PERSPECTIVES rejected by an immune response directed TIMELINE against new antigens that were different for every variant (tum– antigens). Remarkably, Tumour antigens recognized by mice that had rejected tum– variants were also protected against a subsequent injection of the parental tumour cells7, even though T lymphocytes: at the core of this teratocarcinoma was non-immunogenic, similar to the tumours that were described cancer immunotherapy by Hewitt. We concluded that an efficacious response against the tum–­ antigens had an Pierre G. Coulie, Benoît J. Van den Eynde, Pierre van der Bruggen additional effect: it triggered a response and Thierry Boon against antigens that were present on the original tumour but that were apparently Abstract | In this Timeline, we describe the characteristics of tumour antigens that non-immunogenic on their own. In col- are recognized by spontaneous T cell responses in cancer patients and the paths laboration with Hewitt, we treated cells from that led to their identification. We explain on what genetic basis most, but not all, spontaneous tumours with mutagens to – of these antigens are tumour specific: that is, present on tumour cells but not on obtain tum variants, and we observed that these variants were also capable of induc- normal cells. We also discuss how strategies that target these tumour-specific ing immune protection against the parental antigens can lead either to tumour-specific or to crossreactive T cell responses, tumours8. This showed that spontaneous which is an issue that has important safety implications in immunotherapy. mouse tumours do express tumour anti- These safety issues are even more of a concern for strategies targeting antigens gens, albeit poorly immunogenic ones. We that are not known to induce spontaneous T cell responses in patients. became convinced that human tumours might also be susceptible to immunological treatment and that we should first identify Cancer immunotherapy that involves the induced with oncogenic viruses showed the nature of the rejection antigens that were deliberate use of the adaptive immune system that the immune system could reject these observed on the mouse tum– variants and to reject tumours or to prevent their recur- tumours following the recognition of viral their parental tumour. rence is gaining momentum. Interesting antigens1. The first evidence that mouse clinical results have been obtained using tumours that were not induced by viruses Molecular identification of antigens recog- cancer vaccines, adoptive T cell therapies and could also be recognized by the immune nized by T lymphocytes on mouse tumours. antibodies that stimulate the activity of system was obtained by Gross and colleagues After the discovery of T lymphocytes in the T lymphocytes. Moreover, increasing evi- in 1943 (FIG. 1 (TIMELINE)). They induced 1960s9,10, their essential role in graft rejection dence suggests that adaptive immunity con- tumours in mice through the use of chemi- and tumour rejection was soon realized11,12. tributes to the long-term clinical benefits of cal carcinogens and then resected these In the tum– system, we observed that adop‑ anticancer treatments such as chemotherapy tumours. These mice were able to reject the tive transfer of T cells, which were collected and radiotherapy. At the core of these clini- same tumour cells on subsequent exposure2. from mice following rejection of a tum− cal developments lies the fact that cancer Mice that were immunized with lethally variant, protected irradiated mice against patients can produce T lymphocytes that irradiated tumour cells were similarly pro- the growth of the same variant. This clearly recognize tumour-specific antigens. The first tected. These results were confirmed by indicated the involvement of T lymphocytes human tumour-specific antigens that were other groups3, and in the 1960s it became in the tum− phenomenon. Accordingly, for recognized by T cells were discovered about widely accepted that mouse tumour cells and several years, we attempted to obtain specific 20 years ago (FIG. 1 (TIMELINE)). Considering therefore possibly human cancer cells could cytolytic T cells that were directed against the increasing number of clinical studies be recognized by the immune system. tum− variants; this was unsuccessful. We that rely on the presence of tumour-specific In sharp contrast, in 1976, Hewitt4 eventually turned to the P815 mastocytoma antigens that are recognized by T cells, it is reported that a similar analysis carried out cell line, which proved to be remarkably easy worth summarizing the key steps that led to with spontaneous tumours that developed to cultivate and to clone because it prolif- their identification, and it is worth describ- in mice failed to produce any evidence of erated in suspension. Tum− variants were ing the genetic processes that result in their immune control. He concluded that mouse readily obtained for this cell line13. Moreover, presence on tumour cells. A proper under- tumour antigens were artefacts that were excellent cytotoxic T lymphocyte (CTL) standing of the factors that affect the degree induced by the chemical treatment used responses were obtained that showed clear of specificity of the T lymphocyte response to induce experimental tumours and were specificity for each tum− variant14. against tumour antigens is essential to aid therefore unlikely to be present on human We then benefited from a major advance the design of immunotherapy strategies that tumours. in the CTL field: microcultures could be are not only efficient but also free of adverse In the 1970s, we treated a mouse terato­ derived from a single CTL by repeated side effects. carcinoma cell line in vitro with a strong stimulations with irradiated target cells mutagen, and we showed that many cell in the presence of a T cell growth factor Identification of mouse antigens clones that were derived from the mutated that was later identified as interleukin‑2 Initial controversy about the existence of population were incapable of forming (IL‑2)15. These clonal CTL cultures could tumour rejection antigens. From 1940 to progressive tumours when injected into be expanded to large numbers and could be 1960, the study of mouse tumours that were syngeneic mice5,6. These ‘tum–’ variants were maintained indefinitely. These stable CTL NATURE REVIEWS | CANCER VOLUME 14 | FEBRUARY 2014 | 135 © 2014 Macmillan Publishers Limited. All rights reserved PERSPECTIVES clones that were directed against a single The next step was to define the molecular The first gene that encoded a tum– anti- antigen proved to be crucial for a rigorous nature of these antigens. The only available gen was cloned in 1988 (REF. 25). It encoded analysis and dissection of the antigens tools were the stable CTL clones. The exact a ubiquitous protein of unknown function. recognized by T cells on several target cells. molecular nature of the antigens that were Crucially, the coding region contained a With great help from Cerrotini and his recognized by CTLs was unknown at that mutation that changed one amino acid group, who had had a prominent role in time. However, the notion that antigens are in the protein. Small peptides that contained these developments, we obtained stable recognized by T lymphocytes in associa- the mutated residue were shown to sensitize CTL clones that killed the stimulatory tion with major histocompatibility complex parental P815 cells to CTL-induced cell tum– variant but not the other tum– variants (MHC; human leukocyte antigen (HLA) in death, whereas corresponding wild-type nor the parental tumour cells16. These CTL humans) molecules had been known for a peptides did not26. We concluded that the clones clearly recognized a tum– antigen decade21. In 1986, Townsend showed that antigen was a complex between the mutated that was induced by the mutagen treat- antiviral CTLs recognized small peptides of peptide and the presenting MHC class I ment. Other CTL clones killed both the eight to ten amino acids, which were derived molecule. tum– and parental cells, evidently recog- from a viral protein and presented at the The identification of two other tum– nizing an antigen that was present on the surface of infected cells in association with antigens27,28 indicated that each of them also original P815 tumour cells. That these MHC class I molecules22. Soon thereafter, an resulted from a point mutation in a ubiqui- antigens were genuine rejection antigens excellent crystallographic study showed that tously expressed gene. Each mutation cre- was shown by the in vivo observation that MHC class I molecules present small pep- ated a new antigenic peptide. In some cases, some tumours progressed, then nearly com- tides in a groove that is located at the surface the mutation enabled the peptide to bind pletely regressed, then progressed again. of the molecule23. We now know that these to the groove of the presenting MHC mol- These ‘escaping’ tumours had invariably peptides are produced by partial digestion ecule. In other cases, the mutation created lost the antigen that was recognized by one of the parental protein, mainly through the a new epitope in a peptide that was already of the CTL clones17. This was true not only proteasome machinery (BOX 1). These pep- bound to MHC, but the wild-type peptide for tum– antigens but also for antigens that tides then become associated with the MHC was not recognized by T cells because of were present on the parental tumour18. In class I molecule and are displayed at the cell central tolerance (FIG. 3a). Even though fact, these and other studies that were car- surface following a process known as the tum– antigens were artificially induced by ried out in the early 1980s formally showed ‘antigen processing pathway’ (FIG. 2). mutagen treatment, their identification the reality of tumour immune surveillance To identify our antigens, we used a genetic established the principle that rejection anti- and the occurrence of tumour escape after approach that aimed to clone the gene encod- gens can result from mutations in ubiqui- immune selection19, which is a process that ing the antigen.