The most compelling rationale for the use of the immune response as a therapy for cancer is the ability to specifically target the malignant clone while sparing the normal host tissue. This however relies on the ability of the immune system to recognize “non-self” characteristics of a malignancy in the context of appropriately pro-effector environmental cues. Extensive efforts have been made to define antigenic targets that can confer specificity of the immune response against hematological malignancies. The prediction would be that antigens arising from genes essential for cellular transformation and/or conferring a growth advantage (such as “driver” mutations) would represent better antigens than “passenger” mutations (17
) due to obligate expression; indeed it has been noted that the majority of the LAAs identified so far are linked to the cell cycle or proliferation (18
). The last 2–3 years has seen an explosion of information regarding somatic mutations in cytogenetically normal hematological malignancies during first presentation and relapse; we expect this will result in the identification of multiple new antigens within this category.
Cancer-associated antigens can be divided into three main classes. Firstly, true “neo antigens”, defined as molecules uniquely expressed by cancer cells with no normal tissue expression, can be created by somatic genetic events within the leukemia itself. These range from products of chromosomal fusions (eg; BCR:ABL in CML), to atypically spliced isoforms (19
), to single nucleotide variants (SNV) only discoverable in the first instance via whole genome sequencing (21
). One member of this category of antigens unique to hematological malignancies is the idiotypic determinant of a B cell or T cell receptor expressed by the clonally expanded cells comprising most lymphoid malignancies. In this setting, DJ and VDJ recombination of B and T cell receptor gene segments together with additional genetic mechanisms for generating antigen receptor diversity result in genes encoding amino acid sequences that are unique to the clonal lymphocyte population (22
). Experimental therapy with monoclonal antibodies raised against such antigens have led to durable remissions in lymphoma patients (24
), and active immunization with patient-specific idiotypic sequences has been shown to raise host antibody and T cell responses against idiotypic epitopes which correlate with clinical endpoints such as relapse free survival (25
). Unfortunately however, these promising results have proven challenging to reproduce in the phase III trial setting (26
). It remains to be seen if in the appropriate clinical setting, such biological responses can translate into clinically meaningful outcomes.
The second class of cancer-associated antigens in the hematological malignancies are those antigens with germline (unmutated) sequence, whose expression is limited to the leukocyte subset from which the hematological malignancy is derived. Examples include CD20 expression in most B-cell lymphomas (4
), or CD52 expression in chronic lymphocytic leukemia (CLL) and some lymphomas (3
). Given that members of this class of antigens are also expressed on normal hematopoietic cell lineages, they have been most useful as targets for passive immunotherapy with monoclonal antibodies, either alone or as an adjunct to cytotoxic chemotherapy, rather than as a target for the induction of long-lived immunity via vaccination where self-tolerance limits the host response. B cell lineage surface antigens have also been the target of adoptive T cell immunotherapy in the case of CLL, via the adoptive transfer of autologous T cells transduced with a chimeric antigen receptor (CAR) specific for CD19 (27
). This strategy utilizes an engineered transmembrane receptor that exploits the antigen-binding properties of a monoclonal antibody (extracellular domain) specific for the cell surface antigen CD19, fused to an intracellular domain consisting of the zeta chain of the T cell receptor signal transduction complex along with a co-stimulatory motif from CD137. Such systems enable a self-sustaining, amplifiable cytotoxic effector response with T cells that can migrate to tumor compartments not easily accessed by naked antibodies. As for the rationale for using a target that does not distinguish the malignancy from its normal cellular counterpart, even the complete elimination of normal CD19+ cells and the resulting impairment of humoral immunity may be a manageable and acceptable outcome in some clinical settings. Furthermore, future strategies may seek to only transiently target CD19 expressing cells (e.g. with T cells co-transduced with a suicide gene together with the CAR, or by using mRNA rather than viral vectors for CAR transduction). Since CD19 is not expressed on normal hematopoietic stem cells, normal B cell lymphopoiesis would be expected to recover once the transduced T cells are eliminated. Finally, the potency of linking antibody mediated tumor targeting with T cell activation may be achievable with a more easily exportable approach, i.e. via so-called bi-specific antibodies. As a result of significant progress in antibody engineering, antibodies having multiple valences and two (or more) specificities are now routinely generated (29
). Recently, such a chimeric antibody was tested in patients with CD19+ Acute Lymphoblastic Leukemia (ALL) who had persistence or relapse of molecularly measureable disease following intensive induction and consolidation chemotherapy (31
). The experimental antibody, Blinatumomab, a bispecific single-chain antibody for both CD19 (the leukemia target) and CD3 (the T cell target leading to T cell activation) was well tolerated, resulted in clearance of detectable minimal residual disease (MRD) in 16 of 21 patients treated, and was associated with a relapse-free survival of 78% at a median follow-up of 405 days. Of note 12 of the 16 responders in this study had been molecularly refractory to prior chemotherapy.
The third, and final, category is the one most extensively studied so far; those proteins of wild-type sequence that are over-expressed within the tumor target tissue compared to normal host tissue. This class includes the Wilms tumor protein WT-1 (32
), PR1 derived from azurophil granule proteases proteinase-3 and neutrophil elastase (34
) and the cancer testis antigens (eg: PRAME, MAGE, Cyclin A1, CALR-3) (35
). Given their import in current leukemia vaccination efforts WT1, PR1 and PRAME antigens are all discussed individually below.
The Wilms tumor 1 (WT-1) gene encodes a transcription factor that has an essential role in the normal development of the urogenital, nervous and hematopoietic systems and mesothelium. Its normal expression in post-natal life is thought to be limited to highly differentiated glomerular podocytes in the mature kidney (36
) and CD34+CD38− hematopoietic stem cells (37
). It undergoes a loss of function mutation in a subset of patients with Wilm’s tumors, where it functions as a tumor-suppressor gene (38
). Paradoxically, the unmutated gene is expressed by the vast majority of human acute leukemias (39
) where it can function as an oncogene. The amount of WT1 expression in normal CD34+ cells compared to leukemic blasts is controversial, with most studies limited by the analysis of bulk populations rather than defined subsets. WT-1 expression is found in a high percentage of leukemic blasts, whereas its expression is limited to a small subset (1–2%) of normal CD34+ cells (typically in more primitive progenitors) (42
). Nevertheless, analysis at the single cell level did not reveal significant differences in the level of WT-1 mRNA as measured by qRT-PCR. In contrast, other studies comparing phenotypically defined (CD34+
) hematopoietic stem cells (HSCs) and leukemic stem cells (LSCs) have reported 2–5 fold levels of over-expression by LSCs (37
). Differences in protein expression and turnover have not been fully explored, although the demonstration that human WT-1 specific T cells can distinguish LSCs vs normal HSCs (43
) highlights the potential therapeutic window. In spite of low-level self-antigen expression, WT-1 has been found to be an immunogenic cancer antigen in the mouse (44
) and the human with both T-lymphocyte (45
) and humoral responses being seen (47
). Given the observations it is overexpressed in myeloid leukemia stem cells (49
) and that human CTLs specific for this antigen were able to eliminate engraftment of leukemia initiating, but not normal CD34+, stem cells in a mouse model (43
), much effort has focused on inducing protective immunity to this antigen. These observations are strengthened by the observations that WT1 (but not PR1 or PRAME) specific CTL could frequently be detected after Allo-HSCT (50
), that an apparent graft-versus-leukemia effect was associated with detectable Wilms tumor-1 specific T lymphocytes after allo-BMT for ALL (51
) and that detectable WT1 expression following allo-BMT is an independent prognostic factor for leukemia relapse (52
). One note of caution about WT1 as a therapeutic target however, comes from the recent report that WT1 is one of the antigens that triggers T cell-mediated myelosuppression in myelodysplastic syndrome (53
). Whether this represents T cell targeting (but not eradication) of early transformed cells that are over-expressing WT-1 versus collateral damage to normal hematopoietic stem cells is currently an important unknown. Clinical trials of vaccination with this promising LAA are currently underway at a variety of medical centers including MSKCC, Moffitt, NCI, NHLBI and the University of Southampton (detailed in ).
Recruiting, Active and Recently Completed Vaccine Clinical Trials in Acute Myeloid Leukemia
Proteinase 3 and neutrophil elastase are expressed at high concentrations in the primary granules of myeloid leukemia blasts while normal expression is primarily confined to the early promyelocytic/myelocytic stage of bone marrow development (54
). PR1 is a HLA-A*0201 restricted T cell epitope within these proteins against which CTL lines and clones have been expanded in vitro. Such cells demonstrated highly specific recognition of AML and CML blasts isolated directly ex vivo (34
). This work was extended by the finding that T cell responses to PR1 were strongly correlated with clinical responses in patients with CML treated with IFN-alpha and allogeneic HSCT (56
). Furthermore, in patients with CML treated with allo-BMT, donor-derived PR1 specific T cells demonstrated an effector memory phenotype early post-BMT and expansion of this population was followed by complete remission in a patient with CML (57
). The recent development of an anti-PR1/HLA-A2 TCR-like antibody capable of mediating complement-dependant cytotoxicity of AML progenitor cells is an interesting spin-off from these studies with potential therapeutic utility (58
PRAME was identified in 1997 by Ikeda et. al.
, from a human melanoma cell line derived from a late recurrence metastasis (59
). A CTL clone capable of lysing the melanoma line was generated from autologous lymphocytes and used to screen pools from a cDNA library derived from the line. The single positive pool contained a clone with an open reading frame encoding a 509 amino acid protein nearly identical to sequences expressed by myeloid leukemia cell line K562 and promyelocytic leukemia cells HL-60 (60
) and 97% homologous to a 332 bp cDNA from human testis (61
). The authors named the gene PRAME for “preferentially expressed antigen of melanoma”. It was later shown that this antigen is also over-expressed in at least 25% of acute leukemia cells and almost all of those with the 8:21 translocation (62
). PRAME, which is up-regulated by BCR-ABL in CML, is thought to act via inhibition of retinoic acid signaling to both block myeloid differentiation (63
) and to increase resistance to apoptosis by down-regulating the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) (64
). As a member of the cancer testis family, expression in post-natal life is thought to be limited to germ cells. Within the normal hematopoietic compartment, PRAME expression is below the limits of detection by sensitive techniques. One intriguing observation is that PRAME expression is induced or increased by leukemias treated with DNA methyltransferase inhibitors and histone deacetylase inibitors, both of which are being actively used as “differentiating agents” in the treatment of leukemia (65
). As such, immunotherapies targeting PRAME, either through vaccination or adoptive T cell transfer may benefit from systemic modulation of the target antigen expression, if such maneuvers prove to be selective.
The cancer antigens identified to date in the hematological malignancies have been discovered in one of three ways. “Reverse Immunology” identifies candidate antigens based on gene expression profiling. Proteins that are uniquely or preferentially expressed by cancer cells relative to normal cells are evaluated for amino acid sequences that are predicted by MHC binding algorithms to be potential epitopes. Candidate peptides are synthesized, confirmed to bind to the relevant MHC, and then pulsed onto dendritic cells (DCs), which are used to stimulate polyclonal T cells to generate lines. These are then tested to see if they recognize tumor targets that endogenously express and process this antigen. WT1 and PR1 are examples of antigens discovered using this method (45
). This technique has the advantage that it can limit the search for novel cancer antigens to those proteins known to be overexpressed by cancer cells compared to normal cell types, and can even focus the search to those antigens found in self-renewing cancer stem cells, which are relatively well-defined in hematological malignancies (37
). However the approach is relative inefficient, and as relates to vaccine candidates, it assumes that the ability to generate T cell lines in vitro
(used to screen the candidate) predicts how immunogenic the antigen will be in vivo
The second approach, “Forward Immunology” seeks to identify the antigenic targets of an endogenous (or experimentally induced) host response. In the case of antigens recognized by T cells, tumor-reactive T cell lines or clones are first generated against a primary leukemia or cell line. Once the MHC restriction is determined, the specific antigen and presented peptide epitope can be identified either biochemically (by fractionating and sequencing peptides stripped from tumor MHC that sensitize targets for T cell recognition) or genetically (by transfection of targets sharing the restricting MHC with pools from tumor derived cDNA libraries and screening for T cell recognition). PRAME is an example of an antigen discovered via this methodology (66
). A significant bias of this approach arises from uncontrolled factors that influence which T cell specificities emerge from the in vitro T cell expansion; a process that is notoriously inefficient and does not necessarily reflect the full spectrum (or even the hierarchy) of the host T cell response present in vivo.
The third approach, which we refer to as “Fast-Forward Immunology” is the direct characterization of the antigen specificity of an actual immune response from a cancer patient without the bias introduced by in vitro T cell expansion. The classic example of this is the combined use of “SEREX” (serological analysis of recombinant cDNA expression libraries of human tumors with autologous serum) (67
) to define the specificity of antibodies present in cancer patients, followed by evaluation of T cell responses to the serologically defined candidates. The antigen RHAMM was identified using this technique by screening patient sera against a cDNA library derived from the CML blast phase cell line K562 expressed by phage display, and comparing “hits” to those found in healthy volunteers and autoimmune patients (69
). T cell responses to the antibody targets were then examined with standard techniques. Starting with the humoral response provides a more comprehensive picture of the host response than T cell cloning approaches and avoids some of the educated guesswork and inefficiency embedded in the reverse immunology approach. It can be highly focused by creating the cDNA library from highly purified autologous tumor or subsets such as the stem cell fraction. It additionally offers the opportunity to study patients at defined clinical timepoints, for example, before and after response to immunotherapy. Investigators from the Dana-Farber Cancer Institute have elegantly exploited this feature to study the antigenic targets associated with a clinical remission induced by DLI in a CML patient (70
). The antigen identified, CML66, is expressed in CML blast crisis, AML and on normal myeloid progenitor cells but not other normal tissues (71
) and is the target of both B and T cell immunity after CD4+ donor lymphocyte infusion in CML (72
). Strikingly, CML66 specific immunity was first detectable after DLI, and just prior to the decline of Bcr-Abl positive cells in the index patient, peaking when a complete molecular remission was first documented, and persisting for over a year.
While many individual human LAAs have now been described it is unfortunately not yet possible to integrate this information into a complete systems-level understanding of the character of the immune response in those patients whose leukemia is well controlled. Given that the immune system can be effective in controlling leukemia, and that the T cell appears to be an important component of this response, it is reasonable to hypothesize that the functional T lymphocyte repertoire against leukemia antigens would differ for patients having different clinical outcomes (e.g. differences should be able to be observed in remission versus relapsed disease settings) and it is on this basis that clinical trials of vaccination to induce leukemia-specific immunity have proceeded.