechanism by which disease control is achieved after allogeneic transplantation. This has led to the use of reduced-intensity conditioning allogeneic transplants and donor lymphocyte infusions (DLI) to utilize CTLs without the toxicity of conventional allogeneic transplantation. However, the use of allogeneic transplantation and DLI is still limited to a small number of suitable patients, and is complicated by the negative effects of alloreactive CTLs causing graft-vs-host disease due to their lack of specificity for the malignant clone. The unique efficacy of allogeneic transplantation in preventing disease relapse has inspired research to develop other immunotherapeutic strategies that selectively recognize and destroy leukaemia cells with the aim of reducing relapse rates without the need for allogeneic transplantation. Immunotherapeutic techniques in acute leukaemia include dendritic cell (DC) vaccine therapy, adoptive T-cell transfer, gene transfer and peptide vaccination, but this chapter will focus on the key role of professional antigen-presenting DCs to stimulate CTL responses. ADVERSE IMMUNE FUNCTION IN THE LEUKAEMIC STATE There is little evidence for spontaneous anti-leukaemic immune activity in patients with acute leukaemia. This may be due to inadequate activation of the immune system by leukaemia cells and a tumour micro-environment that does not favour anti-leukaemic immunity. For T-cell activation by a DC to occur, there is a two-step process that must take place. Firstly, the T-cell receptor (TCR) must recognize and bind to the human leukocyte antigen (HLA)/peptide complex on the surface of the DC. Secondly, binding of B7 on the DC to CD28 on the T cell provides a co-stimulatory signal allowing the T cell to activate and proliferate. The absence of B7 expression and other co-stimulatory molecules4–6 on leukaemic blasts results in the engagement of T cells at the HLA/TCR complex in the absence of co-stimulation, such that the T cells are rendered anergic (Figure 1). The micro-environment in the leukaemic state is immunosuppressive.5,7 This is due to factors such as direct production of inhibitory cytokines such as tumour growth factor beta and interleukin (IL)-10 by the leukaemia cells. These cells also produce soluble factors which indirectly inhibit T-cell activation5 and T-cell signalling.8 Soluble factors in tumour supernatant from leukaemia cells generate an anti-apoptotic environment aiding survival of myeloblasts whilst inhibiting the normal apoptosis of immune cells.9 Regulatory T cells (Tregs) also suppress anti-leukaemic cytotoxic responses and are elevated in malignant states including AML.10 The adverse effects of Tregs are discussed under the section ‘Limitations of DC vaccine therapy’. Techniques to reduce Treg numbers or inhibit their actions will be discussed in the section entitled ‘Immune augmentation in vaccine therapy’. ROLE OF DCs IN IMMUNOTHERAPY DCs arise from myeloid or lymphoid precursors and circulate in the peripheral blood in immature forms where they take up antigens such as invading pathogens or leukaemia-associated

Dendritic cell vaccines in acute leukaemia 523

CD80 CD28

CTL T-cell activation

TCR

DC HLA/peptide complex

CD28

Leukaemia cell

TCR

CTL T-cell anergy

HLA/peptide complex

Figure 1. Dendritic cell (DC)/T-cell interaction is a two-step process requiring co-stimulatory molecules which are lacking on leukaemia cells.TCR, T-cell receptor; CTL, cytotoxic T lymphocyte.

antigens (LAAs) by phagocytosis. Unlike other phagocytic cells, they then process the antigens efficiently11, mature and migrate to the lymph nodes where they present antigen complexed with HLA to na€ıve T cells. The maturation process involves upregulation of HLA class I and co-stimulatory molecules, allowing the ‘two-step’ activation of T cells to occur; this second step is lacking in the unmodified leukaemic cell.4 DCs have the capacity to stimulate CD4þ T cells via class II HLA and CD8 T cells via interaction of class I HLA (via cross-priming). The activation of CD8 memory cells by DCs12 is crucial in the prevention of leukaemia relapse. DCs also stimulate natural killer cells and B cells, making them highly efficient activators of both innate and adaptive immunity.13 Theircapacity for diverse immunostimulation, their production of T-cell chemokines and their migratory abilities make DCs ideal for use in immunotherapy.12 One injection of DCs is sufficient to generate broad T-cell activity.12 In the setting of active malignancy, DCs in vivo are inadequate at stimulating CTLs effectively. This may be due to insufficient numbers of DCs, production of cytokines such as vascular endothelial growth factor14 and IL-1015 by tumour cells that inhibit maturation and activation of DCs, and low immunogenicity of tumour antigens even when presented by DCs to CTLs. DC vaccine therapy therefore relies on either generating sufficient numbers of DCs ex vivo to prime CTLs or using immunomodulatory agents to overcome deficiencies in DC and CTL function in vivo. This will be discussed in more detail in the sections on ‘Limitations of DC vaccine therapy’ and ‘Immune augmentation in vaccine therapy’. DCs for vaccine therapy can be derived ex vivo from peripheral blood monocytes using the cytokines granulocyte macrophage colony-stimulating factor (GM-CSF) and IL-4 to stimulate differentiation to DCs.16–18 DCs can also be differentiated from CD34þ cells derived from bone marrow or the harvest product from leukapheresis. Leukapheresis or large-volume venesections can also be used to harvest sufficient numbers of the scarce CD11c precursor DCs19 for use in DC vaccine therapy. PRECLINICAL TRIALS IN ACUTE LEUKAEMIA USING DCs There are many different ways to exploit the properties of DCs to stimulate the immune system. DCs can be loaded or fused with leukaemia cells resulting in

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presentation of LAAs in the context of DC-mediated co-stimulation. Alternatively, leukaemia blasts can be differentiated into AML-DCs ex vivo. These cells potentially express the features of both leukaemic blasts and immature DCs, and have been shown to stimulate CTLs in vitro. A central area of leukaemia immunotherapy research concerns whether the use of DC-based vaccines results in the effective stimulation of leukaemia-specific CTLs in vivo. DC vaccines can be divided into those where DCs are loaded with tumour antigens (either defined or undefined tumour antigens), and those where leukaemia cells are modified to express DC properties (by differentiation, fusion or genetic transfer). These DC vaccination techniques will be reviewed in more detail, with examples from preclinical trials and comparison between different forms of DC loading, differentiation and fusion. Other immunotherapy techniques in AML use direct vaccination of patients with tumour antigens or generation of specific anti-leukaemic CTLs ex vivo ready for infusion back to the subject (adoptive T-cell transfer).20 These immunotherapeutic steps do not involve direct DC vaccination and are therefore outside the scope of this chapter. The majority of research in DC vaccination is at the preclinical phase. DCs loaded with defined tumour antigens DCs can be loaded with peptides derived from specific LAAs such as MUC121 or WT1.22 These are usually synthetically manufactured after the peptide sequence has been identified. Use of LAAs in immunotherapy relies on identification of leukaemia antigens, ideally with high levels of expression on malignant cells which are either absent from nonmalignant cells or have a low level of expression. LAAs can be overexpressed in leukaemic cells (e.g. WT1), and can be neo-antigens (i.e. derived from fusion genes) or aberrantly expressed self-antigens. DCs can be loaded with the fusion product of the abnormal genetic translocations which feature in some forms of leukaemia, e.g. PML-RARA23 or DEK-CAN.24 Transfer of mRNA encoding for leukaemia antigens such as WT125 can be used. Peptides derived from LAAs such as PR126,27, WT128,29 and RHAMM30 can be vaccinated directly to subjects without a DC loading step in the hope that immature DCs take up, process and present these peptides to CTLs in vivo. Brossart et al.21 investigated the expression of MUC1 in haematological malignancies and found it to be overexpressed in 67% of AMLs (43 samples analysed) but only 33% of common ALL. Two different MUC1 peptides have been generated synthetically. DCs of healthy donors were loaded with these peptides to stimulate MUC1 peptidespecific CTLs. These CTLs showed toxicity against leukaemic cell lines and patientderived AML blasts. It was concluded that DCs loaded with MUC1 may demonstrate efficacy in clinical vaccination studies. In one study, DCs from healthy donors were loaded with WT1 peptide then co-cultured with autologous CD8þ lymphocytes to generate WT1-specific T-cell clones.22 These CTLs demonstrated lysis of leukaemia cell lines and fresh leukaemia blasts from patients in vitro. Alternatively, DCs loaded with mRNA encoding for WT125 ex vivo were also effective in stimulating an antigen-specific response, and are being studied in clinical trials for AML patients.31 Advantages and disadvantages of DCs loaded with defined tumour antigens (Table 1) Use of single peptides derived from LAAs loaded on to DCs has the disadvantage that as the disease progresses, antigen expression may change (antigen drift). Peptide-based strategies are also restricted to patients of the appropriate HLA type. Transfer of

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Table 1. Advantages and disadvantages of different generation techniques for dendritic cell (DC)-based vaccines. DC loading technique

Advantages

Disadvantages

 Induces CTL responses against leukaemia  Antigen drift/loss cells alone, not stem cells  HLA restriction (for LAA-derived peptides)  Differing responses/avidity of CTLs to peptides  T-cell exhaustion if peptide already strongly expressed DCs loaded with  Broad range of tumour antigens expressed  Difficult to induce pure apoptosis undefined tumour including undefined antigens or necrosis e overlap in stages of antigens  Less likely for antigen drift to be relevant cell death  May stimulate tolerogenic DCs/ danger signals  Broad range of antigens may induce autoimmunity AML-DCs/fusion  Combines features of both leukaemic cells  Low conversion efficiency hybrids and DCs  Broad range of antigens may induce  Broad range of tumour antigens expressed autoimmunity including unknown antigens  Less likely for antigen drift to be relevant DCs loaded with defined tumour antigens

CTL, cytotoxic T lymphocyte; AML, acute myeloid leukaemia; HLA, human leukocyte antigen; LAA, leukaemia-associated antigen.

mRNA encoding specific antigens avoids this HLA restriction. The frequency of LAA expression on malignant cells is important for determining their relevance for cellular immunotherapy. Specific LAAs such as MUC121, PR126,27,32,33 and WT122,28,29 have been identified as useful LAAs in AML; however, recently there has been more interest in newly identified antigens such as RHAMM. RHAMM is highly expressed in different leukaemias – 80% of cases of AML, chronic myeloid leukaemia (CML) and chronic lymphocytic leukaemia (CLL)30,34,35 – and is likely to be broadly applicable in immunotherapy for different types of leukaemia. The immunogenicity of peptide antigens is associated with their binding affinity to the HLA complex. The strength of the immune signal from tumour antigens affects the T-cell response; however, this is not straightforward. ‘Strong’ tumour antigens may have already been activating T cells in vivo, but their response is inadequate to prevent progression of malignancy, and further expansion of this population may not be productive.36 In contrast, vaccination strategies directed against a weaker antigen that is not induced by native immunity may incite novel T-cell responses on vaccination. DCs loaded with undefined leukaemia-cell-derived antigens DCs can be loaded with a wider range of leukaemia-cell-derived antigens derived from apoptotic37–41 or necrotic leukaemia cells, leukaemic cell lysates40,42,43 or acid-eluted peptides.44 Transfer of tumour mRNA into DCs has been used38 with mRNA derived from leukaemia cell lines. Double-loading techniques using loaded DCs with both leukaemic

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cell lysate and AML-specific tumour mRNA compared with DCs loaded with lysate or mRNA alone have been investigated.45 DCs loaded with apoptotic cells In 1999, Fujii et al. were the first to load DCs with apoptotic leukaemic blasts rather than LAAs.37 Using the knowledge that DCs could acquire antigen from apoptotic cells, thus inducing class-I-restricted CD8þ CTL responses46, Fujii et al. derived clusters of DCs from CD34þ cells of the leukapheresis product of three AML patients in remission undergoing autologous stem cell harvesting. The DCs were loaded with autologous, irradiated blasts then co-cultured with autologous CD4þ and CD8þ T cells, and the stimulated CTL populations demonstrated lysis of autologous leukaemia cells. This system required leukapheresis to obtain adequate numbers of DCs, but gave promise for this approach to DC loading. This principle was also studied by Spisek et al.39 who generated DCs from peripheral blood samples of 10 AML patients in remission without the need for leukapheresis. CTLs against the leukaemic targets were induced in samples derived from three patients in which T cells were stimulated by DCs loaded with autologous leukaemia cells. In a further trial, Jarnjak-Jankovic et al.38 used monocyte-derived DCs from healthy volunteers. The DCs were loaded with Jurkat cell lines rendered apoptotic (early stage) by staurosporin. Non-adherent peripheral blood mononuclear cells (PBMNCs) were co-cultured with the loaded DCs before separation into CD4 or CD8 cells, and assessed in enzyme-linked immunospot (ELISpot) assays using aliquots of the original loaded DCs as targets. Although this was an allogeneic set-up and used cell lines, it demonstrated that both CD4 and CD8 cells could produce interferon gamma (IFNg) when stimulated by leukaemic targets. Galea-Lauri et al.40 and Klammer et al.41 assessed DCs loaded with apoptotic leukaemia cells in comparative studies with fusion hybrids. DCs loaded with lysates The immunological potency of DCs loaded with lysate were compared40,42,43 with that observed with other DC-loading techniques. Lysates were obtained by freeze–thaw cycles and removal of cell debris by centrifugation, leaving the supernatant containing tumour lysate. All three of these authors found that other loading strategies were more effective in propagating CTL responses. DCs loaded with acid-eluted peptides Acid elution methods were originally used for identification of tumour antigens and then as a means to provide tumour antigens for DC loading in other malignancies. Delluc et al. first demonstrated a specific anti-leukaemic T-cell response by DCs loaded with acid-eluted peptides.44 DCs loaded with antigens eluted by two different acids were compared, first in a mouse model then with DCs derived from AML patients in remission.44 Using trifluoracetic acid to elute peptides, the authors were able to generate anti-leukaemic T-cell specificity without evidence of autoimmunity (i.e. no responses against mononuclear cells). The use of citrate phosphate to elute peptides was less effective and was associated with evidence of self-reactivity. Transfer of tumour mRNA into DCs Electroporation can be used to transfer total mRNA from leukaemia cells into DCs. This method of loading was assessed by Jarnjak-Jankovic et al.38 They concluded that it was an effective loading strategy and could be used for DC vaccine therapy.

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Double loading Decker et al.45 loaded DCs with both AML tumour lysate and AML-specific tumour mRNA. This double-loading technique generated more efficient CTLs compared with single loading, utilizing both CD4 and CD8 (by cross-priming) responses. Advantages and disadvantages of loading with undefined leukaemia-cell-derived antigens (Table 1) Using a wider repertoire of leukaemia antigens, such as those found in apoptotic cells, lysates or eluted peptides, may appear to be more attractive to avoid limitations of antigen expression, HLA restriction and antigen drift. However, there is concern that the resulting broader T-cell response may also target self-antigens and ultimately precipitate auto-immune disease. Of note, clinical studies to date have not found any evidence of significant, clinical autoimmunity. Uptake of apoptotic or necrotic cells may affect the maturation process of DCs, leading to so-called ‘tolerogenic DCs’.47 Many different reviews have looked at apoptosis in comparison with necrosis and effects on DC function, maturation48,49 and migration49 with contradictory results. It is also difficult to interpret these results because different authors use various terms and methods to define apoptosis or necrosis. Some authors refer to double anexin/PI-positive cells as being in ‘late apoptosis’39, whereas this PI-positive population would be deemed ‘necrotic’ by others. Larmonier et al. compared apoptosis, necrosis or fusion hybrids50 loaded on to DCs in a rat/cell-line model and found equivalent DC uptake, DC maturation and T-cell immunity regardless of the loading technique used. In contrast, acid-eluted peptides provide a wide range of antigens without inducing the immunosuppressive cytokines found in tumour cell lysates.44 Modification of leukaemia cells to express DC properties AML cells can be induced to differentiate down a dendritic-like maturation pathway (using the cytokines GM-CSF and IL-4) to become AML-DCs.51,51–59 These differentiated cells maintain expression of leukaemic antigens whilst exhibiting some of the immunostimulatory capacities of DCs. Alternatively, AML blasts can be fused with DCs creating fusion hybrids40–43 to create a hybridoma that demonstrates properties of both the leukaemia cells and the DCs. The transfer of DNA encoding co-stimulatory molecules can be used to bestow DC-like properties on to leukaemic blasts36,60,61 This technique does not use DCs directly and will not be reviewed further in this chapter. AML-DCs Santiago-Schwartz et al.62 were the first to differentiate the blasts of one patient with AML into AML-DCs. Choudhury et al. had already demonstrated the DC differentiation ability of CML cells63, and went on to successfully differentiate AML blasts into AML-DCs in a series of patients.51 PBMNCs from 19 patients with AML were cultured in the presence of GM-CSF, IL-4 and tumour necrosis factor alpha (TNFa). By Day 10–14, DC-like morphology was observed in 18 of the 19 AML patient samples. Upregulation of CD80, CD86, CD40 and CD54 was observed in all five of the samples in which flow cytometry was undertaken. Fluorescence in-situ hybridization analysis was performed on samples where there was an original cytogenetic abnormality. These abnormalities were still present in the AML-DCs. The AML-DCs were able to stimulate both allogeneic CTLs and,

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crucially, autologous CTLs derived from remission samples. The CTLs stimulated by coculture with AML-DCs were able to lyse autologous leukaemic targets but not autologous remission PBMNCs. In a further study, it was possible to differentiate the blasts of eight out of 10 patients with AML-M5 into cells with the morphological, phenotypic and cytokine production profile of DCs, whilst maintaining the initial cytogenetic and phenotypic features of the AML blasts. Again, these AML-DCs could stimulate na€ıve, allogeneic CD4 and CD8 T cells plus autologous CD8 cells with cytotoxicity demonstrated against the original leukaemic blasts.53 Kohler et al.56 investigated the differentiation ability of blasts of 98 AML and five ALL patients; successful AML-DCs were derived in 68% of the AML patients, but the ALL blasts did not differentiate. Blasts of 24 out of 40 AML patients became AML-DCs, with demonstrable, autologous, antileukaemic cytotoxicity in just 27% of patients in another study.54 This was lower than previous studies51,53 suggested, and demonstrates the limitations of AML-DCs as potential immunotherapy. This study demonstrated that the capacity of leukaemia cells to differentiate into functionally competent DCs is variable, and the immunological potency of these cells is not uniform. Cignetti et al.55 examined the biological properties of leukaemia-derived DCs and strategies. GM-CSF, IL-4 and TNFa were used to differentiate blasts into immature AML-DCs, and assess them for their immunostimulatory capacity and migratory abilities. CD40L was added to fully mature AML-DCs and their functional abilities were re-assessed. Cignetti et al. reported that the mature AML-DCs generated in this manner had more effective migration ability, as assessed by CCR7 and an improved cytokine profile for activation of T cells. Looking ahead to clinical vaccination protocols, Cignetti et al. irradiated the AML-DCs to prevent malignant proliferation and then assessed their function. Irradiated AML-DCs maintained the capacity to mature, migrate and produce cytokines. They concluded that irradiated, mature AML-DCs should be tested in clinical vaccine studies. A higher percentage of AML-DCs were generated successfully by Houtenbos et al.59 Out of 154 patients, differentiation was successful in 66%. AML-DC fusion hybrids Galea Lauri et al.40 first demonstrated the feasibility of constructing a DC-leukaemia cell line fusion hybrid which could provoke specific anti-leukaemic T cells. Using DCs derived from healthy donors and leukaemia cell lines, DCs loaded with irradiated (apoptotic), whole tumour cell lysate or DC/U937 cell line fusion hybrids were assessed. In one AML patient, remission DCs were loaded in these three ways. The fusion hybrids elicited the strongest anti-leukaemic responses in both the healthy donor T cells and the AML patient’s T cells. Klammer et al.41 generated DCs from the remission samples of six AML patients and fused them to their autologous presentation blasts by addition of polyethylene glycol. Fusion efficiency was confirmed by flow cytometric and membrane dye analyses. The fusion hybrids were then co-cultured with autologous remission lymphocytes and assessed in Cr release assays or IFNg ELISpots. Specific anti-leukaemic T-cell responses were seen in three out of six patients. Advantages and disadvantages of modification of leukaemia cells to express DC properties (Table 1) Leukaemic blasts fail to differentiate into AML-DCs in 30–60% of cases, and specific anti-leukaemic cytotoxicity is lower than hoped for. Could other agents successfully differentiate blasts into AML-DCs, especially those blasts unresponsive to differentiation by GM-CSF and IL-4? This question was asked by Roddie et al.64 Trichostatin,

Dendritic cell vaccines in acute leukaemia 529

azacytidine or bryostatin, which act on genetic transcription, were used to overcome differentiation block when differentiation by the conventional cytokine cocktail had failed. Unfortunately, this strategy did not result in increased capacity to generate leukaemia-derived DCs. Nine of the resistant cases were assessed for differentiation with the alternative agents, with bryostatin alone proving effective (six cases). In the AMLDC papers reviewed above, the leukaemic origin of AML-DCs was only confirmed in a small percentage of cases as the blasts rarely have identifiable cytogenetic abnormalities or aberrant expression of surface markers to confirm leukaemic origin. This raises the question of whether inadequate anti-leukemia responses resulted from the fact that the DCs were not derived from leukaemia cells. Fusion hybrids appear to show the most promise in generating specific anti-leukaemic cytotoxicity in the few comparative trials using hybrids in leukaemia.40,41,43 Klammer et al.41 used six patient leukaemia samples rather than cell lines, and showed advantages over DCs loaded with apoptotic cells. Fusion hybrids are more likely to induce CD8 T-cell responses compared with the processing of exogenous antigen acquired from, for example, apoptotic cells which would push the T-cell response towards CD4 activation.40 mRNA loading will also tend towards class I rather than class II processing. A way to utilize both CD4 and CD8 responses is illustrated by the double-loading technique45 and may be a promising area to explore. As interest moves away from AML-DCs towards loading of DCs with antigen, apoptotic cells, mRNA or using fusion hybrids for use in the remission setting, this chapter highlights the need for further studies in which sufficient samples are generated to assess the best methods for generating DC vaccines and activating specific CTLs. Preclinical trials of DC vaccination in ALL In comparison with AML, the use of DC vaccines in ALL has been studied less extensively. Maggio et al.65 loaded remission DCs with autologous, apoptotic leukaemia cells and found that these DCs could stimulate both autologous and allogeneic leukaemiaspecific CTLs. The loading of DCs with Jurkat cell (T-ALL) lysates was found to be more effective in stimulation of CTLs compared with irradiated or apoptotic ALL blasts.66 This differs slightly from AML-DC vaccination preclinical trials where lysates were suboptimal for loading on to DCs and triggering CTL responses. CLINICAL TRIALS USING DCs There are only a few clinical trials to date where DC vaccines have been administered to patients with acute leukaemia. Lee et al.67 vaccinated two patients who had relapsed with AML following autologous stem cell transplants. Autologous DCs were loaded with leukaemic cell lysate and treated with key hole limpet haemocyanin (KLH), an immunomodulatory agent. Four DC injections were given at 2–3-weekly intervals. An increase in leukaemia-specific CTLs was demonstrated along with clinical delayedtype hypersensitivity skin reactions at the site of vaccination. However, vaccination did not influence circulating leukaemia blast counts in the patients. AML-DCs were utilized in a clinical trial by Li et al.57 Five patients with advanced AML were vaccinated with autologous AML-DCs. AML-DCs were obtained from all five patients and showed a mean viability of 93% mature DCs in the vaccine preparations. The planned vaccination programme comprised of four subcutaneous injections administered in the vicinity of the inguinal node region over an 8-week period. Three out of the five

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patients received all four vaccinations, and two patients died of complications of their leukaemia after one injection. Assessment of immune responses by ELISpot assay revealed evidence of an increase in leukaemia-specific CTLs in one patient. Cytokine profiles were assessed and showed an increase in the production of intracellular IFNg in two patients, representing CD4 activation. In one patient who received all four vaccinations, there was a temporary fall in blast count. It is not clear if this was the same patient who demonstrated a specific anti-leukaemic response as measured by ELISpot. Houtenbos et al.58 planned a clinical vaccination study for AML patients in second CR. Fifteen patients were recruited but vaccination did not go ahead in any of the patients because of failure to achieve second CR, early relapse or refusal (8/15), or failure to differentiate adequate numbers of AML-DCs (7/15). This study highlights some of the logistical difficulties of clinical vaccination studies in AML. Another clinical trial did successfully vaccinate patients using AML-DCs.52 Roddie et al. recruited 22 patients at presentation with AML and attempted to differentiate their blasts to AML-DCs. Vaccination with irradiated AML-DCs was carried out after patients had achieved CR following intensive chemotherapy. Five patients achieved both CR and successful AML-DC differentiation. Four escalating doses of irradiated AML-DCs were administered subcutaneously at weekly intervals after the patients had completed their cycles of chemotherapy. Clinical assessment revealed that delayed-type hypersensitivity responses to an intradermal injection of autologous, irradiated leukaemia cells were minimal or absent in all patients. One patient developed eczema and an increase in the titre of anti-nuclear factor, compatible with autoimmunity. The production of IFNg by CTLs as measured by ELISpot was increased in four patients following vaccination. In one patient, HLA tetramer analysis for WT1 revealed specific anti-leukaemic cytotoxicity. Two of the patients remain in CR more than 3 years after vaccination (Unpublished data68). However, the authors conclude that this approach to DC vaccination is not broadly applicable because of the low success rate of AML-DC differentiation. Greiner et al. reviewed the use of LAAs in clinical trials in AML69 in 2006. Clinical trials using peptide PR-1 and WT-1 were reviewed by Barrett and Rezvani70 in 2007. Schmitt et al.30 have since published a clinical vaccine study using RHAMM peptide injected subcutaneously with evidence of immunological and clinical responses. Rezvani et al.71 combined WT1 and PR-1 peptide vaccination in a clinical trial published in 2008, showing immunological responses. These peptide vaccine studies did not use DCs directly and will not be reviewed further. Vaccination directly with inactivated leukaemia cells along with cytokines (IL-2, IL-6 and GM-CSF) utilizes in-vivo DCs. This has shown promise in a clinical study by Zhang et al.72 ASSESSING THE SUCCESS OF DC VACCINE THERAPY The ultimate aims of DC vaccine therapies are to expand specific anti-leukaemic CTLs and CD8þ memory cells, and to reduce relapse rates. DC vaccination in active leukaemia aims to decrease leukaemia blast numbers and this could be measured by serial bone marrow examinations combined with immunophenotyping and cytogenetic analysis. However, the majority of DC vaccine trials have not demonstrated evidence of clinical responses in the presence of active disease. In the remission state, DC vaccines aim to eliminate MRD and reduce relapse rates. Measures of MRD are needed to assess efficacy in this setting. Laboratory measurements of in-vivo and in-vitro immune responses may serve as biological surrogates to assess the success of vaccination. In

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order to compare trials and improve the efficiency of DC vaccine therapy, immunological endpoints that are clinically meaningful need to be defined. The backbone of laboratory assessment in vaccine therapy is assessment of CTLs and their specific anti-leukaemic reactivity. Chromium release assays were the gold standard for measurement of T-cell cytotoxicity and were used in the majority of preclinical DC vaccine trials. These assays have major practical limitations, particularly when using primary leukaemia cells as targets, and require the use of radiation. Today, most trials assess CTLs by ELISpot assays which measure the production of IFNg, an important cytokine produced predominantly by activated CD8þ cells in response to leukaemic targets. ELISpot uses an enzyme-linked immunosorbent assay (ELISA)-type technique to measure release of IFN at the single cell level, resulting in spot production that can be counted by an ELISpot reader. Many variables affect the final results, including sample integrity, spot counting and antibody dilutions. There is growing interest in the standardization of ELISpot techniques and result analysis. Based on results from a 2005 study, there are now guidelines for the standardization of ELISpot techniques.73,74 An alternative technique to measure specific anti-leukaemic cytotoxicity is the use of the tetramer assay (Figure 2). This depends on identification of a suitable HLA-restricted peptide derived from a known LAA. An example of this is the LAA RHAMM which is widely expressed on leukaemic blasts.34 The RHAMM R3 peptide sequence (ILSLELMKL) binds to HLA A201. For patients who are HLA A201, flow cytometry can be used to detect T cells that are specific for this peptide sequence. HLA A201 is only expressed in 40% of patients, whcih limits this peptide sequence to a minority of patients. Tetramer analysis has been used in clinical trials of DC vaccine therapy, such as detection of WT1-restricted T cells.52 There is growing appreciation of the importance of Tregs in suppressing immune responses to cancer. Several studies have demonstrated that DC-based vaccination may increase circulating levels of Tregs paradoxically. As such, it is necessary to monitor Tregs before and after vaccination in clinical DC vaccine trials to assess their potential impact on vaccine efficacy. This may help to identify the optimal time for vaccine therapy. Identification of a CD4þ/CD25þ (high) population was used to quantify Tregs, which is now aided by other markers such as FoxP3 which can be assessed by flow cytometry or molecular techniques.

CTL Fluorochrome label

TCR

Synthetic HLA tetramer Figure 2. Principles of human leukocyte antigen (HLA) tetramer analysis. CTL, cytotoxic T lymphocyte; TCR, T-cell receptor.

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LIMITATIONS OF DC VACCINE THERAPY There is evidence suggesting that the optimal setting for DC-based vaccine therapy is in the remission setting, which minimizes tumour-mediated immunosuppression. The hostile micro-environment in active leukaemia may limit the success of vaccine therapy, but even if the optimally loaded DC was vaccinated into the patient in the remission setting, why do we still see inadequate CTL responses? This can be the result of factors affecting DCs, tumour cells, T cells, the vaccination procedure itself and the timing of vaccination. DCs Successful vaccination requires sufficient numbers of DCs. This may be difficult following chemotherapy; however, most of the studies mentioned do not cite this as a problem. Royer et al.75 successfully produced monocyte-derived DCs in 22 patients with AML in CR following chemotherapy with the proviso that there was a 3-week gap after the last course of chemotherapy. DCs can be cryopreserved without alteration of function or phenotype76 for ease of use in clinical vaccine protocols. The stage of maturation of DCs may be important, and there is still debate over the optimal maturation stage for effective DC therapy. There are conflicting results from melanoma and other non-haematological tumour vaccine studies (reviewed by Tschoep et al.77). It was thought that immature DCs or semi-mature DCs could induce tolerant T cells.78 However, Rutella et al.79 reviewed the examples where mature DCs induced tolerant T cells and Tregs as well as increasing specific CTLs. The maturation stage of the DC affects its ability to induce T-cell responses. In a study in melanoma, it was found that mature DCs loaded with melanoma antigens were able to induce immunological and clinical responses whereas immature loaded DCs did not.80 The ability of the DC to migrate is important (see section on ‘Vaccination procedure and timings’), and this is influenced by the maturation stage of the DC. Different methods to derive DCs may affect their function and migratory ability. Radford et al.19 compared monocyte-derived DCs with CD11cþ DC precursors, and found that the latter could stimulate CTLs as effectively as monocyte-derived DCs but may have superior migratory capacity. DCs themselves may exert negative effects on the success of vaccine therapy by production of immunosuppressive cytokines such as IL-1081 or vasdcular endothelial growth factor.14 Leukaemia cells Antigen drift is a drawback for use of specific LAAs for immunotherapy. Even when a broader range of leukaemia antigens are used for vaccine therapy, there can be changes in antigen expression over time, leading to inadequate T-cell recognition. If relapse occurs from a state of MRD, the antigen pattern may be different from the time of presentation. If remission DCs are loaded with presentation blasts, the resulting Tcell repertoire may not be directed against the leukaemia cell that arises at relapse. There can be disruption of the antigen-processing mechanisms such as transporter associated with antigen presentation defects, so that as leukaemia cells mutate over time, antigens that were expressed previously are no longer processed or presented on the cell surface.82 Leukaemia cells that remain at low levels when patients are in a state of MRD are likely to have mutated in ways to evade killing by chemotherapy. Defects in their apoptotic pathways may also make them resistant to killing by CTLs after vaccination. Downregulation of HLA expression itself can happen as leukaemia progresses, and

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can correlate with resistant leukaemia cells83 or the phenotype found in relapsed leukaemia cells, e.g. relapsed ALL.84 Apoptosis or necrosis of leukaemia cells (naturally occurring, initiated by chemotherapy or induced ready for DC loading) may be involved in adverse DC responses. ‘Danger signals’ caused by the mechanisms of apoptosis may induce tolerogenic DCs.47,79 T cells The success of vaccine therapy hinges on stimulation of effective, leukaemia-specific lymphocytes. The development of leukaemia-specific CTLs has not always correlated with clinical responses. This may be partly explained by the negative influences of Tregs. T-cell numbers and functions may be lowered after chemotherapy or the vaccination process itself. Tregs are characterized by CD4þ/CD25þ (high) expression. More recently, it has been found that expression of intracellular marker FoxP3, a member of the Forkhead transcription factor family, appears to correlate with suppressive T-cell behaviour, first shown in mice models85 then humans. There is a note of caution, however, in Beyer and Schultze’s review of Tregs86 that some other studies have not confirmed the suppressive nature of FOXP3 cells in humans. Elevated numbers of Tregs have been noted in a wide variety of malignancies including AML.10 It is thought that this contributes to the immunosuppression seen in malignant states. Treg levels decline after chemotherapy treatment such as cyclophosphamide87,88, although this is not a universal finding.89 Theoretically, DC vaccination after chemotherapy when Treg numbers are lower should be more effective. Unfortunately, DC vaccination may itself induce Tregs. Banerjee et al.90 found an increase in regulatory FoxP3 T cells both in vitro and in vivo after injection of mature DCs. Immature DCs were able to induce another group of regulatory cells, CD8þ.78 Production of chemokines such as CLL22 by CD8þ cells recruits Tregs.91 The effects of chemotherapy on all lymphocyte groups may affect postvaccination immune responses. Children treated with standard chemotherapy for ALL still have low CD4 and CD8 levels 1 year after the end of chemotherapy.92 This may cause technical problems in generating sufficient numbers of T cells for use in experiments such as ELISpot assays. Clinical vaccination studies may be hampered by insufficient T-cell numbers in vivo. However, there is evidence that, despite low numbers, an improvement in lymphocyte function may occur. This was illustrated by increased proliferative responses of lymphocytes in leukopenic patients with AML or ALL immediately after chemotherapy when compared with healthy controls.93,94 T cells may lose their initial responsiveness to tumour antigens after continuous stimulation.36 This T-cell exhaustion may be exacerbated by the vaccination process by continually driving T-cell responses. The avidity of CTLs to bind to antigens depends on the frequency of antigen expression. CTLs that recognize antigens at low levels of expression are termed ‘highavidity CTLs’ and are important in clearing malignant cells; however, these high-avidity CTLs are more prone to exhaustion.95 There may be more general effects on the immune system of the postchemotherapy patient, such as malnourishment and ongoing infections, which are likely to hinder both DC and T-cell function.96 Vaccination procedure and timings DC vaccines for clinical use are required to conform to good manufacturing practice regulations. This requires culture media to be free from animal-derived serum, and the

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cytokines must be of clinical grade standard. It appears that serum is required to optimize DC uptake of apoptotic cells and for maintenance of DC markers. DCs cultured with autologous serum or pooled human serum were more efficient than DCs cultured in serum-free medium.97 This provides reassurance that autologous serum could provide an easy and safe way to supplement culture media. The optimal route for vaccination is unclear. The majority of vaccine studies use subcutaneous or intradermal vaccination, although there may be advantages in some malignancies for direct vaccination into the lymph node. Poor migration of DCs may contribute to vaccine failure. Only 5% of mature DCs vaccinated intradermally reach the draining lymph node.80 The number of DCs injected and expression of chemokine receptor CCR7 influences DC migration.98 There is interest in administration of cytokines to provoke inflammation at the vaccination site, thus improving migration.98 Timing of vaccination DC vaccination for patients with acute leukaemia would ideally be carried out when the tumour burden is low, i.e. in a state of MRD with favourable micro-environment and low Treg numbers. At the time of presentation of acute leukaemia, progression is rapid and immunotherapy would not be sufficient to overcome rapid leukaemia growth. There are too few clinical trials in the leukaemia setting to know the optimal timing and dosing schedules for DC vaccination. Even in malignancies such as melanoma and renal cell carcinoma, where good numbers of clinical vaccination trials have been reviewed, there is still no clear guidance on optimal route, dosing or vaccination schedules.99 IMMUNE AUGMENTATION IN VACCINE THERAPY Specific immunomodulatory agents may improve DC vaccine therapy in leukaemia. These agents have to be tolerated clinically and this limits the use of some preparations such as IL-2. There are only a few trials using immune adjuncts in leukaemia, and these are mentioned first before a brief overview of other strategies such as cytokines, up- or downregulation of co-stimulatory molecules and agents to reduce Tregs. Immune adjuncts used to date in leukaemia The co-stimulatory molecule 4-1BB is present on activated T cells and has an important role in anti-tumour immune responses. If targeted by 4-1BB ligand, it enhances proliferation of CTLs. Houtenbos et al. 100 added 4-1BB ligand to AML-DCs co-cultured with T cells to assess CD8þ cells. Increases in IFNg production and specific anti-leukaemic responses were seen as measured by ELISA, cytotoxicity assays and tetramer analysis for WT1. CD40 is crucial for DC/T-cell interactions. CD40 ligand can enhance the efficacy of DCs to act as antigen-presenting cells, thus stimulating CTLs. CD40 ligand upregulates tumour immunogenicity in ALL101, and has been used along with vaccine therapy for other malignancies. 102

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Immunomodulatory agents Cytokines such as IL-2, IL-6 and GM-CSF have all been reported to improve vaccine therapy. IL-12 is a key cytokine produced by DCs and is critical for CTL induction. Toll-like receptor (TLR) ligands and CD40 ligand used as vaccine adjuncts can lead to an increase in IL-12 and CTL priming.102,103 CpG oligodeoxynucleotide, ligand for TLR9, activates DCs and primes CTLs.104 CTLA-4 is a negative regulator of T cells and its blockade may be a useful immune adjunct.105 Non-specific immunomodulatory agents such as KLH and incomplete Freunds adjuvant have been used along with vaccine therapy.67,104,106 Lenalidomide, a thalidomide analogue and immunomodulatory agent, is a powerful potentiator of CTLs and natural killer cells.107,108 It may be useful in DC vaccine therapy as an immune adjunct, and is already in use in a preclinical vaccine trial (personal communication68). It has the advantage of clinical tolerability and is already in use for treating haematological malignancies. There are a number of methods to target Tregs. They can be reduced after chemotherapy such as cyclophosphamide.87 Dannull et al.109 used recombinant IL-2 diptheria toxin to bind to CD25, deliver the toxin, destroy the cells and thus reduce Treg numbers. When used in conjunction with a clinical vaccination study in metastatic renal cell carcinoma, this led to a significant increase in tumour-specific cytotoxicity. Tregs may also be reduced by anti-CD25 antibodies or by targeting FoxP3.110

THE FUTURE OF DC VACCINATION IN ACUTE LEUKAEMIA DC trial protocols and the process of vaccination would benefit from standardization. Guidance on minimal quality criteria for designing clinical trials in DC therapy has been produced.111 There is also a list of the compulsory and desired quality standards that DC vaccines must fulfil before vaccination.111 These include the purity, morphology, phenotype and viability of the DCs with optional assessments such as efficiency of DC uptake and assessment of the T-cell response. DC vaccines plus immune adjuncts delivered in the remission setting hold promise as a novel treatment for acute leukaemia. There are very few clinical trials of DC therapy in leukaemia. Collaborations between different research groups with a unified approach to vaccine therapy could improve the numbers for clinical trials, and help to address the many questions still unanswered in DC vaccine therapy.

Practice points  there is a need for novel treatment for acute leukaemia as relapse rates remain unacceptably high  DC vaccine therapy shows promise in the field of immunotherapy to stimulate leukaemia-specific CTLs  the aim of DC vaccine therapy is to overcome the poor spontaneous anti-leukaemic activity in patients with acute leukaemia  this may be most effective if vaccination is performed in the remission setting and augmented by immune adjuncts

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Research agenda  the majority of research to date of DC vaccine therapy is at the preclinical/invitro stage  there are many limitations to the success of DC vaccine therapy as a result of factors affecting DCs, tumour cells, T cells, the vaccination procedure itself and the timing of vaccination  DC trial protocols and the process of vaccination would benefit from standardization  collaborations between different research groups with a unified approach to vaccine therapy and immune adjuncts could improve the numbers for clinical trials, and help to address the many questions still unanswered in DC vaccine therapy

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