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First Published Online March 16, 2009
The Oncologist, Vol. 14, No. 3, 240-252, March 2009; doi:10.1634/theoncologist.2008-0165
© 2009 AlphaMed Press

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Leukemias

Immunotherapy of Acute Myeloid Leukemia: Current Approaches

Evelien L.J.M. Smitsa, Zwi N. Bernemana,b, Viggo F.I. Van Tendelooa,b

aVaccine & Infectious Disease Institute (VIDI), Laboratory of Experimental Hematology, Faculty of Medicine, University of Antwerp, Antwerp, Belgium; bCenter for Cellular Therapy and Regenerative Medicine, Antwerp University Hospital, Antwerp, Belgium

Key Words. Myeloid leukemia • Active immunotherapy • Passive immunotherapy

Correspondence: Viggo F.I. Van Tendeloo, VIDI, Laboratory of Experimental Hematology, University of Antwerp (UA), Antwerp University Hospital (UZA), Wilrijkstraat 10, B-2650 Antwerp, Belgium. Telephone: 32-3-8213661; Fax: 32-3-8214456; e-mail: viggo.van.tendeloo{at}uza.be

Received August 1, 2008; accepted for publication February 16, 2009; first published online in THE ONCOLOGIST Express on March 16, 2009.

Disclosures

Evelien L.J.M. Smits: None; Zwi N. Berneman: None; Viggo F.I. Van Tendeloo: None.

Section editors Bob Löwenberg and Joseph G. Jurcic have disclosed no financial relationships relevant to the content of this article.

The content of this article has been reviewed by independent peer reviewers to ensure that it is balanced, objective, and free from commercial bias.

Target audience: Physicians who wish to advance their current knowledge of clinical cancer medicine in leukemias.


   Learning Objectives
Top
 Learning Objectives
Abstract
 Introduction
 Passive Immunotherapy Approaches
 Active Immunotherapy Approaches
 Conclusion
 Author Contributions
 References
 

  1. Analyze the reasons for active investigation of immunotherapy of AML.
  2. Compare active and passive immunotherapy approaches of AML.
  3. Summarize strategies for immunotherapy of AML and evaluate their potential for further investigation and/or clinical implementation.

This article is available for continuing medical education credit at CME.TheOncologist.com.


   ABSTRACT
Top
 Learning Objectives
 Abstract
Introduction
 Passive Immunotherapy Approaches
 Active Immunotherapy Approaches
 Conclusion
 Author Contributions
 References
 
Following standard therapy that consists of chemotherapy with or without stem cell transplantation, both relapsed and refractory disease shorten the survival of acute myeloid leukemia (AML) patients. Therefore, additional treatment options are urgently needed, especially to fight residual AML cells. The identification of leukemia-associated antigens and the observation that administration of allogeneic T cells can mediate a graft-versus-leukemia effect paved the way to the development of active and passive immunotherapy strategies, respectively. The aim of these strategies is the eradication of AML cells by the immune system. In this review, an overview is provided of both active and passive immunotherapy strategies that are under investigation or in use for the treatment of AML. For each strategy, a critical view on the state of the art is given and future perspectives are discussed.


   INTRODUCTION
Top
 Learning Objectives
 Abstract
 Introduction
Passive Immunotherapy Approaches
 Active Immunotherapy Approaches
 Conclusion
 Author Contributions
 References
 
Acute myeloid leukemia (AML) is a malignancy of the myeloid lineage of white blood cells, characterized by clonal expansion of abnormal myeloid progenitor cells that accumulate in the bone marrow and interfere with normal hematopoiesis. It is the most common acute leukemia affecting adults and its incidence increases with age (median, 60 years) [1, 2]. In order to prolong remission and survival of AML patients, there is a strong need for the development of novel adjuvant treatment options to use in combination with standard therapy.

Experimental observations indicate that immunotherapy might be a suitable adjuvant treatment method for AML patients, acting mainly through activation of natural killer (NK) cells and leukemia-specific T cells [3]. Leukemia-specific T cells are T cells that respond to antigens that are primarily expressed by leukemic cells, the leukemia-associated antigens (LAAs). Ideally, these antigens are not expressed by normal tissues or are expressed only at very low levels, so that the activated specific T cells do not exert their cytotoxic function against nonleukemic cells. Several tumor-associated antigens (TAAs) have been reported to induce immune responses in tumor patients, including AML patients. These antigens include human telomerase reverse transcriptase, proteinase 3, Wilms' tumor 1 (WT1) protein, preferentially expressed antigen in melanoma (PRAME), survivin, and cancer-testis antigens [4]. Furthermore, both the receptor for hyaluronic acid–mediated motility (RHAMM) and the M-phase phosphoprotein 11 were identified as LAAs by serological screening of myeloid leukemia patients [5]. Other molecules also have been reported as potential targets for specific AML immunotherapy, like CD33, CD45, and minor histocompatibility antigens (mHAgs). In this review, we give an overview of current passive and active immunotherapy strategies for AML. Active immunotherapy involves the induction of a cellular immune response in vivo in the cancer patient to fight cancer cells. In passive immunotherapy, antibodies and other agents (T cells or NK cells) are given to the cancer patient, so that the patient adopts an immune response that has been developed in vitro.


   PASSIVE IMMUNOTHERAPY APPROACHES
Top
 Learning Objectives
 Abstract
 Introduction
 Passive Immunotherapy Approaches
Active Immunotherapy Approaches
 Conclusion
 Author Contributions
 References
 
Passive immunotherapy strategies are developed with the aim of generating, in vitro, high numbers of effector molecules (antibodies) or cells (T and NK cells) that can directly and specifically kill leukemic cells after in vivo transfer. However, by injecting effector molecules or cells, no immunological memory is developed. Nevertheless, killed leukemic cells can theoretically be taken up by antigen-presenting cells (APCs), and in this way immunological memory can possibly develop if the antigens are presented to T cells in an immunostimulatory context. In this way, a specific immune response can still be induced, even when the transferred effectors are already dead or cleared.

For AML, the therapeutic benefits of monoclonal antibodies, as well as adoptively transferred T or NK cells, are currently being explored.

Monoclonal Antibodies
The aim of antibody-mediated immunotherapy is to achieve specific binding of an antibody to leukemic cells, followed by clearance of the leukemic cells via antibody-dependent cell cytotoxicity (ADCC) or complement activation. ADCC, on the one hand, is the process whereby NK cells and other leukocytes bind to antibody-coated cells via Fc receptors and destroy these cells. The complement system, on the other hand, consists of several plasma proteins that are activated by cell-bound antibodies or microbial structures and promotes inflammation and destruction of cells. Antibodies against leukemia cells can be used unconjugated or conjugated to radioisotopes or drugs [6]. Conjugation of an antibody and a toxin is performed to decrease the toxicity of the toxin and to increase the potency of the antibody. If the antibody targets antigens that are expressed on leukemic cells only, the tumor cells can be targeted very specifically. A restriction of this kind of immunotherapy is that only membrane-associated antigens are suitable to be targeted by antibodies.

The most commonly used antibody for treatment of AML patients is anti-CD33 antibody. CD33 (sialic-acid dependent cytoadhesion molecule, cell surface glycoprotein) is expressed on 90% of AML blasts. Its expression in normal tissues is restricted to hematopoietic myeloid progenitor cells. Therefore, CD33 has become the target of antibody-directed therapy in AML patients. While unconjugated antibodies to CD33 had little clinical activity in AML [7], the immunoconjugate gemtuzumab ozogamicin (GO; Mylotarg®; Wyeth Pharmaceuticals, Inc., Madison, NJ) holds more promise for efficient eradication of AML cells. In GO, the anti-CD33 antibody is conjugated to the toxin calicheamicin. Binding of the anti-CD33 antibody portion of GO with the CD33 antigen results in the formation of a complex that is internalized. Upon internalization, the calicheamicin derivative is released inside the lysosomes of the myeloid cell and binds to DNA in the minor groove, resulting in DNA double-strand breaks and cell death. This treatment strategy is especially indicated (and approved by the U.S. Food and Drug Administration) for patients with CD33+ AML in first relapse who are aged ≥60 years and who are not considered candidates for other cytotoxic chemotherapy [8]. Currently, several prospective clinical trials are assessing the therapy's potential in different settings [9]. However, because CD33 is also expressed by normal progenitor cells, anti-CD33 antibody is myelosuppressive and causes prolonged thrombocytopenia [10]. Also, hepatotoxicity has been reported as a side effect [11].

Clinical trials have also been performed in which anti-CD33 antibody was conjugated to radionuclides. The antibodies can be labeled with {alpha}-emitters (e.g., 225Ac, 213Bi, and 211At) and/or β-emitters (e.g., 131I, 90Y, and 188Re). β-particles are high-energy, high-speed electrons or positrons emitted by certain radioactive nuclei. By the created field effect, the β-particles can potentially also irradiate antigen-negative cells. This feature makes them useful in treating bulky disease or in selectively irradiating the entire bone marrow prior to hematopoietic stem cell transplantation (as reviewed in [12]). {alpha}-particles are helium nuclei emitted by radioactive nuclei in a process known as {alpha} decay. This process results in less nonspecific toxicity to normal bystander cells and in more efficient single-cell killing than with β-emitting conjugates. The humanized anti-CD33 antibody HuM195, labeled with the {alpha}-emitter 213Bi, has been studied in clinical trials [13, 14]. Preliminary results indicate that sequential administration of cytarabine and 213Bi-HuM195 can lead to complete remission in AML patients. The administration of this radioimmunoconjugate is safe, although transient minor liver function abnormalities and prolonged myelosuppression were common [13, 14].

In conclusion, antibodies hold promise for the elimination of leukemic cells to achieve complete remission or to fight relapse by antibody-directed chemotherapy or radiotherapy. Several clinical trials will further examine the efficacy of this approach in different settings of combination therapy, also considering the possible toxicity of antibody-mediated immunotherapy for AML.

Adoptive Transfer of T Cells
Donor lymphocyte infusion (DLI) for the treatment of AML relapse after allogeneic hematopoietic stem cell transplantation (allo-HSCT) was introduced in the 1990s [15]. In this process, lymphocytes from the original donor are administered to the AML patient in relapse. Evidence for an allogeneic graft-versus-leukemia (GVL) effect by DLI was provided by different reports on DLI treatment in relapsed AML patients after allo-HSCT [16]. However, clinical benefit was shown to be limited to a minority of patients, possibly as a result of great tumor burden during relapse [16]. Furthermore, allogeneic donor T cells not only mediate the beneficial GVL effect, but can also cause graft-versus-host-disease (GVHD) when attacking nonleukemic host cells. Therefore, adoptive transfer of T cells that are specific for antigens primarily expressed by leukemia cells would reduce the risk for GVHD.

Adoptive T-cell transfer involves the identification and expansion of autologous or allogeneic T cells with antitumor activity, followed by administration to cancer patients [17]. The lymphocytes are often administered along with appropriate growth factors to stimulate their survival and expansion in vivo. T cells specific for LAAs can be isolated out of the leukemia patient or can be generated in vitro using LAA-loaded APCs, such as dendritic cells (DCs) or modified leukemic cells [18–20]. Another method is the isolation of genes encoding LAA-specific T-cell receptor (TCR) out of high-avidity LAA-specific T cell clones, followed by the transfer of these genes into T cells [21]. Difficulties of adoptive T-cell transfer that need to be addressed are the generation of sufficient numbers of high-avidity LAA-specific T cells in vitro and the lack of cytotoxic T lymphocyte (CTL) persistence and migration in vivo.

If these obstacles are cleared away, adoptively transferred LAA-specific T cells could kill leukemic cells in vivo, without the need to induce a cellular immune response by APCs. Therefore, T cells specific for mHAgs and for the LAAs WT1 and CD45 are currently being generated and tested for their potential to kill human leukemic cells [3, 22]. mHAgs are defined as alloantigens capable of eliciting an allogeneic T-cell response between human leukocyte antigen (HLA)-matched people. They are derived from genetically polymorphic proteins, resulting in differential expression of peptides between donor and recipient. Donor allogeneic T cells that recognize hematopoiesis-restricted mHAgs of the recipient induce a specific immune response against malignant hematopoietic recipient cells, whereas hematopoietic cells derived from the donor and nonhematopoietic recipient cells are not affected. Therefore, mHAgs expressed on malignant cells may serve as target structures for T cell–mediated GVL reactivity. Human mHAg-specific T cells have been generated by peptide-pulsed monocyte-derived DCs or by CD80-transfected AML cells, and in vitro responses against AML cells were shown [19, 23]. These mHAg-specific T cells are candidates to be used for adoptive transfer. Furthermore, mHAg-specific T cells can be generated by TCR retroviral gene transfer. This technique was applied to generate T cells specific for the mHAg HA-2, and these HA-2–specific T cells were reactive against chronic myeloid leukemia cells [24]. Although hematopoiesis-restricted mHAgs are promising target antigens, the major disadvantage is that only few have been identified to date.

Experiments were also performed using the LAAs WT1 and CD45. WT1 is a tumor suppressor gene that is generally overexpressed in AML cells, as well as in other types of solid cancers and in hematopoietic malignancies. After birth, a low level of WT1 expression is found in only a few progenitor cell types, whereas overexpression of WT1, as found in cancer cells, has been ascribed a role in leukemogenesis [25]. Therefore, WT1 has been put forward as a candidate universal TAA and as an attractive target for cancer vaccination for a wide range of tumors, including AML [3]. Preclinical mouse studies have shown promising data for the use of WT1 as an LAA, without evidence for autoimmunity [26]. In the human setting, it was demonstrated in vitro that WT1 can serve as a target for CTLs with high specificity for leukemic progenitor cells. WT1-specific T cells were generated using peptides and/or APCs [27–32] or by retroviral or lentiviral TCR gene transfer [21, 33, 34]. These WT1-specific T cells were able to lyse WT1-overexpressing leukemic cells in vitro and to impair engraftment of AML cells in nonobese diabetic severe combined immunodeficient (NOD-SCID) mice, without affecting normal hematopoietic cells or hematopoiesis.

Another target antigen for T-cell therapy is CD45 [20]. Cytotoxic CD45-specific HLA-A2–positive T cells for adoptive transfer were generated by peptide-pulsed APCs. The obtained CTL line showed potent activity against HLA-A2–positive leukemic cells and also against healthy progenitor cells [20]. Therefore, these allorestricted CTLs are only useful in certain situations: (a) to restore a GVL effect after HLA-A2–mismatched haploidentical transplantation (with an HLA-A2–positive graft and an HLA-2–negative donor) or (b) to obtain host myeloablation in an HLA-A2–positive host.

In addition to the antigens described above, it was established, in vitro, that CTLs with the potential to kill leukemic cells also could be generated against other LAAs, such as CD68, human neutrophil elastase, and proteinase 3 [35]. In conclusion, following promising in vitro data using different antigens, clinical trial data are needed to further evaluate the safety and efficacy of this passive AML immunotherapy strategy [35–37].

Alloreactive NK Cells
NK cells are negatively regulated by inhibitory receptors that are specific for self–major histocompatibility complex (MHC) class I molecules. If a cell lacks expression of MHC class I molecules that bind to the inhibitory NK receptors (e.g., tumor cells with downregulated expression of MHC class I molecules), there is no inhibition of NK cell–mediated cytotoxicity. Similarly, NK cells may sense the missing expression of self-MHC class I molecules when they encounter mismatched allogeneic cells. This process, resulting in NK cell–mediated alloreactions, is called "missing self recognition" [38]. NK cells with the potential for alloreaction use the inhibitory killer cell immunglobulin-like receptors (KIRs) to sense the missing expression of self-MHC class I molecules. Therefore, NK cell alloreactions are generated between individuals that are KIR ligand-mismatched. In vitro data show that several hematological malignancies are susceptible to alloreactive NK cell killing, including AML [39]. Also in a preclinical model, transfer of human alloreactive NK cells eradicated human AML engrafted in NOD/SCID mice [40]. In clinical trials with human AML patients, NK cell alloreactivity was studied in patients receiving haploidentical or matched unrelated transplants. It was observed that donor-versus-recipient NK cell alloreactivity in haploidentical transplants reduced the risk for relapse [40, 41] while improving engraftment and protecting against GVHD. Furthermore, a multivariate analysis demonstrated that transplantation from an NK cell alloreactive donor is a strong factor predicting survival [38]. When studying the GVL effect of NK cell alloreactivity in matched unrelated transplants, some studies showed an effect, whereas others did not [42]. In conclusion, to date, the best results of NK cell alloreactivity were obtained in patients who received haploidentical transplantation. Moreover, it is likely that NK cell–mediated immunity is the major source of the observed GVL effect in haploidentical transplantations, because of HLA mismatching and the need for T-cell depletion to diminish GVHD. In certain conditions, matched unrelated transplant patients also benefit from NK cell alloreactivity, but more research is needed to identify which conditions are beneficial.

NK cell–mediated alloreactivity was studied not only following hematopoietic stem cell transplantation but also in a phase I study where NK cells from haploidentical donors were successfully expanded and adoptively transferred into AML patients. Four of five remissions occurred when KIR ligand–mismatched donors were used, suggesting that alloreactive NK cells were involved in obtaining a remission [43].

In conclusion, alloreactive NK cells play a prominent role in the specific eradication of AML cells. Larger clinical trials will now be performed to further assess the importance of KIR ligand–mismatched donors to improve AML therapy outcome.


   ACTIVE IMMUNOTHERAPY APPROACHES
Top
 Learning Objectives
 Abstract
 Introduction
 Passive Immunotherapy Approaches
 Active Immunotherapy Approaches
Conclusion
 Author Contributions
 References
 
Administration of Interleukin-2
Interleukin (IL)-2 is critical for the induction of T- and NK-cell responses by stimulating proliferation and activation of both cell types. Therefore, IL-2 was tested in clinical trials for its potential to induce antileukemia immune responses as a nonspecific active immunotherapy approach (Table 1). A significant increase in activated T cells and NK cells was detected in patients with relapsed or refractory AML following high-dose IL-2 administration, as well as a few complete remissions [44]. However, this treatment regimen was associated with severe toxicity, including thrombocytopenia [45]. Data on the outcome of IL-2 administration are varying, depending on which subgroup is included and which other therapeutics are combined with IL-2 [46–48] (Table 1). Research is still ongoing, but caution is needed for the toxicity of IL-2 administration.


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  		Table 1. Clinical trials testing active immunotherapy strategies of AML immunotherapy

 
Peptides
In mouse models, peptides that represent LAA epitopes were tested for their ability to induce activation of LAA-specific T cells [26, 49]. These CTLs could kill LAA-expressing tumor cells. Therefore, several clinical immunotherapy trials of AML are currently exploring the capacity of peptides to elicit efficient T-cell responses (Table 1), as extensively reviewed by others [50–55]. Immunization is performed with MHC class I–restricted peptides derived from WT1, proteinase 3, and/or RHAMM in combination with an adjuvant (montanide) and/or GM-CSF [56–62]. Preliminary results from ongoing clinical trials indicate that these peptides can induce an increase in LAA-specific T cells, as well as clinical responses, including the induction or maintenance of complete remission. The advantage of the peptide approach is the potential to establish a universal and practical AML vaccine that is relatively cheap, easily manufactured to clinical grade, and easily administered. Factors that limit the use of peptide vaccination are: (a) the restriction of usable peptides to previously defined LAA epitopes, (b) the restriction of patients to those expressing the appropriate haplotype, (c) the short half-life of HLA–peptide complexes, and (d) the possible loss of antigen expression by the leukemic cells, resulting in immune escape [3]. The potency of peptide vaccines can be enhanced both by using a mixture of peptides, so that both CD4+ T cells and CD8+ T cells are stimulated [63, 64], and by using heteroclitic peptides [65]. Heteroclitic peptides are synthetic variants of the natural peptide sequence with the same HLA-binding specificity and avidity, but with enhanced affinity to TCR. These peptides have been developed for the LAA WT1 [66–68]. Moreover, these heteroclitic peptides are now being tested in a phase I trial as part of the immunotherapeutic INNO-305TM at the Memorial Sloan-Kettering Cancer Center in New York (Table 1). INNO-305TM is composed of a mixture of peptides and is able to stimulate both CD4+ and CD8+ T cells. Stimulation of both types of T cells may result in a more robust and ubiquitous immune response with a lower risk for immune escape by the AML cells. In conclusion, peptide vaccines are being developed as a feasible immunotherapeutic approach and are currently extensively being tested in clinical trials with promising preliminary results.

Whole Tumor Cell Vaccines

Unmodified Leukemic Cells
When vaccinating AML patients with inactivated whole tumor cells, immune responses can be induced against multiple TAAs simultaneously, without the need for prior identification of the TAAs. Factors that counteract effective antileukemia immune responses include the immune escape mechanisms of leukemic cells and the noninflammatory leukemia microenvironment, containing immunosuppressive factors and/or lacking danger signals. Therefore, whole tumor cell approaches for leukemia treatment seek to remove these factors by modifying leukemic cells in order to augment their antigen-presenting capacity and/or by increasing the amount of inflammatory signals in the microenvironment.

The first report of active specific immunotherapy for AML patients was made by Powles et al. [69, 70] (Table 1). In that trial, patients received chemotherapy with or without immunotherapy. This latter therapy consisted of intradermal injections of irradiated AML cells in combination with Bacillus Calmette-Guérin (BCG). The BCG served as an adjuvant to elicit an immune response against the LAAs, delivered to competent APCs by irradiated AML cells. This immunotherapy approach resulted in prolonged periods of first remission and longer survival after first relapse. However, there were very few long-term survivors. Another study on the combined administration of human irradiated primary AML cells and exogenous proinflammatory stimuli was reported by Zhang et al. [71] (Table 1). They performed a phase I clinical trial in which autologous irradiated AML cells were administered to relapsed or refractory AML patients in combination with the cytokines IL-2, IL-6, and GM-CSF. This procedure resulted in complete remission in four and in partial remission in five of 25 patients.

However, to date, most reports on the whole tumor cell approach studied the immunostimulatory capacity of AML cells that were modified in vitro to express costimulatory molecules or to secrete proinflammatory cytokines (vide infra).

In Vitro–Modified Leukemic Cells
To date, different approaches have been tested to modify human AML cells by gene transfer. Genes that were transferred encoded: (a) molecules to test gene transfer efficiency (e.g., enhanced green fluorescent protein [EGFP], β-galactosidase) [72–80], (b) costimulatory molecules to facilitate T-cell priming (e.g., CD80, CD137 ligand [L]) [23, 73, 77, 81–86], (c) proinflammatory cytokines (e.g., IL-2, GM-CSF, and tumor necrosis factor [TNF]-{alpha}) [77, 79, 83–89], and (d) immunomodulators that promote the differentiation of AML cells into effective APCs (e.g., IL-4, CD40L) [85]. Factors that limited gene transfer efficiencies included the lack of in vitro proliferation of AML cells [76], the rapidly declining viability of AML cells in culture [74], and the lack of fiber receptors for the entry of adenoviruses [74]. However, following optimization of gene transfer protocols, transduced (retro-, adeno-, or lentiviral transduction) or transfected (plasmid DNA electroporation) human AML cells were used to test their T-cell stimulatory capacity. Furthermore, other techniques to modify AML cells were tested [90–92]. Human CD80 IgG fusion protein was used by Notter et al. [91], consisting of the extracellular domain of CD80 fused to the Fc portion of IgG1. This fusion protein was captured by human AML cells through their Fc receptor. The result was a 20-fold increase in CD80 on the cell surface of primary AML blasts. Vereecque et al. [90] showed that {gamma}-irradiation alone, without other modifications, upregulated CD80 expression in 90% of human primary AML samples. Furthermore, protein transduction was examined by Lea et al. [93], using a novel protein transduction sequence coupled to GFP. This resulted in a transduction efficiency in human AML cells of almost 100%.

In all (in vivo) mouse and (in vitro) human models of T-cell activation, the in vitro modification of AML cells (by gene transfer, by fusion proteins, or by {gamma}-irradiation) resulted in greater allogeneic or autologous T-cell responses. Factors that were introduced into AML cells to improve their T-cell stimulatory capacity are CD80, CD86, GM-CSF, IL-4, TNF-{alpha}, IL-12, IL-2, and CD40L. In some reports, two or more of these factors were compared for their ability to induce effective antileukemia immune responses. Using plasmid DNA electroporation in humans, it was shown that CD80-expressing AML cells were superior to CD86-expressing AML cells in protecting mice against wild-type tumor challenge and in eradicating minimal residual disease [94]. However, in a study by Nakazaki et al. [95], GM-CSF–secreting AML cells induced strong immunoprotection, in contrast to CD80-expressing AML cells. Dunussi-Joannopulos et al. [96] confirmed the superiority of GM-CSF. In their mouse model, only GM-CSF–secreting AML cells induced long-lasting immunity, although AML cells that expressed CD86 or secreted IL-4 or TNF-{alpha} could induce protective immunity similarly to GM-CSF–secreting AML cells. In a study by Ling et al. [97], AML cells expressing membrane-bound GM-CSF were produced, and these cells induced a superior protective immune response to CD40L-expressing AML cells. Because of the preclinical efficacy of AML cells secreting GM-CSF in mouse models, a clinical trial has now been started to evaluate the potential of GM-CSF–secreting AML cells (GVAXTM; adenoviral gene transfer) after allogeneic stem cell transplantation [98, 99] (Table 1).

In addition to examining which immunostimulatory factors are superior to others for the induction of antileukemia immune responses, combinations of factors are worth being investigated. Therefore, Chan et al. [86] transduced human AML cells with a lentiviral vector encoding a fusion protein of CD80 and IL-2 that is postsynthetically cleaved. They recently obtained approval to perform a phase I study [100] (Table 1).

Although AML cells could be successfully modified by viral transduction, caution is needed for the use of viral vectors. Drawbacks for the use of viral vectors are the risk for insertional mutagenesis and the fact that viral vectors are immunogenic per se [101, 102]. Therefore, the presentation of dominant viral epitopes might mask the LAAs that are more weakly immunogenic, resulting in an immune response that is directed against the vector rather than against the AML cells. This can be circumvented by the use of transfection techniques.

In conclusion, AML cells can be modified in vitro to express factors that increase their T-cell stimulatory capacity. To date, studies have focused on the optimization of the modification efficiency of AML cells and the quest to find the most potent factors to increase their T-cell stimulatory capacity. In addition to these experiments, ongoing and future clinical studies are required to reveal the potential of modified AML cells to break T-cell anergy in leukemia patients.

Leukemia-Derived DCs
Because AML cells originate from the same precursor as DCs, it is possible to direct the differentiation of AML cells towards DCs by culturing AML cells for 10–14 days with a mix of cytokines (GM-CSF, IL-4, TNF-{alpha}, IL-3, stem cell factor, and/or FLT3 ligand) or for 2 days with calcium ionophores and IL-4 [103]. AML-derived DCs are thought to maintain the expression of patient-specific LAAs, because of the observation that known LAAs (WT1, proteinase 3, RHAMM, and PRAME) were still expressed and chromosomal abnormalities were still present after differentiation of the AML cells [104, 105]. In this way, it is advantageous that AML-derived DCs are able to express several known and unknown LAAs. It was shown that AML-derived DCs are better stimulators of in vitro T-cell proliferation than AML cells, at least partly because of their greater expression of costimulatory and MHC molecules [104]. Furthermore, by using AML-derived DCs, autologous CTLs could be generated that were able to lyse leukemic cells in vitro [106–111]. In a phase I clinical trial (Table 1), it was shown that autologous AML-derived DC vaccination was well-tolerated and that it could result in an enhanced and specific CTL response in AML patients [112, 113]. It is also possible to combine the administration of AML-derived DCs with modification of the microenvironment, for example, by gene transfer of IL-12 in AML-derived DCs [114], by the addition of CD137L (4–1BBL) [115], or by the simultaneous addition of AML-derived DCs with IL-2–producing stromal marrow cells [116], to further improve T-cell responses. The latter method was tested in one patient (Table 1), resulting in greater autologous AML cell–specific T-cell responses in vitro, after expansion of T cells from postinjection skin biopsies following multiple injections [116].

In conclusion, the use of AML-derived DCs for active specific AML immunotherapy is under active investigation. Drawbacks of this approach are related to the generation of the AML-derived DCs. If the protocol using different cytokines is applied, there is an extensive use of expensive cytokines and a long period of in vitro culture to obtain differentiation and maturation of AML-derived DCs. Furthermore, there is the problem of low recovery (DCs obtained comprise 25% of the initial AML cells) and the difficulty of generating AML-derived DCs in 30%–40% of AML patients. This latter problem is circumvented by the identification of molecular features that counteract the generation of AML-derived DCs, like internal tandem duplications in the FLT3 gene or the lack of CD14 expression [103]. In this way, AML cells containing these features can be excluded from culture protocols.

Monocyte- or Bone Marrow–Derived DCs
Instead of modifying LAA-expressing leukemic cells to increase their antigen-presenting capacities, another approach is to load nonleukemic APCs (mostly DCs) with LAAs. There are several strategies to load DCs with LAAs by: (a) pulsing with peptides derived from LAAs, (b) transferring DNA or RNA molecules encoding LAAs into DCs, (c) loading DCs with leukemic cell lysates, apoptotic leukemic cells, or necrotic leukemic cells, and (d) generating hybrids of tumor cells and DCs.

DCs Loaded with LAAs by Peptide Pulsing
DCs pulsed with peptides derived from LAAs were used in a study by Osman et al. [117]. In that study, monocyte-derived DCs from normal HLA-matched donors were pulsed with a peptide obtained from the promyelocytic leukemia–retinoic acid receptor {alpha} (PML–RAR-{alpha}) fusion protein. These peptide-pulsed DCs were able to stimulate T cells and to lyse cells from acute PML patients in vitro. In a mouse study by Ramírez et al. [118], bone marrow–derived DCs were pulsed with two different epitopes of WT1 (pWT126 and pWT330). The two peptides stimulated strong peptide-specific CTL responses when loaded onto DCs. However, WT1-specific CTLs were not able to kill WT1-expressing tumor cells in vitro or to protect against tumor growth in vivo. Mice immunized with DCs that were pulsed with peptides derived from the strong immunogen chicken ovalbumin (OVA) were protected against a challenge with OVA-expressing tumor cells, showing that DC vaccination can be effective for tumor protection. The quality of the WT1-specific CTL response was probably affected by immunological tolerance. The limitation of the use of peptide-pulsed DCs as APCs is its one-antigen approach, thereby limiting the number of leukemia-specific T cells that can be activated, potentially paving the way to immune escape by the leukemic cells. This can possibly be circumvented by pulsing DCs with multiple peptides.

DCs Loaded with LAAs by mRNA Electroporation
The use of mRNA-transfected DCs to induce leukemia-specific T cells is currently being investigated in our laboratory in a phase I/II trial for AML patients in remission [3] (Table 1). For this, mRNA encoding WT1 is introduced in vitro into mature monocyte-derived DCs by electroporation, followed by administration of the WT1-loaded DCs to AML patients in remission. This resulted in greater WT1-specific CD8+ T-cell responses and less WT1 expression [119]. Electroporation of DCs with mRNA was also applied in a study by Zeis et al. [120], in which DCs transfected with mRNA encoding survivin were used for the in vitro generation of CTLs able to kill AML cells in vitro. Survivin is a member of the inhibitors of apoptosis family. It is overexpressed by AML cells and a suitable candidate antigen for leukemia immunotherapy [121].

The advantages of the technique of mRNA electroporation to load DCs with LAAs are the presentation of multiple LAA-derived epitopes simultaneously and its safety for clinical use, because the expression of mRNA is only transient and there is no risk for insertional mutagenesis, as opposed to when viral vectors are used [122]. If whole tumor cell mRNA could be isolated from the primary AML cells and used for electroporation, the additional advantage would be the expression of multiple patient-specific LAAs simultaneously [123].

DCs Loaded with LAAs by Pulsing with Cell Lysates, Pulsing with Apoptotic Cells, or Fusion of DCs with AML Cells
Loading of DCs with LAAs using AML cell lysates was tested in a clinical trial [124] (Table 1), following demonstration of the induction of leukemia-specific CTL responses in vitro [125] and protective antitumor responses in mouse models [126, 127]. After four DC vaccinations, the two AML patients who had relapsed after autologous HSCT showed immunological responses but no hematological responses in terms of decreased bone marrow blast percentage [124]. Next to AML cell lysates, apoptotic leukemic blasts also were examined for their potential to load DCs with LAAs. Several studies indicated that DCs loaded with apoptotic leukemic blasts induced a leukemia-specific cytotoxic response in vitro [128–130]. Furthermore, different approaches to load nonleukemic DCs with LAAs were compared. It was shown that both human DCs pulsed with apoptotic leukemic blasts and human DCs fused with AML cells could elicit antileukemia immune responses in vitro [130–132]. In a mouse model, it was observed that DC–leukemic cell hybrids could induce a potent cytotoxic antileukemia immune response equally well as DCs pulsed with AML cell lysates [133]. In a human model, however, Galea-Lauri et al. [134] showed that DC–leukemic cell hybrids were more efficient in eliciting LAA-specific CTLs than DCs pulsed with apoptotic AML cells or AML cell lysates. Combinations of loading methods also can be applied, as was tested by Decker et al. [123]. Those authors used human AML cell lysates and total tumor mRNA electroporation to obtain superior antigen presentation, and this resulted in enhanced cytotoxic T-cell responses as compared with the use of only one loading strategy.

In conclusion, loading DCs by pulsing with AML cell lysates, pulsing with apoptotic AML cells, and/or fusion with AML cells is a promising strategy to elicit effective antileukemia immune responses in vivo. The advantages of these strategies are the multiepitope approach and the fact that there is no need for previous identification of patient-specific LAAs.


   CONCLUSION
Top
 Learning Objectives
 Abstract
 Introduction
 Passive Immunotherapy Approaches
 Active Immunotherapy Approaches
 Conclusion
Author Contributions
 References
 
A general concern when treating patients with AML is the persistence of residual leukemic cells after standard therapy, ultimately resulting in relapse of the disease. Adjuvant therapeutic strategies are urgently needed to fight refractory disease and to prevent relapse, in order to prolong the survival of AML patients. Therefore, immunotherapy is now being thoroughly investigated for its potential to fight residual leukemic cells through activation of the immune system.

Allogeneic stem cell transplantation and donor lymphocyte infusions have revealed the potential of allogeneic T cells to eradicate leukemic cells. However, this procedure is associated with the development of GVHD and a high treatment-related mortality. Therefore, immunotherapy strategies that are more targeted to leukemic cells are actively being investigated, depending on the identification of LAAs. Promising targeted passive immunotherapy approaches are the administration of anti-CD33 antibodies and adoptive transfer of LAA-specific T cells or KIR ligand–mismatched NK cells. As far as the active strategies are concerned, presumably peptide vaccination is currently being tested in clinical trials. However, whole tumor cell vaccines or DC-based vaccines also hold promise in boosting the immune system for the induction of antileukemia immune responses. Although in vitro antileukemia activity was observed for several immunotherapeutic strategies, only clinical trials will reveal if active and passive immunotherapy for AML can induce antileukemic immune responses in vivo and prolong the survival of AML patients, acting as targeted adjuvant therapy.


   AUTHOR CONTRIBUTIONS
Top
 Learning Objectives
 Abstract
 Introduction
 Passive Immunotherapy Approaches
 Active Immunotherapy Approaches
 Conclusion
 Author Contributions
References
 
Conception/Design: Evelien L.J.M. Smits

Financial support: Zwi N. Berneman, Viggo F.I. Van Tendeloo

Manuscript writing: Evelien L.J.M. Smits, Zwi N. Berneman, Viggo F.I. Van Tendeloo

Final approval of manuscript: Zwi N. Berneman, Viggo F.I. Van Tendeloo


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 Learning Objectives
 Abstract
 Introduction
 Passive Immunotherapy Approaches
 Active Immunotherapy Approaches
 Conclusion
 Author Contributions
 References
 

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