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Universal tumor antigens as targets for immunotherapy
Cytotherapy (2002) Vol. 4, No. 4, 317–327
Martin Dunitz
Taylor&Francis healthsciences
Universal tumor antigens as targets for immunotherapy JD Gordan and RH Vonderheide Abramson Family Cancer Research Institute, University of Pennsylvania Cancer Center and Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, USA Background
Clinically successful Ag-specific cancer immunotherapy depends on the identification of tumor-rejection Ags. Historically, tumor Ags have been
apoptosis survivin, the p53-interacting protein MDM2, and the cytochrome P450 isoform 1B1 – each at various levels of preclinical and clinical development.
identified by analyzing cancer patients’ own T-cell or Ab responses. Discussion Methods
The unveiling of the human genome and optimized immunological analytical tools, particularly ‘reverse immunology’, have made it possible to screen any given protein for immunogenic epitopes. These advances enable the immunological characterization of universal tumor-associated gene products that mediate critical functions for tumor growth and development.
The cardinal feature of universal TAA is that they are expressed in (nearly) all tumors and in no normal tissues. They are directly involved in the malignant phenotype of the tumor. Certain peptides derived from such Ags are expressed on the tumor-cell surface, as evidenced by Ag-specific, MHC-restricted T-cell anti-tumor reactivity in vitro. It is hoped that these features imply a pre-existing, high-affinity T-cell pool that can be activated in vivo in patients, without immunoselection of variant tumor cells no longer expressing the Ag of choice.
Results
Four examples of candidate universal tumor Ags reviewed here
Keywords
include the telomerase reverse transcriptase (hTERT), the inhibitor of
hTERT, telomerase, survivin, MDM-2, CYP IBI.
Introduction
and other methods of monitoring patients’ responses to these vaccines [5,6], these findings have enabled the next phase of Ag-specific immunotherapy of cancer. One fundamental technique of identifying human TAA is based on characterizing the autologous anti-tumor response in patients, either by assessing peripheral [7] or tumor-infiltrating lymphocytes [8], or by dissecting the humoral immune response [9]. A fundamental concept in the identification of these TAA is that their recognition by the immune system is an indication of their relevance in an anti-tumor immune response. This idea raises a series of concerns, as many of the patients in which these TAA are identified have metastatic disease and die of their cancer. Furthermore, many of the TAA that have been described come from a small number of highly responsive patients, suggesting some idiosyncrasy in responses. In some, but not all, cases, further assessment has shown that many of these
The last 10 years have been an extremely exciting time in the study of cancer immunology. Work in mouse models has suggested mechanistic explanations for some of the defining early phenomena of the field, including the identification of specific cytotoxic T lymphocyte (CTL) epitope Ags in chemically induced cancers [1] and a model for tumor immunosurveillance [2]. Mouse models have also been used to describe exciting new vaccine and treatment modalities, including cytokines, APC, and gene transfer. Paralleling these findings has been the identification of a broad variety of human tumor-associated Ags (TAA) [3]. Numerous Phase I clinical trials have been performed using these TAA, successfully demonstrating the safety and feasibility of vaccinating human cancer patients. Together with the development of the MHC Class I peptide tetramers [4],
Correspondence to: Robert H Vonderheide, Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, 551 BRB II/III, 421 Curie Blvd, Philadelphia, PA 19104, USA © 2002 ISCT
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TAA are also expressed by other tumor types and recognized by other patients. A key issue is the distinction between a tumor associated Ag and a tumor rejection Ag [10]. Most of the TAA identified from patients’ anti-tumor response are directed at proteins that do not appear to be necessary for ongoing tumor survival and proliferation. Indeed, the classical TAA have been either normal differentiation Ags, such as Melan-A/Mart-1, or tumor-specific yet functionally irrelevant proteins, such as MAGE-1. It is not entirely unexpected then, that tumors might be able to somehow shed TAA and evade immune recognition [11–13]. An approach is in place to resolve some of these concerns, by targeting commonly mutated, functionally relevant Ags like p53 and ras. However, there remains a great deal of variability in the expression and sequence integrity of these genes [14], and the idiosyncrasy of the various mutations found in these patients again becomes problematic. To attempt to circumvent issues of tumor idiosyncrasy and immune escape, we have sought to identify a class of ‘universal tumor Ags’, TAA that are likely to be present in the vast majority of tumors, and whose continuing expression is necessary to the oncogenic process. A prototype for these TAA is human telomerase reverse transcriptase (hTERT). Pursuing these TAA allows us to benefit from the extensive work in cancer-cell biology that has identified the requisite pathways involved in tumor development and progression. It is hypothesized that the use of such tumor-‘required’ TAA may help avoid immune escape. In this review, we briefly discuss different approaches to T-cell anti-tumor therapy, and describe the overall classes of TAA. We also describe the ideal components of a universal TAA, the methods employed in identifying universal TAA, and review four candidate Ags that have been described. Finally, we describe the pre-clinical experience with these Ags, and attempt to address the question of whether they are tumor associated or tumor rejection Ags.
T cells as cancer therapy Scientific advances in T-cell biology and tumor immunology over the last decade have greatly facilitated efforts to develop novel T-cell-based therapy for the treatment of cancer [3]. Results from work in both animal models and clinical trials support the hypothesis that T cells can be triggered to induce clinically meaningful antitumor responses. Two main approaches are envisioned:
n n
Tumor-specific adoptive T-cell therapy Tumor-specific vaccination.
In the former strategy, tumor-specific T cells are expanded ex vivo and subsequently re-infused into cancer patients. The latter strategy includes the injection of modified autologous cancer cells, as well as the injection of tumor Ag protein, peptide, or nucleic acid delivered alone, cellularly, or genetically with or without adjuvant. Here, we focus our discussion on vaccination with peptide and with APC. In Ag-specific vaccination strategies, patients are immunized against tumor-specific or tumor-associated targets in order to activate specific cellular and/or humoral immunity against cancer. Early trials using this approach tended to use only synthetic peptide, and few responses were seen [15,16]. Combinations of peptides with adjuvant, cytokines (especially GM-CSF, IL-2 and IL-12) or both, have subsequently been tried. These trials have mostly been performed in melanoma and hematologic malignancies [17–19] although, more recently, this approach has been applied to other tumors, including those of the pancreas, breast, and ovary [20,21]. Peptide vaccination has been favored because it is highly feasible, can be well monitored, and has posed a low risk of treatment-related adverse events. Thus, many newly discovered Ags are being assessed in peptide vaccination trials before they are studied in other formulations. The use of APC as a delivery mechanism for therapeutic vaccines is in rapid development, and is driven by our increasing knowledge of the function and regulation of DC. Patients have, for example, received DC pulsed with peptide cocktails or with tumor-cell lysate [18,22]. Many studies have also capitalized on the ability to pulse DC with tumor lysate to attempt treatment of tumor types for which there are few known Ags, including gastrointestinal tumors [23], glioma [24], and prostate cancer [25]. The initial results suggest that DC may be highly effective at initiating immune responses, although the optimal method for their preparation is not yet known. This modality will undoubtedly be extremely useful as an ‘immune adjuvant’ for peptide vaccines.
Classes of TAA Following the initial groundbreaking findings of CTL Ags in melanoma [7,26] the application of a variety of different techniques has increased the number of TAA to
Universal tumor antigens as targets for immunotherapy
at least 100. Generally, these Ags can be broken down into four general classes, with some overlap. These classes are tissue-specific differentiation TAA, cancer neo-TAA, mutated/over-expressed protein TAA, and ‘universal’ TAA.
Differentiation TAA The first class of TAA is exemplified by melanoma Ags, such as Melan-A/MART-1, gp100, and tyrosinase. These are TAA that are expressed by normal and tumor cells, but restricted to the tissue type in question. Other examples of this type of TAA include the prostatespecific Ag in prostate cancer, and proteinase 3 in CML. A fundamental assumption in targeting these TAA is that activation of an autoimmune response is tolerable. Indeed, there are many reports of autoimmune destruction of melanocytes (vitiligo) in patients receiving immunotherapy targeting melanoma TAA [27]. These Ags are among the best described in humans, and have created an interesting controversy about the available effector cells to target them, particularly in the case of Melan-A/MART-1. Early studies of melanoma patients revealed high levels of recognition [28]; unexpectedly, many healthy controls also showed antiMelan-A/MART-1 reactivity [29]. It has since been shown that the Melan-A/MART-1 specific cells in healthy controls are naïve, in part by assessing the length of their telomeres [30]. However, some controversy exists as to the functional status of the pre-existing anti-MelanA/MART-1 cells in melanoma patients, with some investigators suggesting that these cells are functionally inactivated, possibly anergic [31]. To summarize, differentiation Ags are a class of TAA that is promising for tumors of non-vital cell types, such as melanocytes and prostatic epithelia, but may be less advantageous for other tumor types, such as lung and renal cancers. It is not clear from the available data the extent to which they function as tumor rejection Ags, although early intriguing reports of objective clinical responses have been made following therapy.
Neo-TAA Prototypes of the cancer neo-TAA include MAGE family genes and NY-ESO-1. This group might also include the ‘oncofetal Ags’, such as carcinoembryonic Ag. However, the presence of many similar and potentially crossreactive related Ags in adult tissue makes TAA like
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carcinoembryonic Ag closer to the differentiation Ags than true neo-Ags [32]. The MAGE family of genes, and related genes such as the BAGE, GAGE and ESO families, are all expressed in cancerous tissue, and in no normal tissue except testis. MAGE family genes, for example, are expressed in a minority of melanomas, and also esophageal, lung, prostate, colon, and breast cancers, and certain sarcomas [33]. A more widely expressed Ag, NYESO-1 can be found in more than a quarter of lung cancers, bladder cancers and melanoma [34], as well as in some other less common tumors, such as cutaneous Tcell lymphomas [35]. Studies of non-vaccinated cancer patients and healthy controls reveal no detectable humoral reactivity in the absence of NY-ESO-1 expression, suggesting that these TAA may truly be encountered as neo-Ags [36]. The clinical experience with these Ags has just begun, but there is cause for concern that these tumors will escape immune responses directed against them by loss of expression. Furthermore, pathological studies have noted heterogeneity in expression of these Ags, even within a single tumor mass [34,37]. The significance of these effects should become clear as additional clinical studies are undertaken.
Mutated and over-expressed TAA Mutated TAA are appealing targets as they are also neoAgs, in that epitopes containing mutated sequence appear to be able to stimulate a ‘non-self ’ immune response. Clinically relevant mutations can be expected to be in either tumor suppressor genes, such as the Rb protein, or proto-oncogenes such as src. Since the latter mutations are an essential aspect of tumor progression, it can be hypothesized that the mutated gene products are unlikely to be immunoselected out from the tumor. Although there are many reports of specific, idiosyncratic, mutations recognized, such as b -catenin and caspase 8 [3], two mutated gene products have received great attention in this area – p53 and ras. p53 has been suggested to be a universal TAA in its own right. However, it is a complicated example: although it is over-expressed in 50% of tumors, it is also present in normal tissues [14,38]. As a tumor suppressor, once targeted in tumor cells it could be silenced to evade the immune response, and it is not clear whether targeting the increased turnover of p53 in tumor cells, or specific mutations, is more effective in eliciting responses. Early
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reports have shown significant tolerance of p53 in mice [39], with a specific elimination of high-affinity TCR [40]. However, pre-clinical studies have shown that it might be possible to activate anti-p53 responses ex vivo [41,42], although it is not clear how patients would respond to in vivo immunization. There is also the concern of additional mutation or silencing of the gene, since p53 expression is not usually necessary for cancer survival. The immunoselection of additional mutations in p53 has been documented [43]. There are a variety of different approaches that have been considered for such trials, using consensus mutations of p53 or wild type sequences, and a variety of delivery approaches [38]. Ras is a more straightforward TAA for cancer immunotherapy, since its over-expression or mutation both can lead to accelerated growth. Reports of the frequency of ras mutations in pancreatic adenocarcinoma can be as high as 87%, and 50% for lung cancers [44], with certain mutations being of particularly high frequency [45], and there have been reports of immune recognition of these mutations [46]. Vaccination with normal and mutated ras peptides has been performed in patients with pancreatic adenocarcinoma [20], melanoma [47], and colorectal cancers [48]. Results of these studies are encouraging, although it is not clear what the effects of anti-ras autoimmunity would be, if T-cell responses are activated against non-mutated epitopes. A final example of this class of Ag is HER-2/neu. HER-2/neu is well known as a target of the therapeutic antibody trastuzumab [49,50], and is over-expressed in 15–30% of breast adenocarcinomas. For the same reasons that it is a target for Ab therapy, HER-2/neu is also a target for CTL immunotherapy. Some concerns over targeting HER-2/neu for therapeutic immunization are autoimmune damage (particularly cardiac toxicity), and a somewhat low proportion of tumors expressing this Ag [51]. Clinical trials targeting CD4 and CD8 reactivity have been performed in patients with breast and ovarian cancer, with more than three-quarters of patients showing HER-2/neu specific T-cell responses [21,52]. No autoimmune phenomena have been reported in these patients.
Universal tumor Ags The search for ‘universal’ tumor Ags can be seen as an attempt to combine the desirable elements of the differ-
entiation TAA, neo-TAA, and mutated TAA. That is, a universal tumor Ag should be expressed by all tumors and by no normal tissues, and its expression should be necessary for the progression of that tumor. It is hypothesized that combining these features will result in the clinical activation of naïve, high-affinity T cells to target a homogeneously expressed Ag, possibly avoiding low T-cell receptor (TCR) affinities that have been seen with differentiation TAA. This would avoid the heterogeneity of expression seen with neo-TAA and the variety of antigenic epitopes (and risk of escape) seen with mutated TAA. In an optimal situation, universal TAA would be so necessary for cancer progression/survival that immunoselection of non-expressing cells would be impossible. Clearly, these are high expectations. Even with the prototype universal TAA, hTERT, it is not yet known how many of these expectations can be met. Three other known universal TAA – CYP1B1, survivin, and MDM2 – are also still being evaluated.
The bioinformatics of universal TAA One of the unique features of universal TAA is one method by which they may be described. Unlike most of the Ags listed above, which were described following the identification of reactive T cells, universal TAA are more typically identified by ‘reverse tumor immunology’ [53]. The classical approach for this involves extensive in silico studies performed to identify Ags that might meet the criteria of a TAA, followed by MHC binding predictions to identify peptide epitopes derived from candidate Ags. CTL reactivity and function are then evaluated in normal donors and patients (Figure 1). Breakthroughs in genomics have significantly eased the identification of cancer restricted genes. In silico searches, for example, may be performed on the Cancer Genome Anatomy Project (CGAP), which contains over 6000 genes from more than 30 000 combined tumor and normal libraries [54]. CGAP cannot prove cancerrestricted expression, but it is a very helpful first step in the identification of a universal TAA. A recent report described genes found in three tumor types but not in normal tissues, suggesting that many different universal TAA can be identified and pursued [55]. Once a gene of interest has been identified, its protein expression pattern is evaluated. The use of immunohistochemistry is extremely valuable, and has led to unexpected findings on some candidate universal TAA.
Universal tumor antigens as targets for immunotherapy
Following expression analysis, epitope deduction – an algorithm-driven method – can be used to predict binding affinities of peptides for MHC. Different programs are available, some of which apply to MHC Class II [53]. Depending on the stringency of criteria used, at least 10 epitopes may be identified for any one protein.
Candidate gene
T-cell repertoire analysis
Predictions
Generate peptidespecific CTL
Antigen processing MHC affinity
Cytotoxic function
Candidate peptides
Evaluation by tetramer analysis
Confirming predictions experimentally
Repeat analysis in cancer patients
Typically, the next step is to use MHC Class I-restricted epitopes to generate Ag-specific CTL. These may be tested for lysis of tumor cells expressing the Ag (and HLA allele) of choice, also providing information about Ag processing. An additional factor that must be considered in the identification of potential epitopes is that not all clinically relevant peptides have high MHC-binding affinity. Indeed, some of the epitopes in Melan-A/MART-1 [56] and gp100 [17] have been modified for greater binding affinity and presentation. Thus, epitope deduction can be a valuable analytic tool in the identification of clinically relevant epitopes in candidate universal TAA, as well as a way of fine tuning Ags discovered by other methods.
hTERT, CYP1B1, survivin, and MDM2 as universal TAA
Candidate antigen
Figure 1. The essential steps of reverse immunology for the identification of immunogenic epitopes from universal TAA.
Some of the qualities outlined in the above description of an ideal universal TAA have been identified in four identified universal TAA – hTERT, CYP1B1, survivin and MDM2 (Figure 2). The cardinal feature of a universal TAA is that it be expressed in all tumors, and no normal tissues, and that it be directly (universally) involved in the malignant phenotype of the tumor. If peptides derived from these TAA are expressed on the tumor-cell surface in association with MHC, it is hoped
Ag receptor
Candidate universal TAA
Ag processing
Ag
T cell
Peptide– MHC complex
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hTERT survivin MDM2 CYP1B1
Tumor cell
Lysis
Figure 2. For four universal TAA, it is hypothesized that peptides are derived from proteosomal cleavage of intracytoplasmic proteins and are folded together with MHC molecules for presentation on the tumor-cell surface. Unique T-cell receptors bind to these peptide/MHC complexes and initiate an immune response culminating in target lysis.
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that a pre-existing, high-affinity T-cell pool exists, and that its activation will not lead to selection of variant tumor cells that no longer express the Ag of choice. It is already clear that some of the universal TAA do not meet these requirements, and this information is not yet known for many of the more recently described universal TAA. However, some exciting possibilities do exist, and are described further below. hTERT, the human telomerase reverse transcriptase, is the prototypical and best described universal TAA [57]. It has also been recently and extensively reviewed [58]. From the standpoint of expression profile, hTERT is nearly optimal. Telomerase activity is found in . 85% of all human cancers, making telomerase one of the most highly expressed [59–61]. The role of telomerase in human cancer growth and development is well known [62], as is the observation that telomerase inhibition in telomerase-positive human tumors leads to cell death in vitro without any tumor-escape variants [63–65]. Thus, it is our prediction that hTERT down-regulation would be a lethal form of tumor escape. Although absent from heart, lung, liver, kidney and brain, hTERT has been detected in a few rare but important cell types. These include hematopoetic stem cells, activated lymphocytes, basal keratinocytes, gonadal cells, and some epithelial cells [66–69]. This raises some concern of autoimmunity following vaccination. However, in vitro studies show that hTERT-specific CTL do not lyse either telomerase-positive CD341 HPC or activated T lymphocytes, although they do lyse activated B cells [57,70,71]. Fortunately, these findings have held up in vivo, as neither mice vaccinated with mTERT nor patients receiving hTERT peptide loaded autologous DC has shown any BM toxicity [72,73]. The final characteristic that should be considered for hTERT is the type of hTERT-specific CTL that might be elicited by vaccination. Given the endogenous expression of hTERT, it is possible that only tolerating or low-affinity hTERT recognizing T cells can be activated by vaccination. A specific test of TCR affinity has not yet been performed with any of the deduced hTERT epitopes. However, the CTL elicited by hTERT stimulation in vitro are able to lyse a variety of hTERT-expressing cell lines and primary tumors. Using MHC-peptide tetramers, a brightly staining population (more than two logs above background) of CD81 cells was identified in ex vivo stimulated cells. These cells formed 1–3% of the
population, and staining was predictive of cytolytic activity [73]. In addition, ex vivo stimulation of lymphocytes from patients with renal disease with mRNA transfected DC was able to elicit in vitro lysis of the patients’ own tumors [72]. Thus, hTERT is likely to be a proof of concept for the universal TAA, in that its selective expression correlates with lytically active CTL and little autoimmunity. A second characterized universal TAA, survivin, was first identified as an inhibitor of apoptosis [74], and found to interact with the mitotic spindle during cell division [75]. When initially characterized, it was observed that survivin is absent from normal tissues and expressed in most tumor types, including lung, colon, pancreas, prostate, and breast cancer [74], thus making it an excellent candidate universal TAA. A comprehensive analysis of a group of SAGE studies showed highly elevated survivin expression in tumors of the colon, lung, breast, and brain [76], although there was the possibility of some expression in normal tissues. Interference with survivin function leads to cell death in vitro [77] and in an in vivo mouse melanoma model [78]. In contrast to hTERT, there are reports of humoral and cellular responses to survivin in non-vaccinated cancer patients. An initial study of humoral immunity in 50 patients with lung and colorectal cancer showed a 21.6% and 8.2% respective rate of response [79]. A later study looking at CTL showed in situ tetramer staining of survivin-specific CD81 T cells in a primary melanoma and breast cancer lesion. Peripheral blood lymphocytes (PBL) were tested for reactivity by enzyme-linked immunospot (ELISPOT), and showed reactivity in 14 of 20 melanoma patients and in no normal controls [80]. The epitopes studied were deduced in an earlier study, which also showed anti-survivin reactivity in melanoma and CLL patients, but not in controls [81]. Thus, it appears that a naïve population of survivin-reactive CTL exist in healthy controls, and these can be activated by survivin-expressing tumors, as well as by ex vivo activation with DC [82]. There is no report of autoimmune phenomena associated with immune recognition of survivin. Thus, survivin also meets the criteria to be a universal TAA, and is also a quite promising target for specific immunotherapy. MDM2, on the other hand, represents a candidate universal TAA that seems less likely to be therapeutically efficacious in humans. MDM2 is a proto-oncogene, well
Universal tumor antigens as targets for immunotherapy
known to inactivate p53 by targeting it for proteasome degradation [83]. MDM2 is frequently over-expressed in tumors, including breast cancer and many hematologic malignancies [84]. MDM2 is also ubiquitously expressed, although at lower levels than in tumors. It is unclear whether MDM2 can be considered to be necessary for tumor proliferation. As with p53, it seems plausible that a compensatory mutation could lead to immune escape. A study of MDM2 as a universal TAA reported a high level of self-tolerance to the MDM2 protein, such that the investigators were unable to elicit MDM2 specific cytotoxicity with in vitro activated human CTL. However, it was possible to circumvent this problem with HLAA*0201 transgenic mice from which an MDM2 reactive mouse TCR was identified and then transferred into human T cells. Analysis of CYP1B1 as a fourth candidate universal TAA came in part from previously published literature noting the gene product’s marked over-expression in cancer [55]. CYP1B1 is a member of the cytochrome P450 family of enzymes, and is known to play a role in cancer development by metabolizing certain toxins [85]. Based on histochemical studies, CYP1B1 protein was found in . 95% of tumors and in no normal tissue [86]. CYP1B1 has been implicated in carcinogenesis caused by environmental carcinogens, as well as endogenous estrogen-related carcinogenesis in human breast and uterine tumors. Although additional immunohistochemical and functional analysis is clearly needed, pre-clinical immune studies of CYP1B1 are promising. Epitope
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deduction revealed two candidate HLA-A*0201-binding peptides. Using PBL from healthy controls and cancer patients, it was demonstrated that cytolytic CYP1B1 specific T cells could be expanded by in vitro stimulation, although the baseline precursor frequency among PBL is well below the threshold of detection for tetramer analysis (similar to observations with hTERT-specific CTL). Table 1 summarizes the four candidate universal tumor Ags, in terms of differential expression, role in oncogenesis, the available immune repertoire for targeting these TAA, and the risk of autoimmunity in activating those repertoires — with comparisons to prototype Ags from the other classes. With the exception of MDM2, where the pre-existing tolerance would be likely to impede immunotherapeutic efforts, all of these TAA show significant promise for cancer treatment. They are all expressed in a significant majority of tumor types, and seem less prone to tumor escape than many of the other known Ags. Having noted this, however, there is no basis currently for predicting whether any of these universal TAA will be functional tumor rejection Ags. None of them was directly derived based on patient reactivity (the data on surviving notwithstanding), nor is there extensive clinical experience with them at this time. One way to answer this question would be to determine the affinity of the T-cell receptors found on specific CTL from in vitro stimulations. These studies have not yet been extensively performed.
Table 1. Universal tumor antigens in comparison to other prototypic antigens
Available T-cell repertoire
Immune recognition by patients at baseline
Antigen
Expressed in .50% of tumors
Expressed in normal tissue
Required for oncogenesis or carcinogenesis
hTERT
Yes
Rare cells
Yes
Yes
No
Survivin
Yes
Rare cells
Yes
Yes
Yes
MDM2
Yes
Low
Possibly
No
No
CYP1B1
Yes
Rare cells
Yes
Yes
No
Melan-A/MART-1
No
Pigmented cells
No
Yes
Yes
NY-ESO-1
No
Gonadal tissue
No
Yes
Yes
p53
Yes
Yes
Possibly
Yes
Yes
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The in vivo experience with these Ags has shown some efficacy in mouse models. The experience using MDM2 has been described above. In the case of TERT, mice given DC transfected with mTERT mRNA were able to mount specific CTL responses without autoimmunity, and reject transplanted tumors [72]. This shows that antiTERT CTL are sufficient to reject a tumor, and that mice have a sufficient T-cell repertoire to produce tumorrejecting CTL. In a recently completed Phase I clinical trial at the Dana-Farber Cancer Institute, seven patients with refractory, advanced prostate and breast cancer were immunized with hTERT peptide-loaded autologous DC [73]. A total of 34 vaccinations were given and patients showed minimal toxicity. Four of the patients showed hTERT responses detectable by tetramer analysis. One minor clinical response was observed, and stable disease was noted in four of the patients. One patient had progressive disease. One patient was not evaluable. In an ongoing trial at the Dana-Farber Cancer Institute and the University of Pennsylvania, CYP1B1 is also being targeted in advanced cancer patients. These and subsequent studies will further define whether hTERT or CYP1B1 are effective tumor rejection Ags.
have a diagnosis of cancer, a truly preventive approach for these modalities is therefore more difficult to envisage. In many ways, utilizing a universal TAA represents a shift in the approach of tumor immunotherapy. These represent the first TAA to be selected as biologically rational therapeutic targets. Even p53, which would certainly seem to be a highly rational target for tumor immunotherapy, was first characterized immunologically as a serological target in chemically induced murine tumors [87]. With Ags like hTERT, CYP1B1 and survivin, it is now possible to define the desirable ‘target’ characteristics of a TAA, and then seek these candidates in genomic databases. As experience with these TAA extends, it is possible that new desirable qualities will emerge, but the refinement of strategies that target a combination of universal tumor Ags will remain an important goal in the hunt for clinically meaningful Agspecific anti-tumor immunotherapy.
Conclusions
1
With the efforts of clinical and basic science investigators over the last 40 years having clarified many of the issues of tumor vaccine delivery, monitoring, and T-cell modulation, it is now possible to build on this wealth of experience to pursue new pathways for the successful immunotherapy of cancer. Targeting universal TAA is one of several novel approaches currently being pursued, and clinical studies are now underway. The greatest potential of universal TAA rests in the ability to consider preventive immunotherapy. Regardless of the clinical scenario, it remains an important observation that postexposure vaccination is rarely, if ever, clinically effective. The identification of widely applicable immunological targets in cancer might eventually open the avenue to truly preventive strategies. It could be envisioned that the development of a preventive cancer vaccine targeting genes that are expressed in the vast majority of all cancers might be possible, if it can be shown that a panel of universal TAA can be delivered safely and effectively. The use of narrowly restricted TAA or autologous tumor cells as part of a vaccine requires that patients already
Acknowledgements We thank Drs JL Schultze and LM Nadler for helpful discussions.
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Universal tumor antigens as targets for immunotherapy
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39 Theobald M, Biggs J, Hernandez J et al. Tolerance to p53 by A2.1-restricted cytotoxic T lymphocytes. J Exp Med 1997;185:833–41. 40 Hernandez J, Lee PP, Davis MM, Sherman LA. The use of HLA A2.1/p53 peptide tetramers to visualize the impact of self-tolerance on the TCR repertoire. J Immunol 2000; 164:596–602. 41 Nikitina EY, Clark JI, Van Beynen J et al. Dendritic cells transduced with full-length wild-type p53 generate antitumor cytotoxic T lymphocytes from peripheral blood of cancer patients. Clin Cancer Res 2001;7:127–35. 42 Umano Y, Tsunoda T, Tanaka H et al. Generation of cytotoxic T cell responses to an HLA-A24 restricted epitope peptide derived from wild-type p53. Br J Cancer 2001; 84:1052–7. 43 Theobald M, Ruppert T, Kuckelkorn U et al. The sequence alteration associated with a mutational hotspot in p53 protects cells from lysis by cytotoxic T lymphocytes specific for a flanking peptide epitope. J Exp Med 1998;188:1017–28. 44 Minamoto T, Mai M, Ronai Z. K-ras mutation: early detection in molecular diagnosis and risk assessment of colorectal, pancreas, and lung cancers — a review. Cancer Detect Prev 2000;24:1–12. 45 Andreyev HJ, Norman AR, Cunningham D et al. Kirsten ras mutations in patients with colorectal cancer: the ‘RASCAL II’ study. Br J Cancer 2001;85:692–6. 46 Gjertsen MK, Bjorheim J, Saeterdal I et al. Cytotoxic CD41 and CD81 T lymphocytes, generated by mutant p21-ras (12Val) peptide vaccination of a patient, recognize 12Valdependent nested epitopes present within the vaccine peptide and kill autologous tumour cells carrying this mutation. Int J Cancer 1997;72:784–90. 47 Hunger RE, Brand CU, Streit M et al. Successful induction of immune responses against mutant ras in melanoma patients using intradermal injection of peptides and GMCSF as adjuvant. Exp Dermatol 2001;10:161–7. 48 Gjertsen MK, Gaudernack G. Mutated Ras peptides as vaccines in immunotherapy of cancer. Vox Sang 1998; 74:489–95. 49 Shepard HM, Lewis GD, Sarup JC et al. Monoclonal antibody therapy of human cancer: taking the HER2 protooncogene to the clinic. J Clin Immunol 1991;11:117–27. 50 Perez EA. The role of adjuvant monoclonal antibody therapy for breast cancer: rationale and new studies. Curr Oncol Rep 2001;3:516–22. 51 Disis L, Cheever MA. HER-2/neu oncogenic protein: issues in vaccine development. Crit Rev Immunol 1998; 18:37–45. 52 Knutson KL, Schiffman K, Disis ML. Immunization with a HER-2/neu helper peptide vaccine generates HER-2/neu CD8 T-cell immunity in cancer patients. J Clin Invest 2001;107:477–84. 53 Schultze JL, Vonderheide RH. From cancer genomics to cancer immunotherapy: toward second-generation tumor antigens. Trends Immunol 2001;22:516–23.
54 Strausberg RL, Greenhut SF, Grouse LH et al. In silico analysis of cancer through the Cancer Genome Anatomy Project. Trends Cell Biol 2001;11:S66–S71. 55 Maecker B, Sherr D-H, Shen C et al. Targeting universal tumor antigens with cytotoxic T cells: potential of CYP1B1 for broadly applicable antigen-specific immunotherapy. Blood 1999;94S:438a. 56 Romero P, Dunbar PR, Valmori D et al. Ex vivo staining of metastatic lymph nodes by class I major histocompatibility complex tetramers reveals high numbers of antigenexperienced tumor-specific cytolytic T lymphocytes. J Exp Med 1998;188:1641–50. 57 Vonderheide RH, Hahn WC, Schultze JL, Nadler LM. The telomerase catalytic subunit is a widely expressed tumorassociated antigen recognized by cytotoxic T lymphocytes. Immunity 1999;10:673–9. 58 Vonderheide RH. Telomerase as a universal tumorassociated antigen for cancer immunotherapy. Oncogene 2002;21:674–9. 59 Kim NW, Piatyszek MA, Prowse KR et al. Specific association of human telomerase activity with immortal cells and cancer. Science 1994;266:2011–15. 60 Shay JW, Bacchetti S. A survey of telomerase activity in human cancer. Eur J Cancer 1997;33:787–91. 61 Ramakrishnan S, Eppenberger U, Mueller H et al. Expression profile of the putative catalytic subunit of the telomerase gene. Cancer Res 1998;58:622–5. 62 Hahn WC, Counter CM, Lundberg AS et al. Creation of human tumour cells with defined genetic elements. Nature 1999;400:464–8. 63 Hahn WC, Stewart SA, Brooks MW et al. Inhibition of telomerase limits the growth of human cancer cells. Nat Med 1999;5:1164–70. 64 Herbert B, Pitts AE, Baker SI et al. Inhibition of human telomerase in immortal human cells leads to progressive telomere shortening and cell death. Proc Natl Acad Sci USA 1999;96:14276–81. 65 Zhang X, Mar V, Zhou W et al. Telomere shortening and apoptosis in telomerase-inhibited human tumor cells. Genes Dev 1999;13:2388–99. 66 Harle-Bachor C, Boukamp P. Telomerase activity in the regenerative basal layer of the epidermis in human skin and in immortal and carcinoma-derived skin keratinocytes. Proc Natl Acad Sci USA 1996;93:6476–81. 67 Kolquist KA, Ellisen LW, Counter CM et al. Expression of TERT in early premalignant lesions and a subset of cells in normal tissues. Nat Genet 1998;19:182–6. 68 Norrback KF, Roos G. Telomeres and telomerase in normal and malignant haematopoietic cells. Eur J Cancer 1997; 33:774–80. 69 Yasumoto S, Kunimura C, Kikuchi K et al. Telomerase activity in normal human epithelial cells. Oncogene 1996; 13:433–9. 70 Minev B, Hipp J, Firat H et al. Cytotoxic T cell immunity against telomerase reverse transcriptase in humans. Proc Natl Acad Sci USA 2000;97:4796–801.
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Martin Dunitz
Taylor&Francis healthsciences
Universal tumor antigens as targets for immunotherapy JD Gordan and RH Vonderheide Abramson Family Cancer Research Institute, University of Pennsylvania Cancer Center and Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, USA Background
Clinically successful Ag-specific cancer immunotherapy depends on the identification of tumor-rejection Ags. Historically, tumor Ags have been
apoptosis survivin, the p53-interacting protein MDM2, and the cytochrome P450 isoform 1B1 – each at various levels of preclinical and clinical development.
identified by analyzing cancer patients’ own T-cell or Ab responses. Discussion Methods
The unveiling of the human genome and optimized immunological analytical tools, particularly ‘reverse immunology’, have made it possible to screen any given protein for immunogenic epitopes. These advances enable the immunological characterization of universal tumor-associated gene products that mediate critical functions for tumor growth and development.
The cardinal feature of universal TAA is that they are expressed in (nearly) all tumors and in no normal tissues. They are directly involved in the malignant phenotype of the tumor. Certain peptides derived from such Ags are expressed on the tumor-cell surface, as evidenced by Ag-specific, MHC-restricted T-cell anti-tumor reactivity in vitro. It is hoped that these features imply a pre-existing, high-affinity T-cell pool that can be activated in vivo in patients, without immunoselection of variant tumor cells no longer expressing the Ag of choice.
Results
Four examples of candidate universal tumor Ags reviewed here
Keywords
include the telomerase reverse transcriptase (hTERT), the inhibitor of
hTERT, telomerase, survivin, MDM-2, CYP IBI.
Introduction
and other methods of monitoring patients’ responses to these vaccines [5,6], these findings have enabled the next phase of Ag-specific immunotherapy of cancer. One fundamental technique of identifying human TAA is based on characterizing the autologous anti-tumor response in patients, either by assessing peripheral [7] or tumor-infiltrating lymphocytes [8], or by dissecting the humoral immune response [9]. A fundamental concept in the identification of these TAA is that their recognition by the immune system is an indication of their relevance in an anti-tumor immune response. This idea raises a series of concerns, as many of the patients in which these TAA are identified have metastatic disease and die of their cancer. Furthermore, many of the TAA that have been described come from a small number of highly responsive patients, suggesting some idiosyncrasy in responses. In some, but not all, cases, further assessment has shown that many of these
The last 10 years have been an extremely exciting time in the study of cancer immunology. Work in mouse models has suggested mechanistic explanations for some of the defining early phenomena of the field, including the identification of specific cytotoxic T lymphocyte (CTL) epitope Ags in chemically induced cancers [1] and a model for tumor immunosurveillance [2]. Mouse models have also been used to describe exciting new vaccine and treatment modalities, including cytokines, APC, and gene transfer. Paralleling these findings has been the identification of a broad variety of human tumor-associated Ags (TAA) [3]. Numerous Phase I clinical trials have been performed using these TAA, successfully demonstrating the safety and feasibility of vaccinating human cancer patients. Together with the development of the MHC Class I peptide tetramers [4],
Correspondence to: Robert H Vonderheide, Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, 551 BRB II/III, 421 Curie Blvd, Philadelphia, PA 19104, USA © 2002 ISCT
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TAA are also expressed by other tumor types and recognized by other patients. A key issue is the distinction between a tumor associated Ag and a tumor rejection Ag [10]. Most of the TAA identified from patients’ anti-tumor response are directed at proteins that do not appear to be necessary for ongoing tumor survival and proliferation. Indeed, the classical TAA have been either normal differentiation Ags, such as Melan-A/Mart-1, or tumor-specific yet functionally irrelevant proteins, such as MAGE-1. It is not entirely unexpected then, that tumors might be able to somehow shed TAA and evade immune recognition [11–13]. An approach is in place to resolve some of these concerns, by targeting commonly mutated, functionally relevant Ags like p53 and ras. However, there remains a great deal of variability in the expression and sequence integrity of these genes [14], and the idiosyncrasy of the various mutations found in these patients again becomes problematic. To attempt to circumvent issues of tumor idiosyncrasy and immune escape, we have sought to identify a class of ‘universal tumor Ags’, TAA that are likely to be present in the vast majority of tumors, and whose continuing expression is necessary to the oncogenic process. A prototype for these TAA is human telomerase reverse transcriptase (hTERT). Pursuing these TAA allows us to benefit from the extensive work in cancer-cell biology that has identified the requisite pathways involved in tumor development and progression. It is hypothesized that the use of such tumor-‘required’ TAA may help avoid immune escape. In this review, we briefly discuss different approaches to T-cell anti-tumor therapy, and describe the overall classes of TAA. We also describe the ideal components of a universal TAA, the methods employed in identifying universal TAA, and review four candidate Ags that have been described. Finally, we describe the pre-clinical experience with these Ags, and attempt to address the question of whether they are tumor associated or tumor rejection Ags.
T cells as cancer therapy Scientific advances in T-cell biology and tumor immunology over the last decade have greatly facilitated efforts to develop novel T-cell-based therapy for the treatment of cancer [3]. Results from work in both animal models and clinical trials support the hypothesis that T cells can be triggered to induce clinically meaningful antitumor responses. Two main approaches are envisioned:
n n
Tumor-specific adoptive T-cell therapy Tumor-specific vaccination.
In the former strategy, tumor-specific T cells are expanded ex vivo and subsequently re-infused into cancer patients. The latter strategy includes the injection of modified autologous cancer cells, as well as the injection of tumor Ag protein, peptide, or nucleic acid delivered alone, cellularly, or genetically with or without adjuvant. Here, we focus our discussion on vaccination with peptide and with APC. In Ag-specific vaccination strategies, patients are immunized against tumor-specific or tumor-associated targets in order to activate specific cellular and/or humoral immunity against cancer. Early trials using this approach tended to use only synthetic peptide, and few responses were seen [15,16]. Combinations of peptides with adjuvant, cytokines (especially GM-CSF, IL-2 and IL-12) or both, have subsequently been tried. These trials have mostly been performed in melanoma and hematologic malignancies [17–19] although, more recently, this approach has been applied to other tumors, including those of the pancreas, breast, and ovary [20,21]. Peptide vaccination has been favored because it is highly feasible, can be well monitored, and has posed a low risk of treatment-related adverse events. Thus, many newly discovered Ags are being assessed in peptide vaccination trials before they are studied in other formulations. The use of APC as a delivery mechanism for therapeutic vaccines is in rapid development, and is driven by our increasing knowledge of the function and regulation of DC. Patients have, for example, received DC pulsed with peptide cocktails or with tumor-cell lysate [18,22]. Many studies have also capitalized on the ability to pulse DC with tumor lysate to attempt treatment of tumor types for which there are few known Ags, including gastrointestinal tumors [23], glioma [24], and prostate cancer [25]. The initial results suggest that DC may be highly effective at initiating immune responses, although the optimal method for their preparation is not yet known. This modality will undoubtedly be extremely useful as an ‘immune adjuvant’ for peptide vaccines.
Classes of TAA Following the initial groundbreaking findings of CTL Ags in melanoma [7,26] the application of a variety of different techniques has increased the number of TAA to
Universal tumor antigens as targets for immunotherapy
at least 100. Generally, these Ags can be broken down into four general classes, with some overlap. These classes are tissue-specific differentiation TAA, cancer neo-TAA, mutated/over-expressed protein TAA, and ‘universal’ TAA.
Differentiation TAA The first class of TAA is exemplified by melanoma Ags, such as Melan-A/MART-1, gp100, and tyrosinase. These are TAA that are expressed by normal and tumor cells, but restricted to the tissue type in question. Other examples of this type of TAA include the prostatespecific Ag in prostate cancer, and proteinase 3 in CML. A fundamental assumption in targeting these TAA is that activation of an autoimmune response is tolerable. Indeed, there are many reports of autoimmune destruction of melanocytes (vitiligo) in patients receiving immunotherapy targeting melanoma TAA [27]. These Ags are among the best described in humans, and have created an interesting controversy about the available effector cells to target them, particularly in the case of Melan-A/MART-1. Early studies of melanoma patients revealed high levels of recognition [28]; unexpectedly, many healthy controls also showed antiMelan-A/MART-1 reactivity [29]. It has since been shown that the Melan-A/MART-1 specific cells in healthy controls are naïve, in part by assessing the length of their telomeres [30]. However, some controversy exists as to the functional status of the pre-existing anti-MelanA/MART-1 cells in melanoma patients, with some investigators suggesting that these cells are functionally inactivated, possibly anergic [31]. To summarize, differentiation Ags are a class of TAA that is promising for tumors of non-vital cell types, such as melanocytes and prostatic epithelia, but may be less advantageous for other tumor types, such as lung and renal cancers. It is not clear from the available data the extent to which they function as tumor rejection Ags, although early intriguing reports of objective clinical responses have been made following therapy.
Neo-TAA Prototypes of the cancer neo-TAA include MAGE family genes and NY-ESO-1. This group might also include the ‘oncofetal Ags’, such as carcinoembryonic Ag. However, the presence of many similar and potentially crossreactive related Ags in adult tissue makes TAA like
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carcinoembryonic Ag closer to the differentiation Ags than true neo-Ags [32]. The MAGE family of genes, and related genes such as the BAGE, GAGE and ESO families, are all expressed in cancerous tissue, and in no normal tissue except testis. MAGE family genes, for example, are expressed in a minority of melanomas, and also esophageal, lung, prostate, colon, and breast cancers, and certain sarcomas [33]. A more widely expressed Ag, NYESO-1 can be found in more than a quarter of lung cancers, bladder cancers and melanoma [34], as well as in some other less common tumors, such as cutaneous Tcell lymphomas [35]. Studies of non-vaccinated cancer patients and healthy controls reveal no detectable humoral reactivity in the absence of NY-ESO-1 expression, suggesting that these TAA may truly be encountered as neo-Ags [36]. The clinical experience with these Ags has just begun, but there is cause for concern that these tumors will escape immune responses directed against them by loss of expression. Furthermore, pathological studies have noted heterogeneity in expression of these Ags, even within a single tumor mass [34,37]. The significance of these effects should become clear as additional clinical studies are undertaken.
Mutated and over-expressed TAA Mutated TAA are appealing targets as they are also neoAgs, in that epitopes containing mutated sequence appear to be able to stimulate a ‘non-self ’ immune response. Clinically relevant mutations can be expected to be in either tumor suppressor genes, such as the Rb protein, or proto-oncogenes such as src. Since the latter mutations are an essential aspect of tumor progression, it can be hypothesized that the mutated gene products are unlikely to be immunoselected out from the tumor. Although there are many reports of specific, idiosyncratic, mutations recognized, such as b -catenin and caspase 8 [3], two mutated gene products have received great attention in this area – p53 and ras. p53 has been suggested to be a universal TAA in its own right. However, it is a complicated example: although it is over-expressed in 50% of tumors, it is also present in normal tissues [14,38]. As a tumor suppressor, once targeted in tumor cells it could be silenced to evade the immune response, and it is not clear whether targeting the increased turnover of p53 in tumor cells, or specific mutations, is more effective in eliciting responses. Early
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reports have shown significant tolerance of p53 in mice [39], with a specific elimination of high-affinity TCR [40]. However, pre-clinical studies have shown that it might be possible to activate anti-p53 responses ex vivo [41,42], although it is not clear how patients would respond to in vivo immunization. There is also the concern of additional mutation or silencing of the gene, since p53 expression is not usually necessary for cancer survival. The immunoselection of additional mutations in p53 has been documented [43]. There are a variety of different approaches that have been considered for such trials, using consensus mutations of p53 or wild type sequences, and a variety of delivery approaches [38]. Ras is a more straightforward TAA for cancer immunotherapy, since its over-expression or mutation both can lead to accelerated growth. Reports of the frequency of ras mutations in pancreatic adenocarcinoma can be as high as 87%, and 50% for lung cancers [44], with certain mutations being of particularly high frequency [45], and there have been reports of immune recognition of these mutations [46]. Vaccination with normal and mutated ras peptides has been performed in patients with pancreatic adenocarcinoma [20], melanoma [47], and colorectal cancers [48]. Results of these studies are encouraging, although it is not clear what the effects of anti-ras autoimmunity would be, if T-cell responses are activated against non-mutated epitopes. A final example of this class of Ag is HER-2/neu. HER-2/neu is well known as a target of the therapeutic antibody trastuzumab [49,50], and is over-expressed in 15–30% of breast adenocarcinomas. For the same reasons that it is a target for Ab therapy, HER-2/neu is also a target for CTL immunotherapy. Some concerns over targeting HER-2/neu for therapeutic immunization are autoimmune damage (particularly cardiac toxicity), and a somewhat low proportion of tumors expressing this Ag [51]. Clinical trials targeting CD4 and CD8 reactivity have been performed in patients with breast and ovarian cancer, with more than three-quarters of patients showing HER-2/neu specific T-cell responses [21,52]. No autoimmune phenomena have been reported in these patients.
Universal tumor Ags The search for ‘universal’ tumor Ags can be seen as an attempt to combine the desirable elements of the differ-
entiation TAA, neo-TAA, and mutated TAA. That is, a universal tumor Ag should be expressed by all tumors and by no normal tissues, and its expression should be necessary for the progression of that tumor. It is hypothesized that combining these features will result in the clinical activation of naïve, high-affinity T cells to target a homogeneously expressed Ag, possibly avoiding low T-cell receptor (TCR) affinities that have been seen with differentiation TAA. This would avoid the heterogeneity of expression seen with neo-TAA and the variety of antigenic epitopes (and risk of escape) seen with mutated TAA. In an optimal situation, universal TAA would be so necessary for cancer progression/survival that immunoselection of non-expressing cells would be impossible. Clearly, these are high expectations. Even with the prototype universal TAA, hTERT, it is not yet known how many of these expectations can be met. Three other known universal TAA – CYP1B1, survivin, and MDM2 – are also still being evaluated.
The bioinformatics of universal TAA One of the unique features of universal TAA is one method by which they may be described. Unlike most of the Ags listed above, which were described following the identification of reactive T cells, universal TAA are more typically identified by ‘reverse tumor immunology’ [53]. The classical approach for this involves extensive in silico studies performed to identify Ags that might meet the criteria of a TAA, followed by MHC binding predictions to identify peptide epitopes derived from candidate Ags. CTL reactivity and function are then evaluated in normal donors and patients (Figure 1). Breakthroughs in genomics have significantly eased the identification of cancer restricted genes. In silico searches, for example, may be performed on the Cancer Genome Anatomy Project (CGAP), which contains over 6000 genes from more than 30 000 combined tumor and normal libraries [54]. CGAP cannot prove cancerrestricted expression, but it is a very helpful first step in the identification of a universal TAA. A recent report described genes found in three tumor types but not in normal tissues, suggesting that many different universal TAA can be identified and pursued [55]. Once a gene of interest has been identified, its protein expression pattern is evaluated. The use of immunohistochemistry is extremely valuable, and has led to unexpected findings on some candidate universal TAA.
Universal tumor antigens as targets for immunotherapy
Following expression analysis, epitope deduction – an algorithm-driven method – can be used to predict binding affinities of peptides for MHC. Different programs are available, some of which apply to MHC Class II [53]. Depending on the stringency of criteria used, at least 10 epitopes may be identified for any one protein.
Candidate gene
T-cell repertoire analysis
Predictions
Generate peptidespecific CTL
Antigen processing MHC affinity
Cytotoxic function
Candidate peptides
Evaluation by tetramer analysis
Confirming predictions experimentally
Repeat analysis in cancer patients
Typically, the next step is to use MHC Class I-restricted epitopes to generate Ag-specific CTL. These may be tested for lysis of tumor cells expressing the Ag (and HLA allele) of choice, also providing information about Ag processing. An additional factor that must be considered in the identification of potential epitopes is that not all clinically relevant peptides have high MHC-binding affinity. Indeed, some of the epitopes in Melan-A/MART-1 [56] and gp100 [17] have been modified for greater binding affinity and presentation. Thus, epitope deduction can be a valuable analytic tool in the identification of clinically relevant epitopes in candidate universal TAA, as well as a way of fine tuning Ags discovered by other methods.
hTERT, CYP1B1, survivin, and MDM2 as universal TAA
Candidate antigen
Figure 1. The essential steps of reverse immunology for the identification of immunogenic epitopes from universal TAA.
Some of the qualities outlined in the above description of an ideal universal TAA have been identified in four identified universal TAA – hTERT, CYP1B1, survivin and MDM2 (Figure 2). The cardinal feature of a universal TAA is that it be expressed in all tumors, and no normal tissues, and that it be directly (universally) involved in the malignant phenotype of the tumor. If peptides derived from these TAA are expressed on the tumor-cell surface in association with MHC, it is hoped
Ag receptor
Candidate universal TAA
Ag processing
Ag
T cell
Peptide– MHC complex
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hTERT survivin MDM2 CYP1B1
Tumor cell
Lysis
Figure 2. For four universal TAA, it is hypothesized that peptides are derived from proteosomal cleavage of intracytoplasmic proteins and are folded together with MHC molecules for presentation on the tumor-cell surface. Unique T-cell receptors bind to these peptide/MHC complexes and initiate an immune response culminating in target lysis.
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that a pre-existing, high-affinity T-cell pool exists, and that its activation will not lead to selection of variant tumor cells that no longer express the Ag of choice. It is already clear that some of the universal TAA do not meet these requirements, and this information is not yet known for many of the more recently described universal TAA. However, some exciting possibilities do exist, and are described further below. hTERT, the human telomerase reverse transcriptase, is the prototypical and best described universal TAA [57]. It has also been recently and extensively reviewed [58]. From the standpoint of expression profile, hTERT is nearly optimal. Telomerase activity is found in . 85% of all human cancers, making telomerase one of the most highly expressed [59–61]. The role of telomerase in human cancer growth and development is well known [62], as is the observation that telomerase inhibition in telomerase-positive human tumors leads to cell death in vitro without any tumor-escape variants [63–65]. Thus, it is our prediction that hTERT down-regulation would be a lethal form of tumor escape. Although absent from heart, lung, liver, kidney and brain, hTERT has been detected in a few rare but important cell types. These include hematopoetic stem cells, activated lymphocytes, basal keratinocytes, gonadal cells, and some epithelial cells [66–69]. This raises some concern of autoimmunity following vaccination. However, in vitro studies show that hTERT-specific CTL do not lyse either telomerase-positive CD341 HPC or activated T lymphocytes, although they do lyse activated B cells [57,70,71]. Fortunately, these findings have held up in vivo, as neither mice vaccinated with mTERT nor patients receiving hTERT peptide loaded autologous DC has shown any BM toxicity [72,73]. The final characteristic that should be considered for hTERT is the type of hTERT-specific CTL that might be elicited by vaccination. Given the endogenous expression of hTERT, it is possible that only tolerating or low-affinity hTERT recognizing T cells can be activated by vaccination. A specific test of TCR affinity has not yet been performed with any of the deduced hTERT epitopes. However, the CTL elicited by hTERT stimulation in vitro are able to lyse a variety of hTERT-expressing cell lines and primary tumors. Using MHC-peptide tetramers, a brightly staining population (more than two logs above background) of CD81 cells was identified in ex vivo stimulated cells. These cells formed 1–3% of the
population, and staining was predictive of cytolytic activity [73]. In addition, ex vivo stimulation of lymphocytes from patients with renal disease with mRNA transfected DC was able to elicit in vitro lysis of the patients’ own tumors [72]. Thus, hTERT is likely to be a proof of concept for the universal TAA, in that its selective expression correlates with lytically active CTL and little autoimmunity. A second characterized universal TAA, survivin, was first identified as an inhibitor of apoptosis [74], and found to interact with the mitotic spindle during cell division [75]. When initially characterized, it was observed that survivin is absent from normal tissues and expressed in most tumor types, including lung, colon, pancreas, prostate, and breast cancer [74], thus making it an excellent candidate universal TAA. A comprehensive analysis of a group of SAGE studies showed highly elevated survivin expression in tumors of the colon, lung, breast, and brain [76], although there was the possibility of some expression in normal tissues. Interference with survivin function leads to cell death in vitro [77] and in an in vivo mouse melanoma model [78]. In contrast to hTERT, there are reports of humoral and cellular responses to survivin in non-vaccinated cancer patients. An initial study of humoral immunity in 50 patients with lung and colorectal cancer showed a 21.6% and 8.2% respective rate of response [79]. A later study looking at CTL showed in situ tetramer staining of survivin-specific CD81 T cells in a primary melanoma and breast cancer lesion. Peripheral blood lymphocytes (PBL) were tested for reactivity by enzyme-linked immunospot (ELISPOT), and showed reactivity in 14 of 20 melanoma patients and in no normal controls [80]. The epitopes studied were deduced in an earlier study, which also showed anti-survivin reactivity in melanoma and CLL patients, but not in controls [81]. Thus, it appears that a naïve population of survivin-reactive CTL exist in healthy controls, and these can be activated by survivin-expressing tumors, as well as by ex vivo activation with DC [82]. There is no report of autoimmune phenomena associated with immune recognition of survivin. Thus, survivin also meets the criteria to be a universal TAA, and is also a quite promising target for specific immunotherapy. MDM2, on the other hand, represents a candidate universal TAA that seems less likely to be therapeutically efficacious in humans. MDM2 is a proto-oncogene, well
Universal tumor antigens as targets for immunotherapy
known to inactivate p53 by targeting it for proteasome degradation [83]. MDM2 is frequently over-expressed in tumors, including breast cancer and many hematologic malignancies [84]. MDM2 is also ubiquitously expressed, although at lower levels than in tumors. It is unclear whether MDM2 can be considered to be necessary for tumor proliferation. As with p53, it seems plausible that a compensatory mutation could lead to immune escape. A study of MDM2 as a universal TAA reported a high level of self-tolerance to the MDM2 protein, such that the investigators were unable to elicit MDM2 specific cytotoxicity with in vitro activated human CTL. However, it was possible to circumvent this problem with HLAA*0201 transgenic mice from which an MDM2 reactive mouse TCR was identified and then transferred into human T cells. Analysis of CYP1B1 as a fourth candidate universal TAA came in part from previously published literature noting the gene product’s marked over-expression in cancer [55]. CYP1B1 is a member of the cytochrome P450 family of enzymes, and is known to play a role in cancer development by metabolizing certain toxins [85]. Based on histochemical studies, CYP1B1 protein was found in . 95% of tumors and in no normal tissue [86]. CYP1B1 has been implicated in carcinogenesis caused by environmental carcinogens, as well as endogenous estrogen-related carcinogenesis in human breast and uterine tumors. Although additional immunohistochemical and functional analysis is clearly needed, pre-clinical immune studies of CYP1B1 are promising. Epitope
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deduction revealed two candidate HLA-A*0201-binding peptides. Using PBL from healthy controls and cancer patients, it was demonstrated that cytolytic CYP1B1 specific T cells could be expanded by in vitro stimulation, although the baseline precursor frequency among PBL is well below the threshold of detection for tetramer analysis (similar to observations with hTERT-specific CTL). Table 1 summarizes the four candidate universal tumor Ags, in terms of differential expression, role in oncogenesis, the available immune repertoire for targeting these TAA, and the risk of autoimmunity in activating those repertoires — with comparisons to prototype Ags from the other classes. With the exception of MDM2, where the pre-existing tolerance would be likely to impede immunotherapeutic efforts, all of these TAA show significant promise for cancer treatment. They are all expressed in a significant majority of tumor types, and seem less prone to tumor escape than many of the other known Ags. Having noted this, however, there is no basis currently for predicting whether any of these universal TAA will be functional tumor rejection Ags. None of them was directly derived based on patient reactivity (the data on surviving notwithstanding), nor is there extensive clinical experience with them at this time. One way to answer this question would be to determine the affinity of the T-cell receptors found on specific CTL from in vitro stimulations. These studies have not yet been extensively performed.
Table 1. Universal tumor antigens in comparison to other prototypic antigens
Available T-cell repertoire
Immune recognition by patients at baseline
Antigen
Expressed in .50% of tumors
Expressed in normal tissue
Required for oncogenesis or carcinogenesis
hTERT
Yes
Rare cells
Yes
Yes
No
Survivin
Yes
Rare cells
Yes
Yes
Yes
MDM2
Yes
Low
Possibly
No
No
CYP1B1
Yes
Rare cells
Yes
Yes
No
Melan-A/MART-1
No
Pigmented cells
No
Yes
Yes
NY-ESO-1
No
Gonadal tissue
No
Yes
Yes
p53
Yes
Yes
Possibly
Yes
Yes
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The in vivo experience with these Ags has shown some efficacy in mouse models. The experience using MDM2 has been described above. In the case of TERT, mice given DC transfected with mTERT mRNA were able to mount specific CTL responses without autoimmunity, and reject transplanted tumors [72]. This shows that antiTERT CTL are sufficient to reject a tumor, and that mice have a sufficient T-cell repertoire to produce tumorrejecting CTL. In a recently completed Phase I clinical trial at the Dana-Farber Cancer Institute, seven patients with refractory, advanced prostate and breast cancer were immunized with hTERT peptide-loaded autologous DC [73]. A total of 34 vaccinations were given and patients showed minimal toxicity. Four of the patients showed hTERT responses detectable by tetramer analysis. One minor clinical response was observed, and stable disease was noted in four of the patients. One patient had progressive disease. One patient was not evaluable. In an ongoing trial at the Dana-Farber Cancer Institute and the University of Pennsylvania, CYP1B1 is also being targeted in advanced cancer patients. These and subsequent studies will further define whether hTERT or CYP1B1 are effective tumor rejection Ags.
have a diagnosis of cancer, a truly preventive approach for these modalities is therefore more difficult to envisage. In many ways, utilizing a universal TAA represents a shift in the approach of tumor immunotherapy. These represent the first TAA to be selected as biologically rational therapeutic targets. Even p53, which would certainly seem to be a highly rational target for tumor immunotherapy, was first characterized immunologically as a serological target in chemically induced murine tumors [87]. With Ags like hTERT, CYP1B1 and survivin, it is now possible to define the desirable ‘target’ characteristics of a TAA, and then seek these candidates in genomic databases. As experience with these TAA extends, it is possible that new desirable qualities will emerge, but the refinement of strategies that target a combination of universal tumor Ags will remain an important goal in the hunt for clinically meaningful Agspecific anti-tumor immunotherapy.
Conclusions
1
With the efforts of clinical and basic science investigators over the last 40 years having clarified many of the issues of tumor vaccine delivery, monitoring, and T-cell modulation, it is now possible to build on this wealth of experience to pursue new pathways for the successful immunotherapy of cancer. Targeting universal TAA is one of several novel approaches currently being pursued, and clinical studies are now underway. The greatest potential of universal TAA rests in the ability to consider preventive immunotherapy. Regardless of the clinical scenario, it remains an important observation that postexposure vaccination is rarely, if ever, clinically effective. The identification of widely applicable immunological targets in cancer might eventually open the avenue to truly preventive strategies. It could be envisioned that the development of a preventive cancer vaccine targeting genes that are expressed in the vast majority of all cancers might be possible, if it can be shown that a panel of universal TAA can be delivered safely and effectively. The use of narrowly restricted TAA or autologous tumor cells as part of a vaccine requires that patients already
Acknowledgements We thank Drs JL Schultze and LM Nadler for helpful discussions.
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