Human Tumor Antigens Are Ready to Fly

ADVANCES IN IMMUNOLOGY, VOL 62

Human Tumor Antigens Are Ready to Fly ROBERT A. HENDERSONAND OLlMRA 1. FINN h p a ~ noft Molecular Genefics and Biochemishy, Universivof Pitkbugh School of Medicine, Pitkburgh, Pennsylvania 15260

1. introduction

The immune system is fully capable of successfully eliminating harmful or infectious microbes through both humoral and cellular mechanisms. Its function may also be broadened to include detection of any sort of “danger” that threatens the integrity of an organism (Matzinger, 1994). Why is it then that tumors, which are clearly detrimental to survival, appear to go unchecked by the immune system? It is now known that the reason is not the lack of antigens recognized by either antibodies or T cells (Urban and Schreiber, 1992). Many factors influencing the immune response to tumors have begun to be appreciated, including the quality of the antigens themselves. From studies in mice, it has been known for many years that the immune system mounts a response to tumors and that immunization against certain tumors is indeed possible. Early studies in humans also demonstrated the presence of lymphocytes that were reactive against or inhibited the growth of autologous tumors (Hellstrom & Hellstrom, 1969; Vanky and Klein, 1982). Despite these observations, it was clear that most tumors had only low or undetectable immunogenicity. Their immunogenicity, however, could be enhanced by various means including, but not limited to, infection with virus (for review see Schirrmacher, 1992), the use of adjuvants, or the insertion of genes encoding either cytokines or T cell costimulatory molecules (for review see Pardoll, 1993; Hellstrom et al., 1995). The important observation derived from this line of investigationwas that tumor antigens did exist, although they were not necessarily strongly immunogenic. Molecular characterization and isolation of tumor antigens that are potentially immunogenic in humans became an important goal of tumor immunologists. The incentive was the ability to use purified antigens to create effective immunogens and thus increase the immune response to a level necessary to reject the tumor. We will review our current knowledge of tumor antigens recognized by both humoral and cellular arms of the 217 Copright 0 1996 by Academic Press, Inc.

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immune system and the promise that some of these antigens currently hold for cancer immunotherapy. II. Tumor Antigens Defined by Antibodies

The search for cell surface molecules that may immunologically distinguish tumor cells from their normal counterparts has been a constant process in tumor immunology, which greatly intensified in the late 1970s with the development of the hybridoma technology (Kohler & Milstein, 1976). To the dismay of many tumor immunologists, after almost two decades of experiments this powerful technology yielded very few molecules that showed tumor-specific expression and could be justifiably called “tumor-specific antigens” (Lloyd, 1990; Old, 1981). Moreover, the majority of these tumor-specific molecules were unique to a single tumor (for review see Schreiber et al., 1988) thus being of limited value for examining tumorspecific immune responses in general. Nevertheless, they confirmed the notion that tumor specific-antigens did exist and a further search might in fact uncover some that are shared by many tumors. When “shared tumor antigens” began to be identified, they were found to be less than tumor specific. They showed high-level expression on tumors, whereas on normal tissues they were either expressed very weakly or with a very restricted distribution; thus, these antigens were referred to as “tumor-associated’ antigens. The immunogenic potential of such antigens and the ability of the immune system to target them on tumors and not on normal tissue has not been fully evaluated to this day. Furthermore, inasmuch as these antigens on tumors were almost always detected with monoclonal antibodies generated in another species, for example, human tumor antigens detected by mouse monoclonal antibodies, the immunogenic potential in the species of tumor origin of many of these antigens has remained unknown. For a small number of them, however, these questions have been addressed and information concerning their immunogenicity is available. We will limit this review to discussing only these more fully characterized molecules. They are primarily human tumor antigens. They are also good representative examples of distinct classes of molecules into which the majority of antigens described so far can be placed. The knowledge about tumor-associated antigens detected by antibodies can be and has been used in several ways: (a) the presence of the antigen has been used for detection and diagnosis of malignancy; (b) the antibody specific for the antigen has been conjugated to toxins, drugs, or radioisotopes and used for tumor therapy; (c) the antibodies have been made bispecific or multispecific and used to bring other effector mechanisms, like T cell, natural killer (NK) cells, and macrophages, to the tumor site;

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and (d) tumor-associated antigens identified as growth factor receptors or other types of signaling molecules have been used to induce tumor cell death or differentiation when bound by the specific antibody. These uses are all grouped under the term “passive immunotherapy” and have the drawback that repeated treatments are necessary and very high concentrations of tumor-specific antibodies are required for each treatment to see a limited effect (Roth, 1986; Kumer and Staerz, 1993; Waldman, 1991; Vitetta and Uhr, 1994). “Active immunotherapy” is perceived to be more desirable based on the hypothesis that induction of an immune response to the tumor antigen, if it could be achieved, would be long lived and would involve multiple components of the immune system. Active immunotherapy, however, is contingent on the immunogenicity of the tumor antigen in its host. Considering that the best known tumor antigens are “tumor associated’ rather than “tumor specific,”and that the host’s immune system has seen the antigen on normal tissue either during development or chronically throughout life, the magnitude of the immune response that can be generated to these molecules is hard to predict. Common immunological wisdom would in fact predict full tolerance to such molecules, but the experimental data appear to show otherwise. A CARBOHYDRATE ANTIGENS Carbohydrate antigens are present on tumor cells as either glycolipids or glycoproteins with the anti-tumor antibody specificity residing in their sugar moiety (Lloyd and Old, 1989).The best studied carbohydrate antigens are gangliosides and blood group antigens (Hakamori, 1985). 1 . Gangliosides These cell surface molecules are neuraminic acid-containing glycosphingolipids that consist of an oligosaccharide chain linked to the ceramide moiety that anchors them into the plasma membrane. They are abundantly expressed on a number of tumors, especially those of neuroectodennal origin-most notably melanomas, astrocytomas, and neuroblastomas-but are also expressed in the brain and other neural crest-derived tissues. As an example, malignant melanomas express GM3 (NeuAca2+ 3GalP1+ 4GlcCer) and GD3 (NeuAccr -+ 8NeuAccu + 3GalP1 + 4Glc-Cer) as their predominant gangliosides and, in addition, varying amounts of GM2 (GalNacPl + 4NeuAccu2 + 3GalPl + 4Glc-Cer) and GD2 (GalNacPl + 4NeuAca2 4 8NeuAccr2+ 3GalP1+ 4Glc-Cer), whereas normal melanocytes express mostly GM3 (Puke1et al., 1982; Carubia et al., 1984). These molecules are expressed on tumor cell membrane at very high density and for that reason deemed potentially good targets of an immune response. Although not tumor specific, their restricted distribution on normal tissue,

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or in an immunopriviledged site (brain), appeared to allow at least an operational window of specificity. The potential of these molecules to serve as targets of an immune response was initially investigated by looking for the presence of anti-ganglioside antibodies in sera from cancer patients and healthy individuals. Antibodies against GM2 and GD2 gangliosides were found in some healthy donors and in some melanoma patients (Lloyd, 1991), indicating that, at least at the B cell level, there was no tolerance to these molecules. The IgM isotype predominated in these responses indicating a T cell-independent response, not unexpected for carbohydratespecific antibodies. Furthermore, some patients immunized with whole melanoma cell vaccine generated anti-ganglioside antibodies (Tai et al., 1985). Based on these results, there has been an ongoing effort to evaluate ganglioside-based cancer vaccines. GM2 has received most attention (Livingston et aZ.,1987),even though GD2 and GD3 have also been considered. A number of clinical trials have been conducted to evaluate the conditions under which their immunogenicity could be enhanced (for review see Livingston, 1995). The most exciting results were derived from vaccination trials involving GMWBCG vaccine. Most patients developed IgM antibodies to GM2, and those with the highest antibody titer also showed longer disease-free interval and overall survival (Livingston et al., 1989, 1994). Despite this success, there is a perception that the predominantly IgM response induced with this vaccine may be an inferior response, and that induction of IgG antibodies may enhance the effect of GM2 vaccines. For that reason, GM2 has been conjugated to a carrier protein keyhole limpet hemocyanin (KLH) and administered with several other adjuvants (Helling et al., 1995). Initial studies indicate that conjugation to KLH induced a T helper cell response, the result of which was the antibody isotype switch to IgG. This is expected to provide additional effector mechanisms to the anti-tumor response, and a longer lasting antibody response. In turn, the expectation is that a more impressive clinical response will follow. 2. Blood Group Antigens These were primarily defined as tumor-associated antigens by antibody reactivity with epithelial tumors. Blood group antigens are expressed on hematopoietic and epithelial cells and their expression depends on glycotransferase activity, which in turn depends on the stage of cell differentiation or on malignant transformation (Lloyd, 1987; Cordon-Cardo et al., 1988; Gold and Mattes, 1988; Schuessler et al., 1991; Springer, 1984; Springer et al., 1995).The tumor-associated epitopes are created by carbohydrate chains decorating the polypeptide core primarily of the mucin family of glycoprotein molecules. The best studied have been the T (Thomsen-Friendenreich) and sialylated Tn (sTn) antigens that have been shown

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to be immunogenic in cancer patients (O’Boyle et al., 1992). Based on serum antibody reactivity with these molecules and a number of murine studies, clinical trials have been initiated to explore the use of chemically synthesized forms of these antigens as cancer vaccines (MacLean et al., 1992, 1993). The ability to make synthetic forms of these carbohydrates is an advantage as long as the synthetic versions mimic faithfully the naturally occurring epitope on the tumor cell (Zhang et al., 1995). As is the case with gangliosides, these molecules also require conjugation to carrier proteins and the use of strong adjuvants to elicit antibody responses. One drawback that is attributed to carbohydrate vaccines is the general difficulty in inducing carbohydrate-specific memory responses, mostly due to the lack of T cells that can recognize carbohydrates. This is presented as a serious problem considering that cancer may recur several years after vaccination, and a secondary response capable of dealing with the tumor recurrence would be the only desirable outcome of a tumor vaccine protocol. Evidence suggests that the ganglioside GD2 is a target for cytotoxic T lymphocytes (Zhao and Cheung, 1995). It is possible that helper T lymphocytes capable of reacting with oligosaccharides will be identified eventually. This may imply that induction of memory in this cell population may indeed be possible.

B. PROTEIN ANTIGENS 1 . Carcinoemby o n i c Antigen (CEA) CEA is one of the most broadly expressed molecules on human malignancies. It is also a tumor antigen that has been most extensively studied in terms of both its molecular characteristics and its potential application for diagnosis and treatment of cancer (Shievely and Beatty, 1985; Thomson et al., 1991).It is a cell surface glycoprotein of 180-kDa molecular weight, and numerous antibodies have been generated that react with either the polypeptide core or the carbohydrate determinants on this molecule. Expression of CEA is most often associated with colon, breast, and lung adenocarcinomas, but it is likely that most tumor cells of epithelial origin express this antigen. It is also expressed on normal epithelia, most abundantly in the colon, but also on other normal epithelial and endothelial cells (Majuri et al., 1994). However, in addition to drasticdy different levels of expression, there are often differences in the processing of this molecule between normal and malignant cells (Muraro et al., 1985) or between tumors originating from different organs (Hernando et al., 1994). Taking advantage of these differences may increase tumor specificity of this antigen. Even though a lot has been done with this molecule to test its immunogenicity and the consequence of the anti-CEA immune response

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on the growth of CEA-expressing tumor cells, the majority of the work has utilized human CEA in species other than humans (Hand et al., 1993; Kantor et al., 1992; Conry et al., 1994, 1995). Despite a relatively high degree of conservation between species, the human CEA is also considerably different from that of other species and those differences could be entirely responsible for its immunogenicity. Derivation of CEA transgenic mice may provide a better system for evaluating CEA immunogenicity in an animal model (Eades-Perner and Zemmerman, 1995).Concurrently, a clinical trial under way at the University of Alabama is showing preliminary evidence that CEA is immunogenic in cancer patients (A. LoBuglio and J. Schlom, personal communication). Somewhat surprisingly, all the immunization protocols have utilized some form of a recombinant molecule, expression system, or simply the CEA DNA. In all of them, the CEA is expected to be expressed by normal cells, most likely muscle cells, and yet it is hoped that the immunity generated will preferentially target tumor cells. The reasons for choosing this unaltered form of CEA as an immunogen are difficult to appreciate. It would appear more rational to re-create as an immunogen tumor-like CEA, considering that differences in CEA expression between tumor and normal cells are not simply quantitative but also qualitative. Quantitative differences may be important in the elicitation phase of an immune response, in which perhaps low levels of CEA on normal tissues are ignored by the immune system. Once the immune system is fully activated by the CEA vaccine, it is questionable if quantitative differences will remain important. On the other hand, any difference in the processing of CEA that may yield a new tumor-specific epitope, if re-created in the vaccine, might provide a required degree of tumor specificity to the activated immune response.

2. Mucins Advantages and disadvantages of CEA as a tumor antigen are very similar to those of another family of molecules, tumor-associated mucins. These molecules are expressed at very high levels on the surface of epithelial cell tumors, primarily breast, pancreas, colon, ovary, and lung adenocarcinomas (Zotter et al., 1988), and detected by numerous mouse monoclonal antibodies generated by immunization with these human tumors (TaylorPapdimitriou, 1991). They are also expressed on normal tissues, usually at lower densities, and with a different repertoire of specific epitopes (Burchell et al., 1983). Some of the epitopes appear exquisitely tumor specific (Girling et al., 1989). Mucins are very-high-molecular-weight glycoproteins that share the structural characteristic of being composed of numerous tandem repeats of varying lengths, depending on the particular mucin, and being heavily glycosylated on the repeats with O-linked carbohydrates.

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The best studied, and perhaps most promising as a tumor antigen, is the mucin MUC-1, also known as episialin, polymorphic epithelial mucin, or human tnilk fat globule, and encoded by the gene MUC-1 (Gendler et al., 1990). This is the only mucin molecule that is an integral membrane protein, others being secreted, and its normal tissue expression is confined to the apical surface of ductal epithelial cells. It is thus sequestered from the immune system, especially the humoral arm, throughout adult life. Malignant transformation of the ductal epithelial cells that gives rise to the previously mentioned tumors changes the normal pattern of MUC-1 expression. Tumor cells express this molecule in high amounts on the cell surface, the expression is not polarized, and furthermore, processing differences begin to uncover tumor-specific epitopes (for review see Finn et al., 1995). Some of these new epitopes are carbohydrate in nature and belong to the previously discussed blood group antigens. The MUC-1 unique epitopes preferentially expressed on tumor cells are located on the polypeptide core and exposed as a result of incomplete or aberrant glycosylation. These polypeptide epitopes are apparently immunogenic, as antibodies against them have been detected in cancer patients (Rughetti et al., 1993; Kotera et al., 1994). As we will discuss later, there is also evidence of a T cell response against the same or a similar polypeptide core epitope. This has prompted a number of attempts at induction of an efficient antitumor immune response using mucin immunogens. These attempts have been successful, although it is not clear how informative. With the exception of one study in which immune response to human MUC- 1,thought to be identical to the chimpanzee molecule,was generated in chimpanzees using autologous B cells transfected with the human MUC-1 cDNA (Pecher and Finn, submitted for publication), all other studies have been performed in mice (Hareuveni et al., 1990; Acres et al., 1993; Ding et nl., 1993; Apostolopoulos et nl., 1994).These studies have led to induction of immunity and protection from MUC-l-expressing tumors. Whether similar results can be achieved in cancer patients is unknown, and it is currently being tested in several clinical trials.

3. HER-2lneu This cell surface protein is a product of a protooncogene expressed in breast, ovarian, and several other types of epithelial tumors. It has homology to the epidermal growth factor receptor, and there is considerable evidence that this molecule can also function as a target for negative signaling (Stancovaski et nl., 1991; Drebin et al., 1988). Anti-HER-Yneu antibodies are thought to mimic the natural ligands of this molecule, several of which have been described (Peles et nl., 1992; Lupu et al., 1992). In addition, there is great interest in immunological targeting of tumors via this mole-

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cule. Similar to the previously discussed tumor antigens, HER-Yneu is characterized by only an increased rather than specific expression on tumors. It is weakly expressed on normal tissues (Press et al., 1990) and the challenge remains to use this molecule as an immunogen in a way that will induce tumor immunity and not autoimmunity. Induction of immunity in rats by immunization with purified rat HER-Z’neu does not appear to exert any untoward effect on normal rat tissues that express this antigen (Cheever et al., 1995). Although these experiments convincingly show the lack of tolerance to this autoantigen, they cannot appropriately evaluate the quality of the immune response. Rat tumor-rejection studies attempting to show that tumor rejection can be effected without damage to normal tissue have not yet been attempted. Expression of this antigen on normal human tissues also fails to induce tolerance, as both antibodies and T cells specific for this molecule can be found in cancer patients and, albeit more rarely, in healthy subjects (Disis et al., 1994). This encourages further manipulations of this antigen or its gene to be used as an anticancer vaccine, and several clinical trials based on this immunogen are pending. 4. Prostate-Spec@ Antigen (PSA) PSA was thought to be produced exclusively by the epithelial cells of the prostate, and its presence in the circulation at elevated levels has been used for diagnosis of prostate cancer (Wanget al., 1981). It is a glycoprotein of 33-kDa molecular weight and it is a member of the serine protease family with trypsin-like and chymotrypsin-like protease activity (Wat et al., 1986). It has become clear that PSA is a marker of steroid hormone action in many normal tissues and in tumors derived from these tissues. Androgenic hormones increase PSA transcription in prostate tumor cell lines (Hentu et al., 1992), and glucocorticoids, progestins, as well as androgens increase PSA in breast cancer cell lines (Yu et al., 1994). Moreover, PSA can be found in milk of lactating women and in amniotic fluid, and its production by normal breast can be stimulated by oral contraceptives (Yu and Diamandis, 1995a,b; Yu et al., 1995a). PSA expression has now been documented in various other tumors in addition to prostate-most frequently in breast, and some tumors of the skin, ovary, and salivary gland (Levesque et al., 1995).There is much interest in this molecule as apossible immunogen but no information is yet avadable on that subject. There is also no evidence yet of possible differences in the PSA molecules expressed on tumors versus the normal tissues. An interesting observation was made that the expression of PSA is a favorable prognostic factor in women with breast cancer (Yu et al., 1995b). This may be one reason to pursue the study of this molecule at the immunological level, others being that it is

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expressed on some very important human malignancies and its expression can be upregulated, providing the possibility of high levels of antigen.

S. Idiotypes A protein that has a unique property of being a normal molecule and yet absolutely tumor specific is the immunoglobulin receptor on B cell tumors that carries the clonally expressed idiotype, the unique antigen recognition site. This tumor antigen has been the target of passive immunotherapy with anti-idiotypic antibodies (Levy and Miller, 1990).In addition to the ability of the anti-idiotype treatment to engage the conventional effector mechanisms for tumor cell destruction, like complement and ADCC, the anti-idiotypic antibody also induced Ig signal transduction that correlated with tumor regression (Vuist et al., 1994).There are a number of other receptors, some that are members of the Ig signaling complex, like CD19 and CD20, or the growth factor receptors on epithelial carcinomas that, when targeted with monoclonal antibodies, induce tumor regression. This nonimmunological effect is considered to result in the induction of tumor dormancy through negative signaling (for review see Vitetta and Uhr, 1994).If the presence of circulating antibodies against these molecules is necessary to maintain tumor dormancy, then clearly passive immunotherapy involving repeated infusions of antibody cannot remain the treatment of choice. For this reason, the potential of these molecules to be immunogenic and to induce antibodes in vivo becomes very important. This question has been explored for the B cell lymphoma idiotypes in animal models and in patients. Immunization with purified idiotype protein under several different conditions leads to the development of anti-idiotypic immunity in vivo that is associated with tumor regression (Kaminski et al., 1987; Flamand et al., 1994; Kwak et al., 1992). The major drawback to the idiotype as a tumor antigen is the emergence of idiotype variants that lose reactivity with the antibody (Meeker et al., 1985). In that respect, active immunization with idiotype protein may have an advantage over passive therapy with infused anti-idiotypic antibody. Idiotype vaccination would likely generate a polyclonal response that will be less sensitive to single amino acid mutations. In addition to generating anti-idiotype antibodies that may induce tumor cell dormancy through negative signaling, active immunization has also been seen to generate idiotype-specific T cell responses that should provide a second antitumor effector mechanism, hopefully one with long-term memory. Idiotypic DNA vaccines have been developed and their efficacy has been tested in animal models. DNA alone has been shown to induce anti-idiotypic antibodies, but the intensity of the response is increased with the addition of plasmids encoding various cytokines (for review see Stevenson et al., 1995).

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C. MUTATED ONCOGENE PRODUCTS AS TUMOR ANTIGENS Protein products of oncogenes could have been Gods gift to tumor immunologists. Not only were they critically associated with transformation and therefore expected to be present in all tumor cells as targets, but they were also products of mutated genes and the resulting mutation in the protein could be expected to elicit the response from the immune system. Not fully appreciated on their immediate discovery because most of them were not cell surface proteins, they entered center stage when it was realized that T cells could detect their presence by recognizing the mutated peptides bound to class I MHC molecules. Unfortunately, the results of several years of investigation do not support the initial enthusiasm. We will address the reasons for this as we discuss below the three most studied oncoproteins.

1 . p53 The tumor-suppressor gene $3 is located on the short arm of chromosome 17 and serves as a negative regulator of the cell cycle. By arresting the cells in G1, it allows DNA repair (Hollstein et al., 1991; Vogelstein and Kinzler, 1992). It is thought to play an important role in suppression of malignant transformation (Finlay et al., 1989). Mutations in the p53 gene are very frequently found in human tumors, primarily with antibodies which detect high level of expression of the mutated p53 protein. The wild-type p53 protein is mostly undetectable in normal cells. The reason for this difference appears to be that subtle mutations anywhere in the p53 protein induce a profound conformational change resulting in the longer half-life, accumulation to high levels in the cytoplasm, and exposure of epitopes not accessible on the wild-type protein (Steven and Lane, 1992). All these changes are clearly noticed by the immune system as evidenced by the presence of anti-p53 antibodies in patients with breast cancer (Crawford et al., 1982; Davidoff et al., 1992), pancreatic cancer (Marxen et al., 1994), and several other cancers (Angelopoulouet al., 1994; Labrecque et al., 1993). Interestingly, antibodies are directed not only against the mutated portions of the protein (Winter et al., 1992), but also against the wild-type protein epitopes (Schlichtholtz et al., 1992). Moreover, not all patients whose tumors contain p53 mutations develop antibodies. This may be due to different outcomes of different mutations (Winter et al., 1992) or in some instances to the tumor type. Glioblastoma patients, for example, have a characteristic absence of anti-p53 antibodies in their sera despite a high percentage of glioblastoma carrying the p53 mutations (Rainovet al., 1995).There appears to be no clinical correlation between either the absence or the presence of the antibody and the course of the disease. The antibody against this tumor antigen, as in the case of

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the previously dscussed molecules, is for now simply an indication that the immune response can be elicited. The reason to pursue immunity to p53 as a possible antitumor treatment is the hypothesis that anti-p53 responses induced in the patient through the tumor presence only may not be as effective as responses induced by vaccination. Unlike the molecules previously discussed, which are all expressed on the cell surface and could be targets for specific antibodies, p53 is an intracellular protein, and it is not immediately obvious what is the utility of the anti-p53 humoral response. It could be postulated that the presence of antibody is merely diagnostic of another ongoing immune response carried out by T cells that may be causing tumor destruction and the release of the p53 protein. As we will show later, T cells specific for p53, both wild-type and mutated forms, can be generated, but their ability to recognize tumor cells has not yet been convincingly shown. Despite this, not all enthusiasm for p53-based tumor immunotherapy has been extinguished. Immunization with p53 peptides is a part of an ongoing phase I clinical trial at the National Cancer Institute.

2. c-myc and c-myb Two other oncogene products, c-myc and c-myb, both nuclear proteins and both associated with the control of cellular growth and differentiation, have also elevated expression in a wide variety of human leukemias and in some solid tumors. Antibodies specific for these proteins have been found in cancer patients (Ben-Mahrez et al., 1988, Sirokine et al., 1991). As with p53, the significance of this humoral response is not clear, and its generation may be secondary to another antitumor response the target of which may be another tumor antigen. These responses to intracellular or nuclear proteins are reminiscent of humoral responses monitored in autoimmune diseases (Tan, 1989). There is usually a strong correlation between the presence of antibody and the disease. The antibody in most instances appears not to contribute to the pathology of the disease but rather to be a consequence of an ongoing d'isease. Because experiments in animal models appeared to indicate that antibodies alone could not elicit tumor rejection, it became important to identify antigens on tumors that could serve as targets for cellular immunity. This area of research received a great boost with the development of new methods for growing tumor-specific T cell lines and clones and a better understanding of the process of antigen recognition by T cells. 111. Tumor Antigens Defined by T Cells

The majority of tumor antigens identified to date that are targets of cellular immunity have been those recognized by CD8+ tumor-specific

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cytotoxic T lymphocytes (CTLs). Identification of tumor antigens recognized by CD4’ tumor-specific T cells has also been accomplished. A number of approaches have been employed for the identification of these antigens. Most have utilized patient-derived CTLs that specifically recognize various tumors as a tool to identify tumor-specific antigens. Tumor-specific CTLs are usually isolated by culturing in vitro irradiated tumor cells with peripheral blood lymphocytes,tumor-draining lymph node cells, or tumor-infiltrating lymphocytes with the T cell growth factor IL-2. These CTL lines recognize the tumor but display minimal reactivity with “normal” tissues such as autologous B cells or fibroblasts. In addition, these CTLs do not kill the NK cell target, the erythroleukemia cell line K562. The largest number of antigens so far derived is from melanoma tumors using melanoma-specific T cells. This is in part due to the relative ease in establishing melanoma tumor cell lines that can be used for expansion of melanoma-specific T cells in vitro. Only a limited number of T cells specific for other tumors have been developed, and consenquently fewer antigens have been identified in those systems. The antigens and their most important characteristics are listed in Table I. The first and most successful approach to the identification of a gene encoding a tumor antigen utilized melanoma-specific CTLs in combination with gene transfection. This is now known as the “genetic approach.” This approach identified the melanoma tumor cell antigen MZ2-E (van der Bruggen et al., 1991). Briefly, a cosmid library was prepared with the DNA from a melanoma tumor cell line that expressed this antigen. The cosmids were transfected into a melanoma tumor cell line that had lost the expression of the MZ2-E antigen but still expressed the restricting class I MHC molecule. The transfected cells were screened for their ability to be recognized by a CTL clone specific for this antigen. By retrieving the transfected DNA, repeated rounds of transfection using smaller fragments of originally transfected DNA were possible thus leading to the identification of the gene encoding the antigen MAGE-1. By transfecting even smaller fragments of this gene and through the use of synthetic peptides, the antigen MZ2-E was identified as a nine-amino-acid peptide with the sequence EADPTGHSY (Traversari et al., 1992). Based on previous work (Seed and Aruffo, 1987), this approach has been modified to use COS-7 cells transiently transfected with cDNA libraries cloned into expression vectors containing the SV40 origin of replication (Brichard et al., 1993). The plasmid containing the cDNA insert replicates to a very high copy number in the transfected COS-7 cells that have been stably transfected with the appropriate human class I MHC molecule. Transfectants containing the tumor antigen are identified by their ability to stimulate cytokine release (usually TNF-a) from tumor-specific T cells. Once the gene encoding the antigen has

TABLE I TUMOR ANTIGENS RECOGNIZED BY HUMAN T CELLS ~

Name (gene or protein) MAGE-1 MACE3 BAGE GAGE Tyrosinase

HLA restriction A1 Cw16 A1

A2

Cw16 CW6

A2

A2 A24

B44 DR4

MART-UMELAN-A GP100/Pme117

A2 A2 A2 A2 A2

A2 GP75 MUM-I HERWneu

A2 A31 B44

HPV-16

A2 A2 A2 A2 A2

MUC-1

Unrestricted

~~

Peptide sequence

Normal hssue

Tumors

EADPTGHSY SAYGEPRKL EVDPIGHLY FLWGPRALV AARAVFLAL YRPRPRRY MLLAVLYCL YMDGTMSQV AFLPWHRLF SEIWRDIDF ND* AAGIGILTV ILTVILGVL YLEPGPVTA LLDGTATLRL KTWGQYWQV ITDQWFSV VLYRYGSFSV MSLQRQFLR EEKLIWLF IISAWGIL KIFGSLAFL YMLDLQPE'IT LLMGTLGIV TLGIVCPI -PDTRP-

Testis Testis Testis Testis Testis Testis Melanocytes Melanocytes Melanocytes Melanocytes Melanocytes Melanocytes Melanocytes Melanocytes Melanocytes Melanocytes Melanocytes Melanocytes Melanocytes NE" Epithelial cells Epithelial cells NE NE NE NE

Variable" Variable Variable Variable Variable Variable Melanomas Melanomas Melanomas Melanomas Melanomas Melanomas Melanomas Melanomas Melanomas Melanomas Melanomas Melanomas Melanomas Melanomad Breast and ovarian tumors Breast and ovarian tumors Cervical carcinomas Cervical carcinomas Cervical carcinomas Epithelial cell tumors

This antigen is expressed in variable numbers of tumors of different histological origin. Not determined. This antigen is not expressed in normal tissues. This antigen is unique and is expressed in only a single individual's tumor.

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been identified, the epitope recognized by the CTL can be established by transfecting gene fragments and using synthetic peptides to sensitize target cells for lysis by the CTL. This methodology has been very successful in identifying the majority of tumor antigens recognized by T lymphocytes to date. One of the advantages of this approach is that it leads not only to the identification of the peptide antigen but also to the gene that encodes it. However, there are some disadvantagesto this approach as well. Because of the high level of expression of the transfected genes in COS cells, it is possible to identify cross-reacting peptides that are recognized by CTL because of their high density of expression on MHC molecules. Furthermore, as we will discuss later, the structure of the naturally processed peptide antigen presented by the tumor may be post-translationally modified differently in COS cells than in tumors. This may be an important consideration as it has been shown that glycosylation may affect the recognition of both class I- and class II-presented peptides (Haurum et al., 1994; Michaelsson et al., 1994). An alternative approach to the isolation of tumor antigens recognized by T cells has been by direct identification and sequencing of the relevant peptide antigen by biochemical techniques. In this “peptide elution” approach, antigenic peptides are isolated from class I MHC molecules by acid denaturation and fractionated by reverse-phase high-performance liquid chromatography (RP-HPLC).The fractions are then tested for their ability to sensitize target cells expressing the appropriate class I MHC molecule for lysis by the relevant CTL. Those fractions that sensitize target cells are fractionated further by RP-HPLC and are then analyzed by tandem mass spectrometry. The advantage of tandem mass spectrometry, as opposed to Edman degradation, is that it is capable of identifying both the mass and the amino acid sequence of peptides even when they are present in complex mixtures. Thus, a number of candidate peptides that may correspond to the antigenic peptide can be identified and sequenced. These peptides are then synthesized and tested individually to confirm the exact antigenic sequence. This approach has been successfully employed by two groups to identify melanoma tumor antigens (Cox et al., 1994; Castelli et al., 1995). One of the major advantages of this approach is that it clearly identifies what the naturally processed form of the peptide tumor antigen is and can potentially identify post-translational modifications. However, the gene that encodes that antigen is not identified by this approach. In addition, this approach requires large numbers of tumor cells from which to isolate the class I-associated peptides and requires relatively complex technology to carry out the mass spectrometric analysis. One additional approach for the identification of these antigens has been to focus on peptides derived from cellular proteins that have the potential

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of being tumor antigens. Peptides that bind to class I MHC molecules have been shown to have a length of 9-12 amino acids and a characteristic “motif” of conserved amino acids, so-called “anchor residues,” which serve to facilitate the binding of the peptide to a particular MHC molecule. The “anchor motifs” for several class I and class I1 MHC molecules have been identified (Engelhard, 1994). This information allows one to analyze the sequence of proteins and to predict which peptides from the protein may bind to a particular class I MHC molecule. The peptides can then be synthesized and tested in in vitro binding assays for their ability to bind to a particular MHC allele. Those peptides that are shown to be capable of binding, preferentially with high affinity, are then screened for their ability to induce CTL responses in vitro or to reconstitute the epitope of tumor-specific CTL. This approach has been used to screen various oncogenic proteins, tumor and tissue differentiation antigens, as well as viral proteins expressed by virus-transformed tumors. It is important to note that peptide-specific CTLs thus elicited must be further tested for their ability to recognize tumors that express both the appropriate MHC molecule and the gene encoding the antigen for this peptide to be identified as a tumor antigen. The advantage of this approach is that it allows rapid screening of a large number of potential antigens. However, this approach is limited to known proteins and to those class I MHC molecules whose motif is known. In addition, the methods for generating peptide-specific CTLs on peptide-loaded antigen-presenting cells (APCs) may lead preferentially to the generation of low-affinity T cells that may not be able to recognize the relatively low amounts of naturally processed peptide present on the tumor cell surface, even if the peptide is correctly processed and presented by the tumor. A. MAGE, BAGE, AND GAGE These genes encode tumor antigens expressed on melanoma cells and recognized by autologous CTLs. They are expressed in various other tumors in addition to melanomas but not expressed in normal tissues except for testis and placenta. Because of the reactivation of their expression in tumor cells, it has been speculated that these antigens may represent examples of oncofetal antigens, i.e., products of genes that are normally transcribed during embryonal development, silent in adult life, but reexpressed during malignant transformation. 1. MAGE

As discussed above, one of the first human tumor antigens recognized by T cells to be discovered was the melanoma tumor antigen, MZ2-E, which was found to be a nine-amino acid peptide EADPTGHSY encoded

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by the MAGE-1 gene and restricted by the class I MHC molecule HLAA 1 (Traversari et al., 1992). This gene was found to be homologous to at least two other nonidentical genes that were termed MAGE-2 and MAGE3. The exsistence of such similar genes to MAGE-1 implied the exsistence of a family of related genes. At this point in time, there are at least 12 related genes in the MAGE family (De Plaen et al., 1994) and there may be others elsewhere in the genome (Muscatell et at., 1996). MAGE-1 also encodes a second peptide antigen (SAYGEPRKL) recognized by melanoma-specific T cells restricted by HLA-Cw01601 (van der Bruggen et al., 1994). This is not a surprising result because other antigens have been shown to be capable of providing multiple peptides that can be presented by different class I MHC molecules. The novel finding is that it is restricted by an HLA-C molecule. Very few antigens to date have been found that are restricted by HLA-C molecules compared to HLA-A or HLA-B, and this finding underscores the possibility that these molecules may also play a more important role in immunosurveillance than previously suspected, despite their relatively low expression on the cell surface. As we will see below, although this was the first, this is not the only tumor peptide restricted by an HLA-C molecule. The related gene, MAGE-3, encodes several tumor antigens as well. The MAGE-3 antigens are restricted by both HLA-A1 (EVDPIGHLY) (Gaugler et al., 1994) and HLAA2 (FLWGPRALV) (van der Bruggen et al., 1994). Except for these two genes, no other member of the MAGE family has been reported to encode an antigen that can be recognized by CTLs. However, it is possible that there are other immunogenic peptides that can be derived from these two genes as peptides derived from MAGE-1 have been shown to be capable of binding to multiple class I MHC molecules (Celis et al., 1994a). In normal tissues, the expression of the MAGE genes is limited to the placenta and testis as determined by PCR analysis of a large number of normal adult tissues and a few tissues derived from >20-week-old fetuses (Van Pel et at., 1995). In tumor cells, the expression of these genes is somewhat variable between tumors of different histological origins, but it is clear that some proportion of melanomas, bladder carcinomas, mammary carcinomas, squamous cell carcinomas, non-small cell lung carcinomas, sarcomas, and prostatic carcinomas express a number of the MAGE genes. Interestingly, these genes do not appear to be expressed well in colon or rectal carcinomas and do not appear to be expressed at all in leukemias or lymphomas. It is also of interest to note that the frequency of expression of MAGE-1, -2, -3, and -4in cutaneous melanoma is higher in metastatic lesions than in primary lesions (Brasseur et al., 1996), which is important from the prospect of immunotherapy of metastatic disease.

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2. BAGE and GAGE Two other gene families with no sequence homology to any other known genes have been reported to encode tumor antigens recognized by melanoma-specific CTLs. The first of these, BAGE, encodes a nine-amino-acid peptide AARAVFLAL that is restricted by HLA-Cw"l601 (Boel et al., 1995),another example of an HLA-C-restricted peptide. Southern blotting of DNA extracted from normal peripheral blood lymphocytes or from the melanoma tumor from which BAGE was originally isolated with the BAGE coding sequence indicated the presence of multiple hybridizing bands. This result suggests that BAGE belongs to a multigene family similar to that of the MAGE genes. The second gene, GAGE, encodes an eightamino acid peptide YRPRPRRY that is restricted by HLA-Cw6 (Van den Eynde et al., 1995). A total of five homologous genes have been isolated that are 80-98% identical to the original GAGE cDNA. Of these, only GAGE-1 and GAGE-2 encode the antigen recognized by the CTLs. In a similar pattern as the MAGE genes, BAGE and GAGE are not expressed in normal tissues except for testis and are expressed to a variable extent in a number of tumors including melanomas, bladder carcinomas, mammary carcinomas, head and neck squamous cell carcinomas, non-small cell lung carcinomas, and sarcomas. These genes are not expressed in renal or colorectal carcinomas (Boel et al., 1995, Van den Eynde et al., 1995). Additionally, the BAGE genes are not expressed in leukemias or lymphomas (Boel et al., 1995). B. MELANOCYTIC DIFFERENTIATION ANTIGENS These antigens were also identified as tumor antigens expressed on melanoma and recognized by autologous CTLs. However, unlike the antigens discussed previously, these antigens are expressed by tumors and normal cells of the melanocytic lineage but not by other tissues or tumors of different histiologic origin. The existence of these antigens was evident from studies of antimelanoma CTLs restricted by HLA-A2 that were shown to recognize several allogeneic melanomas as well as normal melanocytes thath expressed the HLA-A2 molecule (Anichini et al., 1993). 1. Tyrosinme

The first of these differentiation antigens was found to be encoded by the tyrosinase gene. This gene was identified by cotransfection of COS-7 cells with both a cDNA library derived from a melanoma tumor and a plasmid-encoding HLA-AS. The sequence of the cDNA that encoded this antigen was virtually identical to that of a previously cloned human tyrosinase gene isolated from a melanoma tumor (Brichard et al., 1993). Tyrosinase is a part of the melanin biosynthesis pathway and catalyzes the synthe-

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sis of the melanin precursor dihydroxyphenylalanine. This gene was subsequently shown to encode two antigenic peptides restricted by HLAA2.1 that were recognized by tumor-specific CTLs (Wolfel et al., 1994). Both of these peptides were 9 amino acids in length and were derived from amino acids 1-9 (MLLAVLYCL)and amino acids 368-376 (YMNGTMSQV) of the tyrosinase protein. In addition to these peptide antigens, other peptides derived froin tyrosinase have been reported to be recognized by melanoma-specificCTLs restricted by both HLA-A24 (AFLPWHRLF) (Kang et al., 1995) and HLA-B44 (SEIWRDIDF) (Van Pel et d., 1995). Interestingly, a naturally occurring HLA-A2.l-associated pepide YMDGTMSQV was identified by mass spectrometry from peptides extracted from purified HLA-A2.1 molecules expressed by melanoma cells but not from extracts from lymphoid cells. The sequence of this peptide was identical to residues 368-376 of tyrosinase except that aspartic acid (D) was found in place of the asparagine (N) predicted by the gene sequence (Skipper et al., submitted for publication). Although both forms of this peptide bound equally well to HLA-A2.1, the naturally occurring peptide containing aspartic acid sensitized target cells for lysis by CTLs at a 100-fold lower concentration than the asparagine-containing version of the peptide. Additional studies showed that the naturally occurring peptide corresponding to the tyrosinase epitope was distinct from that deduced from the gene sequence and is the only one of these two peptides to be presented by HLA-A2.1 expressed on the tumor cell surface. It is hypothesized that naturally processed species arise as a result of a post-translational modification that converts asparagine to aspartic acid. This modification is thought to be due to the enzymatic deamidation of asparagine to aspartate through the action of peptide:N-glycanase, which may act on a glycosylated asparagine. This is an important finding because it demonstrates that changes in the post-translational modification in tumor cells may lead to the generation of new antigens that could be relevant to tumor rejection. It also indicates that in some cases it may not be possible to identify certain peptide antigens directly from a DNA sequence. Tyrosinase also contains an antigen that can be presented by class I1 MHC molecules and recognized by CD4' T cells (Topalian et al., 1994). Multiple CD4+ T cell clones restricted by DR4 were shown to recognize autologous EBV-B cells pulsed with lysates of COS-7 cells transfected with tyrosinase but not COS-7 cells transfected with control genes or untransfected COS-7 cells. The ability of tyrosinase to provide antigens restricted by both class I and class I1 MHC molecules makes it an excellent target for immunotherapy or vaccination because it would presumably be able to stimulate both cytotoxic CD8+ and helper CD4' T cells simultaneously.

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2. MAR T-l/MELAN-A An additional gene that encoded a melanocyte differentiation antigen recognized by melanoma-specificHLA-A2.1-restricted CTLs derived from tumor-infiltrating lymphocytes (TILs) was identified and named MART-1 (Kawakami et al., 1994). The same gene was also identified using melanoma-specific CTLs derived from peripheral blood lymphocytes and named MELAN-A (Coulie et al., 1994). This gene encoded a novel transmembrane protein whose expression was virtually identical to that of tyrosinase as it was expressed by most melanoma tumor cell lines and normal rnelanocytes but no other normal tissue except for the retina. It was not found to be expressed in any other tumors except for melanoma. The antigenic peptide from this protein was identified by testing multiple synthetic peptides from the MART-1 protein sequence that fit the HLA-A2.1 binding motif for their ability to sensitize target cells for lysis (Kawakami et al., 1994a). From this analysis, it was determined that the peptide AAGIGILTV was the optimal peptide for T cell recognition. A second antigenic peptide from MART-1 was identified from the analysis of naturally processed peptides isolated from melanoma tumor cells using mass spectrometry (Castelli et al., 1995). This peptide was found to be ILTVILGVL and to overlap with the previously discovered MART-1 peptide in the amino acids ILTV. It was also shown that two CTL clones specific for the MART-1 peptide AAGIGILTV also recognized the MART1peptide ILTVILGVL. MART-1 is a potentially good candidate for vaccination or passive immunotherapy as this antigen was shown to be recognized by 9 of 10 melanoma-specificTIL cell lines (Kawakamiet al., 1994a). However, a recent study indicates that an even better candidate may be gp100 (Kawakami et d., 1995). 3. gplOO/Prnell7 The gene encoding this protein was originally identified as a melanocyte lineage-specificantigen recognized by the antibodies NKI-beteb, HMB-50, and HMB-45, which are used as diagnostic markers for human melanoma (Adema et al., 1993). Analysis of the cDNA of this gene revealed it to be a type I transmembrane glycoprotein that was highly homologous to another melanocyte-specific protein Pme117. Like some of the proteins previously discussed, this protein is expressed in melanoma tumors but not in other tumor cell types or normal cells with the exception of melanocytes and pigmented cells in the retina. The identification of this protein as a melanoma-specific T cell antigen was accomplished when it was demonstrated that the transfection of this gene could reconstitute the epitope recognized by a CD8' HLA-A2.1-restricted TIL cell line (Bakker et al., 1994; Kawa-

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kami et al., 1994b). An antigenic peptide from this protein was identified independently through the mass spectrometric isolation of a naturally processed peptide that was capable of reconstituting the epitope recognized by multiple melanoma-specific CTL lines (Cox et al., 1994). This peptide was restricted by HLA-A2.1 and had the sequence YLEPGPVTA. The second peptide epitope from this protein was identified by screening synthetic peptides based on the HLA-A2.1 binding motif for their ability to sensitize target cells for lysis and to stimulate IFN-.)I secretion from a melanoma-specific CTL, TIL 1200 (Kawakamiet al., 1994b). This peptide was also restricted by HLA-A2.1 and had the sequence LLDGTATLRL. An additional HLA-Ae.l-restricted peptide from gp100, KTWGQYWQV, was identified that was also capable of reconstituting the epitope recognized by TIL 1200 (Bakker et at., 1995). Because the gpl00 peptide KTWGQYWQV was capable of sensitizing target cells for lysis at a 100-fold lower concentration than was LLDGTATLRL, it was considered to be the immunodominant peptide of gpl00 recognized by TIL 1200. Other a100 peptides restricted by HLA-A2.1 have been reported (Kawakami et al., 1995). Although it is not clear which of these peptide epitopes may be immunodominant during an actual immune response, gpl00, like MART1,is potentially a good candidate for immunotherapy of melanoma because one of these peptide epitopes is recognized by five independently derived melanoma-specific CTL lines (Cox et at., 1994). In support of this, TIL 1200 and other TIL lines, which are specific for gpl00 peptides, have been shown be effective in adoptive immunotherapy (Kawakami et al., 1994b, 1995). C. MUTATEDOR ALTERNATEANTIGENS 1. gp75 The gp75 protein, a tyrosinase-related protein (TRP-l), was identified as an antigen recognized by serum IgG antibodies from one melanoma patient (Mattes et al., 1983; Vijayasyradhi et al., 1990). Similar to gpl00 and tyrosinase, gp75 is expressed in human melanocyticcells and melanoma tumors. Through a similar identification and cloning strategy as described previously, this gene was shown to encode a shared melanoma antigen recognized by HLA-A31-restricted TIL (Wang et al., 1996b). However, unlike the other melanoma differentiation antigens, the antigenic peptide recognized by the TIL was not derived from the normal gp75 protein. Instead, it was shown that the antigenic peptide, MSLQRQFLR, was derived from an alternative open reading frame that directs the translation of a small 24-amino acid protein (Wanget al., 1996b).Although the translation of overlapping reading frames has been previously described for viral

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genes (Spiropoulou and Nichol, 19931,this may be the first known example of the same phenomenon in eukaryotic cells. Alternative translation of otherwise normal cellular genes is potentially another way by which new tumor-specific antigens may be derived. 2. MUM-1 Although it has been demonstrated in mouse tumor models that point mutations can generate new antigens recognized by syngeneic CTLs (Lurquin et al., 1989; Sibille et al., 1990; Mandelboim et al., 1994), this had not been shown to occur for human tumor antigens until the identification of the MUM-1 antigen (Coulie et al., 1995). A cDNA cloned from a melanoma tumor was shown to confer the recognition of a HLA-B44restricted melanoma-specific CTL clone. The sequence of this cDNA had no significant homology to any known gene and was expressed in normal tissues including liver, colon, muscle, and heart. Through transfection of truncated cDNA clones and the use of synthetic peptides, the antigenic peptide encoded by this cDNA was identified as a nine-amino-acid peptide with the sequence EEKLIWLF. This peptide conformed to the HLAB44 binding motif (Fleischhauer et al., 1994) and sensitized HLA-B44expressing target cells for lysis. When the sequence of this gene in tumor cells was compared to that in normal cells, it was found that there was a point mutation in the tumor gene such that a serine residue was replaced with isoleucine, EEKL(S 1)WLF. Both the mutated and the normal forms of the peptide bind equally well to HLA-B44, but the normal form is not recognized by the melanoma-specific CTL. This indicates that the mutated isoleucine residue is a critical residue for the recognition of this epitope by the T cell receptor. Furthermore, the cDNA sequence encoding the antigenic peptide is found to be a part of an intron, which suggests that this peptide is derived from the translation product of an incorrectly spliced mRNA. This may be the first example of an antigen generated by a point mutation that is recognized by human CTLs. The recognition of this antigen also has implications for the role of T cells in the surveillance of translated intronic regions and thus the integrity of the genome itself. This could be especially relevant to the immune response to viruses that integrate into the host cell genome. PROTEINS D. ONCOGENIC HER2/neu, ras, and p53 have all been investigated for their ability to serve as tumor antigens. However, despite a number of reports that have shown that peptides derived from both ras and p53 can be recognized by both CD8' and CD4+ T cells (Fossum et al., 1993, 1994; Van Elsas et al., 1995; Houbiers et al., 1993), currently there is no evidence that these

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antigens are naturally processed and presented by human tumor cells. More convincing results have been achieved with the HERWneu oncoprotein. The realization that HERWneu had a role in the recognition of tumor cells came with the demonstration that the recognition of ovarian tumor cell lines by HLA-AS.l-restricted tumor-specific CTLs correlated with the level of expression of the HERWneu protein (Yoshino et al., 1994a) and from CD4' T cell responses of breast cancer patients to HERWneu peptides (Disis et al., 1994).Furthermore, it was also demonstrated that the transfection of HERWneu into HLA-A2.1t melanoma tumor cells conferred on them the ability to be recognized by ovarian-specific CTLs (Yoshinoet al., 1994a). A peptide GP2 from the HERYneu protein sequence (IISAWGIL) that had the same HLA-Ae.l-binding residues as those found in the immunogenic influenza matrix peptide 58-66 was synthesized and shown to reconstitute an epitope recognized by both breast and ovarian HLAA2.1-restricted TILs (Peoples et al., 1995). This epitope was also shown to be recognized by non-small cell lung cancer-specific CTLs (Yoshino et al., 1994b). An immunodominant HERWneu peptide restricted by HLAA2.1 (KIFGSLAFL)was also identified through a more extensive screening of HLA-A2.l-binding peptides derived from the HERWneu protein sequence (Fisk et al., 1995). Clinical trials utilizing HER2/neu peptides as an immunogen are expected to begin shortly at the University of Washington (M. Cheever, personal communication). E. EPITHELIAL ANTIGENS In the section dealing with tumor antigens detected by antibodies we discussed several epithelial antigens including CEA, PSA, and MUC- 1 mucin. Only the mucin has so far been reported to also be recognized by T cells. There is some preliminary evidence that T cells specific for CEA can also be expanded in vitro from patients vaccinated with CEA nucleotide vaccine (J. Schlom, personal communication). No evidence to date has been reported for T cell recognition of PSA. 1. M U G 1 Mucin As described previously, mucin molecules are high-molecular-weight glycoproteins expressed on epithelial cell tumors. Pancreatic, breast, and ovarian tumor cells, among others, express large amounts of one of the mucins, MUC-1, in an aberrantly glycosylatedform that uncovers an immunodominant epitope PDTRP on the polypeptide core of the protein. This epitope is immunogenic not only to B cells, as discussed previously, but also to ceil-mediated responses (for review see Finn et al., 1995). It has been demonstrated that lymph node cells of pancreatic cancer patients when stimulated with allogeneic pancreatic tumors will yield CD8+ CTL lines that will recognize and lyse mucin-expressing pancreatic tumor cell

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lines as well as breast tumor cell lines (Barnd et al., 1989) expressing the same mucin. Similar results have been obtained with lymph node cells of breast cancer patients (Jerome et al., 1991) and in patients with multiple myeloma, a tumor that also expresses mucin (Takahashi et al., 1994). Recognition of the mucin on tumor cells is not MHC restricted and cannot be blocked by antibodies to class I MHC molecules, even though it appears to be T cell receptor mediated. It is hypothesized that the highly repetitive nature of the tandem repeat region of the much protein allows for the direct crosslinlang of T cell receptors on mucin-specific T cells and their activation. This would explain the lack of MHC restriction and suggest that the recognition of this tumor antigen is essentially an aberrant function of the mucin-specific T cell receptor and as such may be more analogous to the T cell recognition of haptens such as fluorescein (Siliciano et al., 1986).In support of this model of recognition, there have also been reports of MHC-unrestricted recognition of HSV-l-infected mononuclear cells by both d/3 and y/S T cells (Maccario et al., 1993). Because of the tumorspecific expression of this T cell epitope and the lack of a need for a particular MHC molecule, this unorthodox tumor antigen may be an excellent target for immunotherapy and an immunogen for vaccination. A nineamino acid peptide was identified in the tandem repeat region on the MUC-1 polypeptide core that binds HLA-A11 and elicits T cell responses in vitro (Domenech et al., 1995). It has not yet been determined if HLAA l l t tumor cells naturally process and present this peptide. A phase I clinical trial has just been completed at the University of Pittsburgh Cancer Center testing the feasibility of a MUC-1 peptide-based vaccine for breast, pancreas, and colon cancer. One hundred micrograms of a mucin peptide composed of five tandem repeats was administered together with BCG every 3 weeks for a total of three injections. No toxicity has been observed attributed to the peptide. Iinmunogenicity of the peptide, even though not the immediate goal of this phase I trial, is currently being evaluated on samples collected from the immunized patients.

F VIRALANTIGENSAS TUMOR ANTIGENS A number of viruses have been associated with cellular transformation including human papillomaviruses (HPV) and Epstein-Barr virus. These viruses may play a role in the development of human tumors and the viral proteins that are still expressed in transfonned cells are potential tumor antigens. Most of the work to date has been done on HPV as a potential tumor antigen. 1. HPV The human papilloma viruses are DNA viruses that infect epithelial tissues. Certain types of these viruses, notably HPV-16 and HPV-18, have

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been associated with squamous cell carcinomas of the human cervix and are thought to have an important role in cervical carcinogenesis (Resnick et ul., 1990; Zur Hausen, 1991).Expression of the HPV genes E6 and E7 is constitutive in cervical tumors (Seedorfet al., 1987, Von Knebel Doeberitz et al., 1991) and required for the maintenance of the transformed state (Howley, 1991). Because of their continued expression in tumor cells, the E6 and E7 proteins are promising targets for immune intervention in cervical cancers. Because they are of viral origin, the immune response against them is expected to be exquisitely tumor specific. The immunogenicity of these proteins has been analyzed extensively (Kast et al., 1994). A set of 240 overlapping nonameric peptides derived from both E6 and E7 proteins was synthesized and tested for binding to several of the most common human HLA-A alleles. From these studies, a number of high-affinity binding peptides were determined and the immunogenicity of these peptides was tested in vivo by immunization of HJAA2.1/Kh transgenic mice and in vitro by stimulation of CTLs from normal HLA-A2.1+ human peripheral blood lymphocytes (Ressing et al., 1995). Four high-affinity binding peptides were immunogenic in the transgenic mice and three of these peptides were also immunogenic to CTLs from normal donors. Human HLA-A2.l-restricted CTL clones specific for these peptides were able to recognize and lyse peptide-pulsed targets as well as the HLA-A2.1t cervical carcinoma cell line CaSki that expresses the HPV16 E6 and E7 genes. These results strongly support these peptides as naturally processed T cell epitopes of HPV-16 and as cervical carinoma tumor antigens. A phase I and I1 immunotherapy trial using these peptides is currently being conducted at the University of Leiden Medical Center (Melief and Kast, 1995). IV. Reflections and Perspectives

When all the best known tumor antigens, discovered either by antibodies or by T cells, are put together under the magnifying glass of a review article, clear patterns emerge. The outcome of many years of searching for tumor antigens can now begin to be evaluated and certain predictions made for the future direction of this line of research. First, despite the great excitement generated by the development of cloned T cells as an additional tool with which to search for tumor antigens, the antigens discovered with this new tool, though numerous, are either exactly the same as those previously discovered by antibodies or belong to the same classes of molecules. This should not have been totally unexpected because it is just another illustration of the way the immune system works. Responses to most antigens, with the exception of some carbohydrates,

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come about through antigen processing and presentation by antigenpresenting cells, including antigen-specific B cells. Amplification of the response is accomplished through cognate T-B cell interactions in which a B cell, having bound antigen through surface immunoglobulin, presents fragments of the antigen bound to MHC to T cells specific for the same antigen (Croft and Swain, 1992). This interaction encourages a response of both arms of the immune system to the same antigen only to different epitopes. Which response i s more important in tumor immunity, humoral or cellular, has been a topic of discussion that has received more attention than necessary. It would be most reasonable to assume that the effective immune response against tumors would need to include both arms of specific immunity, T cells and antibodies. They in turn would uniquely recruit the participation of numerous nonspecific effector mechanisms that would further amplify the response and add to its effectiveness. There is a wealth of data, especiaIly in the mouse models, that show the importance of specific antibodies or specific populations of T cells in tumor immunity. On the contrary, there are absolutely no data that conclusively show that any of these immune effectors can work alone. When induction of an antibody response against a tumor antigen leads to tumor regression or protection from tumor growth, the cause and effect relationship is clear. This is not to say, however, that the destruction of the tumor was antibody mediated and that it did not also include a cellular response, generated in vivo, secondary to the antibody response, either against the same tumor antigen targeted by the antibody or against other tumor antigens that are still unknown. One of the more successful clinical trials to date employed treatment of colorectal cancer patients postoperatively with 17-1A antibody directed against a tumor-associated glycoprotein (Herlyn et al., 1979). Antibody therapy extended life and prolonged remission in treated patients (Riethmuller et al., 1994). It is tempting, therefore, to laud this antibody as a good antitumor effector molecule, except for the fact that the exact effector mechanism responsible for tumor rejection is not really known. In this particular case, there is ample evidence that a group of patients that responded to the antibody treatment with tumor regression all developed T cells specific for the idiotype of the injected antibody (Fagerberg et al., 1995). These T cells also recognize the 17-1A antigen and may be the effector mechanism responsible for tumor rejection. Similarly, when adoptive transfer of a single clone of tumor-specific cytotoxic or helper T cells is credited with tumor eradication (Kahn et al., 1991), one cannot be certain that this clone worked alone. There is always a possibility that a limited effect of the T cell clone on the tumor initiated an in vivo immune

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response that produced tumor-specific antibodies about which we know very little and that may have contributed to tumor elimination. Second, and an unexpected outcome, is the realization that the majority of shared tumor antigens are self-antigens. In retrospect, this outcome should have been expected. Tumor growth is an expansion of cells of a particular tissue bearing tissue-specific antigens beyond their anatomical barriers and to greater than physiological numbers. Immunological tolerance to tissue-specific antigens is in part regulated either by a low level of expression of these antigens or by their sequestration in immunopriviledged sites. Tumor growth changes both parameters and thus tolerance is broken. Or is it? Despite the presence in cancer patients of both antibodies and cytotoxic T cells specific for self-antigens carried on tumor cells, they appear to have no obvious effect either on the tumor that continues to grow and eventually kills the patient or on the normal tissues expressing the same antigen. One possibility to consider is that self-reactive B and especially T cells are rendered unresponsive in the periphery on encounter with low levels of antigen on normal tissues in the absence of costiinulatory molecules. The anti-tumor antibodies and T cells specific for self-antigens may be those that have encountered these antigens on or around the tumor where a certain level of activation was possible. In particular, they may have been activated by higher than normal expression of antigen. The second possibility is that these antibodies and T cells are remnants of an antitumor immune response that was defeated at the start by the large load of antigen on tumor cells that were already too numerous when the tumor broke out of its original site. Due to a large number of tumor cells, heavy antigen load, the chronic presence of antigen, or the hostile environment that the tumor creates by producing immunosuppressive cytokines, amplification of this antitumor immune response did not take place. The frequency of T cells or the titer of the antibodies specific for the antigens we have described is usually very low compared to responses generated against viruses or other foreign antigens. Furthermore, it has now been repeatedly shown that in many cancer patients, not only tumor-specific T cells, but T cells in general are abnormal in the way they process signals through the T cell receptor (Mizoguchi et al., 1992 ). Considering any or a11 of these possibilities, is there a role for the so far identified tumor antigens in tumor immunotherapy? With the disclaimer that the data available for most of the reviewed antigens are still preliminary, and basing our judgment primarily on some longer running studies, we must conclude that these antigens can definitelyparticipate in antitumor immunity, their individual contributions being relative depending on the form of immunity necessary for a particular tumor or stage of the disease.

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Many of the tumor antigens that are cell surface proteins have been successfully targeted by infusion of antibodies specific for those antigensthe process known as passive immunotherapy. Large numbers of tumorspecific cytotoxic T cells have also been infused into patients and this too has resulted in tumor regression. Neither treatment affected in any obvious way the normal tissues. Thus, both sets of antigens, those seen by antibodies and those seen by T cells can be targets of premade effector molecules, antibodies, or T cells (“passive immunity”). Because we limited our review primarily to antigens known to be immunogenic in patients, it is obvious that they are also candidate immunogens for eliciting immunity in patients through vaccination (“active immunity”). The ultimate success of a vaccine depends on the presence of fully immunocompetent T cells and B cells specific for the tumor antigen used as immunogen. Inasmuch as most antigens are normal self-proteins, the extent of the immune response that will be possible to induce against them is still unknown. The most important role, however, that the tumor antigens so far identified may have is as catalysts for in vivo antitumor immune responses against potential new antigens through a process that could be named “provoked immunity.” T cell tolerance is an active process that depends on a certain level of presentation of the self-antigens to the immune system-enough to either induce deletion of specific T cells or anergy (Lanzavecchia, 1995). It can be postulated, and it has been experimentally shown in model antigen systems (Sercarz et al., 1993), that many self-antigens are either not processed and presented or are presented below the threshold of detection. T or B cells specific for these antigens are in the peripheral repertoire but they remain unstimulated. The same situation can be envisioned for many unknown but postulated tumor-specific antigens. Even for some known antigens, like the mutated oncoprotein ras, it appears that the presentation at subthreshold levels on tumors may be responsible for its lack of recognition by T cells. There is ample evidence, however, that cryptic epitopes do get presented under certain conditions (Sercarz et al., 1993), the most recent example involves the HIV-gpl20 molecule (Salemi et al., 1995). It appears that human T cell clones isolated from HIV patients and specific for DR-restricted CD4 epitopes recognize these epitopes only on B cells that have processed and presented engineered CD4, but not on CD4’ T cells that express this molecule and should be expected to present these epitopes. Thus, CD4 is processed differently when captured by B cells. Interestingly, the T cell clones can recognize the CD4 epitopes on T cells when it is downregulated by HIV-gpl20 ligation or anti-CD4 antibodies. This is thus an example of increased processing and presentation of cryptic epitopes on a self-molecule induced by an external ligand. Most

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of the antigens we have discussed that have been defined by antibodies have the potential to present new epitopes to the immune system when bound by the antibody on the tumor cell itself or when bound by the antibody in their soluble form and processed by APCs. HER-Yneu, for example, is a cell surface molecule and antibodies found in cancer patients are directed to the epitopes on the extracellular domain of the molecule. However, in patients treated with anti-HER-Yneu antibody, additional antibodies develop that are specific for the previously cryptic intracellular domain epitopes (Cheever et al., 1995). “Provoked immunity” may be the comon endpoint, and thus a common denominator of a number of approaches that have been used to elicit tumor rejection. One approach has involved heat shock proteins, primarily hsp70 and gp96 (Udono and Srivastava, 1994, Srivastavaet al., 1994).These molecules have been shown to bind numerous peptides and purification of these molecules from tumor cells and their uptake and reprocessing by APCs leads to stimulation of tumor-specific CTLs. In this case as well, one can postulate presentation by APCs of unknown cryptic epitopes carried on peptides bound to the heat shock proteins that on the tumor cell were not presented above the threshold level necessary to activate the immune response. This approach does not depend on the knowledge of a specific tumor peptide bound to the heat shock proteins, but it would be of interest to use the provoked immunity, which is the result of the vaccination with tumor-derived hsp70 or gp96, to identifjr the target antigens involved in the tumor-rejection response. Another approach that leads to provoked imunity has been to use as an immunogen tumor cells modified by transfection with various cytokines (for review see Pardoll, 1993; Cavalo et al., 1994), costimulatory molecules B7-1 and B7-2 (for review see Hellstrom et aZ., 1995), or class I1 antigens (Baskaret al., 1994).All of these methods are independent of the knowledge of a particular tumor antigen and routinely induce tumor immunity in mice. When applied to human tumors, they may help identify new tumor antigens, targets of the in vivo provoked immunity responsible for tumor rejection. The importance of better understanding the provoked immunity is that it apparently leads to tumor rejection. None of the antigens we have reviewed can be called tumor-rejection antigens because they were identified by antibodies and T cells from cancer patients who succumbed to their tumors. When these antigens are used as targets for passive immunotherapy or as immunogens in active immunotherapy and when a rare tumor-rejection response is observed, it will be very important to study the immune system following the tumor rejection. We may find that the tumor-rejection response may indeed be against some of those antigens.

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More likely, we will find that the majority of them only initiated a process of tumor destruction that led to antigen release, cryptic antigen presentation-in other words provoked immunity. The targets of that immunity are the real tumor-rejection antigens. They may turn out to be better at inducing immunity and may replace the antigens we know now. On the other hand, they may all turn out to be unique rather than shared, in which case we can fall back on the shared antigens described here but with a greater understanding of what it takes to achieve tumor rejection.

Drawing by S. Gross; 0 1995 The New Yorker Magazine Inc

ACKNOWLEDGMENTS This work was supported by Grants NIH R 0 1 CA56103 and NIH R 0 1 CA57820 to O.J.F. and an American Cancer Society Fellowship to R.A.H. We are grateful to d l our colleagues who sent manuscripts and shared unpublished observations. We thank Ms. Sonja Finn for help in literature collection and members of the Finn Laboratory for critically reading the manuscript. O.J.F. is a member of the Immunology Program of the Pittsburgh Cancer Institute and Faculty of the American Cancer Society.

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