You are here

Article Text

Article menu

Download PDFPDF

Immune responses to tumour antigens: implications for antigen specific immunotherapy of cancer
Free
  1. D Jäger,
  2. E Jäger,
  3. A Knuth
  1. II. Medizinische Klinik, Hämatologie-Onkologie, Krankenhaus Nordwest, Steinbacher Hohl 2–26, 60488 Frankfurt am Main, Germany
  1. Dr E Jäger EJ200161{at}aol.com

Abstract

Tumour associated antigens recognised by cellular or humoral effectors of the immune system are potential targets for antigen specific cancer immunotherapy. Different categories of cancer antigens have been identified that induce cytotoxic T lymphocyte (CTL) responses in vitro and in vivo, namely: (1) “cancer testis” (CT) antigens, expressed in different tumours and normal testis, (2) melanocyte differentiation antigens, (3) point mutations of normal genes, (4) self antigens that are overexpressed in malignant tissues, and (5) viral antigens. Clinical studies with peptides and proteins derived from these antigens have been initiated to study the efficacy of inducing specific CTL responses in vivo. Immunological and clinical parameters for the assessment of antigen specific immune responses have been defined—delayed type hypersensitivity (DTH), CTL, autoimmmune, and tumour regression responses. Specific DTH and CTL responses and tumour regression have been observed after the intradermal administration of tumour associated peptides alone. Peptide specific immune reactions were enhanced after using granulocyte macrophage stimulating factor (GM-CSF) as a systemic adjuvant by increasing the frequency of dermal antigen presenting Langerhans cells. Complete tumour regression has been observed in the context of measurable peptide specific CTL. However, in single cases with disease progression after an initial tumour response, either a loss of single antigens targeted by CTL or of the presenting major histocompatibility complex (MHC) class I allele was detected, pointing towards immunisation induced immune escape. Cytokines to modulate antigen and MHC class I expression in vivo are being evaluated to prevent immunoselection. Recently, a new CT antigen, NY-ESO-1, has been identified on the basis of spontaneous antibody responses to tumour associated antigens. NY-ESO-1 appears to be one of the most immunogenic antigens known to date, with spontaneous immune responses observed in 50% of patients with NY-ESO-1 expressing cancers. Clinical studies have been initiated to evaluate the immunogenicity of different NY-ESO-1 constructs to induce both humoral and cellular immune responses in vivo.

  • tumour antigens
  • antigen specific T cell response

Statistics from Altmetric.com

    Request Permissions

    If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.

    Spontaneous immune responses against human tumours have been reported in different types of cancer, especially in melanoma and renal cell carcinoma,1,2 but also in other types of cancer, such as non-small cell lung cancer, bladder carcinoma, and breast cancer, indicating the specific interaction of the immune system with antigenic determinants presented by the tumour.3,4 The first evidence for antigen specific immune responses arose from studies with cultured melanoma cells, which were lysed by autologous CD8+ T cells in vitro in a major histocompatibility complex (MHC) class I restricted fashion. As a clinical extension of this observation, single patients with metastatic melanoma were vaccinated with irradiated autologous tumour cells for an extended period of time. Two patients (SK-29/MEL-AV and MZ-2/MEL-GH) with refractory metastatic melanoma have been followed by our group since 1978 and 1982, respectively.5,6 A complete regression of all tumour manifestations was achieved after prolonged immunisation with autologous tumour cells in both patients, which has been maintained for 20 and 15 years, respectively. In a collaborative effort, a systematic search was initiated to identify and characterise the cancer antigens and immune effector mechanisms mediating tumour regression in vivo.6–8

    Tumour antigens defined by specific T cell responses

    CANCER TESTIS ANTIGENS

    The specific lysis of cultured tumour cells by autologous cytotoxic T lymphocytes (CTL) in vitro was first observed in melanoma systems.6 Antigenic peptides presented by MHC class I and II molecules have been identified as the target structures for CTL recognition. The first antigen defined by CTL in the context of histocompatibility leucocyte antigen A1 (HLA-A1) was isolated from a melanoma cell line derived from patient MZ-2, designated MAGE-1.7 Later, a group of related genes (BAGE, GAGE) was identified, encoding antigens expressed in melanomas and several other tumours, but not in normal tissues except for the testis.7,9–12 Therefore, antigens of the MAGE pattern of expression are designated “cancer testis” (CT) antigens. More recently, a new CT antigen, NY-ESO-1, was cloned from an oesophageal cancer by the serological expression cloning method, an approach based on the screening of recombinant tumour cDNA libraries for specific interactions with autologous serum antibodies.13,14 HLA-A2 binding peptides derived from NY-ESO-1 were found that induce strong CTL responses in vitro.15 Because NY-ESO-1 and members of the MAGE gene family are frequently expressed in tumours of different histological type, they are attractive targets for antigen specific immunotherapy of cancer.

    MELANOCYTE DIFFERENTIATION ANTIGENS

    A second group of antigens, first cloned from the SK-MEL-29 system, expressed during melanocyte differentiation was identified as targets for autologous CTL in melanomas.16–19 Epitopes derived from self antigens such as Melan A/MART-1, tyrosinase, gp100/Pmel17, and gp75/TRP-1 have been found to be targets for CTL and tumour infiltrating lymphocytes (TIL) in the context of HLA-A2.1 and other MHC class I molecules.20–22 Phase I clinical trials in patients with melanoma using antigenic peptides injected intradermally have shown that specific delayed type hypersensitivity (DTH) reactions can be elicited.23 Granulocyte–macrophage colony stimulating factor (GM-CSF) used as a systemic adjuvant enhanced peptide related DTH reactions in single patients.24 In contrast to phase I clinical trials with MAGE derived peptides, where peptide specific CTL were rarely identified, the induction of peptide specific CTL was often observed after immunisation with peptides derived from Melan A/MART-1 and tyrosinase.23,24 Furthermore, objective tumour regression was observed in single patients under continued immunisation.23–26

    POINT MUTATIONS

    Several cancer antigens are defined by point mutations of constitutive cellular proteins, leading to strong CTL responses against tumour cells in patients with cancer or experimental animals.27–29 In breast cancer, mutations of the p53 and Ras proteins have been reported. Humoral immune responses to the mutated and the wild-type proteins occurring spontaneously in patients with breast, lung, and gastrointestinal cancer have been detected.30,31 In women with a family history of breast cancer, antibody responses to p53 occur with a higher incidence than in controls (11% v 1%).30 Because most anti-p53 antibodies detected are of the IgG type, a CD4+ T cell response to p53 can be predicted. In single patients with breast cancer with an overexpression of p53 in primary tumours, a proliferative CD4+ T cell response to wild-type p53 was demonstrated.32 These findings suggest that immune responses occur after the mutation of oncoproteins. These may also recognise non-mutated portions of the proteins. It is still unknown whether intracellular p53 is also presented at the cancer cell surface or in the extracellular cancer environment to serve as a target for humoral and/or cellular effectors to mediate tumour regression.

    Mutant p53 has been shown to induce specific CTL responses that mediate lysis of the transformed cells in animal models. In a murine sarcoma model, vaccination with p53 peptides combined with interleukin 12 (IL-12) has led to regression of p53 expressing advanced Meth A sarcomas.33 In many human cancers, the accumulation of wild-type p53 in the cytosol is seen. It is assumed that p53 can be effectively presented by MHC class I molecules to elicit specific CTL responses. Therefore, immune responses against wild-type p53 may be of benefit in the treatment of cancers with p53 accumulation.

    Ras mutations involve single amino acid substitutions, mostly at positions 12 and 61. These are less complex than in p53 and thus easier to evaluate. CD4+ and CD8+ T cell responses mediating tumour cell lysis can be induced by immunisation with mutant Ras peptides in animal models.34 In humans, it remains to be determined whether wild-type or mutant Ras protein is a useful target for active or passive immunotherapy. In a small number of patients with metastatic pancreatic cancer, Ras specific proliferative T cell responses were documented after immunisation with MHC class I restricted Ras peptides.35

    Other mutation induced antigens defined primarily through CTL recognition—MUM-1 and mutated cyclin dependent kinase 4 (CDK4)—have been shown to be new peptide epitopes presented by MHC class I molecules. It remains to be determined whether these antigens will be useful targets for CTL based vaccines in a larger patient population.27,29

    OVEREXPRESSED SELF ANTIGENS

    Many tumours abundantly express normal self proteins. The most extensively studied self antigens that are targets for active and passive immunotherapy are Melan A/MART-1, a melanocyte differentiation antigen present in melanoma and normal melanocytes, and HER-2/neu, a growth factor receptor overexpressed in 30% of breast and ovarian cancers and a variety of other adenocarcinomas.30 Immune reactions directed against these antigens may result in the damage of normal tissues. However, preliminary experiences with peptide immunisation in patients with Melan A/MART-1 expressing melanomas have not shown adverse reactions directed to normal tissues, except for the development of vitiligo in single patients.23 Spontaneous humoral and cellular immune responses in patients with HER-2/neu expressing tumours have been described. They may be amplified by appropriate immunisation strategies, possibly leading to tumour regression.30

    VIRAL ANTIGENS

    Viral diseases are associated with different malignancies in humans—for example, Epstein-Barr virus (EBV) with Burkitts lymphoma,36 hepatitis B and C viruses (HBV, HCV) with hepatocellular carcinoma,37,38 human papilloma virus (HPV) with cervical and anal carcinoma,39 and human T lymphotropic virus (HTLV) with T cell leukaemia. Independent of whether the viral infection is the oncogenic agent, it was shown that viral antigens are expressed in the associated tumours and can be used as targets for preventive or therapeutic vaccination.40

    Development of immunotherapeutic strategies

    PEPTIDES DERIVED FROM CT ANTIGENS

    Peptides derived from MAGE-1 and MAGE-3 have been used alone or combined with different adjuvants—GM-CSF and QS21—for immunisation in HLA-A1 positive patients with MAGE expressing tumours. Tumour regression has been observed in more than 30% of patients with melanoma after immunisation with the MAGE-3 derived, HLA-A1 restricted peptide.41 However, MAGE-3 specific CTL were not detected in response to the vaccine in these patients.42 In a subsequent study with systemic GM-CSF to improve antigen presentation by enhancement of CD1a+ dermal Langerhans cells, followed by intradermal administration of MAGE-1 and MAGE-3 peptides, a partial regression of liver and lung metastases was achieved in a patient with melanoma within three months of immunisation (E Jäger et al, unpublished data). Correlating with this remarkable clinical development, MAGE-1- and MAGE-3 specific CTL were detected in this patient, and these cells showed an increased frequency after immunisation. Currently, a subsequent phase I study is being initiated to evaluate immune reactions to peptide vaccination in patients with other MAGE expressing carcinomas, such as breast, bladder, non-small cell lung, and head and neck cancer.

    MAGE-1 and MAGE-3 specific CTL were repeatedly detected in the peripheral blood of patient MZ2, whose melanoma cell line gave rise to the discovery of the MAGE gene family.7,10 This suggests that antigen specific CTL may be effective mediators of tumour regression because this patient experienced a complete regression of metastatic, MAGE-1, and MAGE-3 positive melanoma after repeated immunisation with irradiated autologous, MAGE-1/MAGE-3 expressing tumour cells. During the course of continued tumour cell vaccination, increased frequencies of CTL against autologous tumour cells were detected in the peripheral blood of this patient.43 However, the specificity of CTL responses could not be determined at that time because the structure of the antigenic determinant(s) was unknown. The infrequent detection of CTL against MAGE genes in patients with MAGE expressing melanoma may be a consequence of either a low immunogenicity of MAGE genes, or a frequency of CTL precursors below the level of detection. Different methods for the assessment of MAGE specific CTL responses are being evaluated. A sensitive approach appears to be the ELISPOT assay, an enzyme linked immunosorbent assay that visualises direct antigen–T cell receptor interaction by staining of the spot-like release of interferon γ (IFN-γ) or other cytokines by the T cell, interacting specifically with its target antigen.

    TARGETING MELANOCYTE DIFFERENTIATION ANTIGENS

    Tumour regression in single patients with melanoma has been achieved after adoptive transfer of TIL lines with specificity for gp100/Pmel17, tyrosinase, and gp75 derived epitopes, suggesting that melanocyte differentiation antigens are tumour rejection antigens.16,44,45 To study the effects of T cell interactions with melanocyte differentiation antigens in vitro and in vivo we undertook the following investigations. We compared the baseline CTL reactivity against HLA-A2 restricted peptides derived from melan A/MART-1, tyrosinase, and gp100/Pmel17 in HLA-A2 positive patients with melanoma and healthy individuals,46 and determined CTL responses to melanoma associated peptides injected intradermally as a vaccine in HLA-A2 positive patients with melanoma.23,24 In addition, we compared changes of expression of melanoma associated antigens and peptide presenting MHC class I molecules in melanoma tissues showing regression or progression in the presence or absence of detectable antigen specific CTL responses in vivo.47

    First, the spontaneous CTL reactivity against melanoma associated peptides was determined in patients with melanoma and in healthy individuals. Baseline CTL reactivity against the differentiation antigens Melan A/MART-1, tyrosinase, and gp100/Pmel17 is frequently detected in patients with melanoma and in healthy individuals, without significant differences in intensity and frequency of CTL responses.46,48,49 In healthy individuals, Melan A/MART-1 specific CTL, which lysed Melan A/MART-1 positive melanoma cells were isolated from depigmented skin (vitiligo areas).50 These findings indicate that CTL responses against self antigens may occur spontaneously, and might be amplified by appropriate vaccination.

    Peptides derived from Melan A/MART-1, tyrosinase, or gp100/Pmel17 can induce DTH reactions and specific CD8+ CTL responses after intradermal immunisation. Objective clinical responses were found to be associated with measurable CTL responses to the vaccine. Major toxicity of the vaccine was not observed. However, some patients with favourable clinical picture developed vitiligo.23,24 In a single patient, a clonal expansion of a Melan A/MART-1 specific T cell receptor Vβ16 was identified in T cell cultures stimulated with Melan A/MART-1 peptide, from Melan A/MART-1 specific DTH reactions, and from vitiligo areas after continued immunisation with Melan A/MART-1 peptide for five years.51

    Dermal CD1a+ antigen presenting cells (APCs), such as Langerhans cells, can be enhanced and activated by GM-CSF in vivo.52 Combined administration of melanoma associated peptides and GM-CSF resulted in the amplification of DTH reactions and CD8+ CTL responses. Immunohistochemical characterisation of DTH reactions showed infiltrates of CD4+ and CD8+ T cells and a strong expression of IL-2 and IFN-γ, suggesting the activation of CD4+ T helper type 1 (Th1) cells and CD8+ CTL by the immunisation peptides presented by MHC class I molecules of dermal APCs.24

    AUTOLOGOUS AND ALLOGENEIC WHOLE TUMOUR CELL VACCINES

    Despite the increasing number of tumour antigens defined in different types of tumours, many investigators have returned to the approach of active immunisation using autologous or allogeneic tumour cells to mount immune responses in patients with cancer without knowing the antigenic repertoire of the individual disease. Tumour cell lysates, irradiated whole tumour cells, and fusion products of tumour cells and autologous or allogeneic dendritic cells have been used for the immunisation of patients with melanoma, breast, and renal cell cancer. Clinical responses of metastatic disease were reported in single cases, but detectable immune responses against the vaccines were difficult to document.53,54

    Immunoselection of antigen and MHC class I loss variants

    Monoclonal antibodies used for immunohistochemical staining of melanocyte differentiation antigens expressed in melanoma tissues are an important prerequisite for studying the microheterogeneity of defined antigens ín tumour lesions.55,56 In HLA-A2 positive patients with melanoma immunised with Melan A/MART-1, tyrosinase, and gp100 derived peptides combined with GM-CSF, we observed after an initial phase of tumour regression in some patients, progressive disease in the presence of detectable peptide specific CTL.47 When compared with the initially described homogeneous antigen expression, biopsies taken from lesions in the phase of progressive tumour growth showed a highly heterogeneous distribution of antigens in response to increased peptide specific CTL reactivity. Furthermore, a loss of MHC class I molecules, as detected by immunohistochemistry, was found in single cases, and this is an additional mechanism of immune escape from antigen specific immunosurveillance.

    Future clinical studies involving antigen specific T cell reactions in patients with cancer will consider the prognostic implication of the heterogeneity of MHC class I and tumour associated antigen expression in tumours for T cell based immunotherapy. Cytokines, such as IFN-γ or IL-12, will be evaluated in future clinical trials to show whether they can modulate the expression of antigens and antigen presenting molecules in tumour tissues.

    Immunotherapy in cancer: perspectives

    Different types of cancer expressing defined tumour associated antigens may become targets for immunotherapeutic interventions. The growing number of tumour antigens detected and the experience with peptide vaccination in malignant melanoma have set a solid basis for the development of more effective immunotherapeutic strategies in patients with cancer. CT antigens are thought to be promising targets for specific CTL induced by peptide or protein vaccines. Spontaneous antibody responses to CT antigens detected in the sera of patients with cancer,57 and the correlation of antibody titres with the course of the disease,58 suggest the presence of antigen specific CD4+ T cells against peptides presented by MHC class II molecules on the surface of tumour cells.59 The characterisation of these antigens as targets for CD4+ T cell responses will allow combined immunisation with MHC class I and II binding epitopes, potentially eliciting more effective immune responses.

    Targeting viral antigens expressed by different types of cancer, such as cervical and hepatocellular carcinoma, by active immunisation is a strategy currently being evaluated in clinical trials. Although there is some evidence for specific immune responses to the vaccine, major clinical responses have not been achieved yet.60 Because viral infection is thought to be a tumorigenic factor, immunisation against viral epitopes may have a preventive benefit in stages of premalignancy or even earlier after infection.61

    Future perspectives of tumour vaccination are focused on the definition of more potent strategies of immunisation. Whole tumour proteins containing multiple, possibly relevant, antigenic epitopes may increase the chance of polyvalent B and T cell activation. Adjuvants might enhance the immunogenicity of peptides and proteins by activating costimulatory factors and mediating the production of cytokines.62 Dendritic cells loaded with peptides or proteins in vitro, or transduced with the relevant genes, might effectively stimulate both MHC class I and II restricted T cells in vivo.63,64 Cytokines have been found to play a key role in T cell activation. GM-CSF has been shown to induce long lasting Th1 and CD8+ T cell responses by the efficient induction of dendritic cells in vivo.65 IL-12 is a potent activator of Th1 and CD8+ T cells. At low doses, it has been shown to mediate complete tumour regression when used as an adjuvant to immunisation with a mutant peptide of p53 in an animal model.33 The identification of new tumour antigens will provide a broader basis for polyvalent immunisation to prevent the escape of antigen loss variants.66 As the clinical effectiveness of cancer vaccination becomes more established, antigen specific immunotherapy might be considered as an alternative modality for adjuvant treatment of patients with cancer at high risk for recurrence.

    References

    1. ↵
      Gromet MA, Epstein WL, Blois MS. The regressing thin malignant melanoma: a distinctive lesion with metastatic potential. Cancer 1978;42:2282–92.
      OpenUrlCrossRefPubMedWeb of Science
    2. ↵
      Balch CM, Houghton AN, Milton GW, et al. Cutaneous melanoma. Philadelphia: JB Lippincott, 1992.
    3. ↵
      Knuth A, Wölfel T, Klehmann E, et al. Cytolytic T-cell clones against an autologous human melanoma: specificity study and definition of three antigens by immunoselection. Proc Natl Acad Sci U S A 1989;86:2804–8.
      OpenUrlAbstract/FREE Full Text
    4. ↵
      Wölfel T, Klehmann E, Müller C, et al. Lysis of human melanoma cells by autologous cytolytic T cell clones. Identification of human histocompatibility leukocyte antigen A2 as a restriction element for three different antigens. J Exp Med 1989;170:797–810.
      OpenUrlAbstract/FREE Full Text
    5. ↵
      Knuth A, Danowski B, Oettgen HF, et al. T-cell-mediated cytotoxicity against autologous malignant melanoma: analysis with interleukin-2-dependent T-cell cultures. Proc Natl Acad Sci U S A 1984;81:3511–15.
      OpenUrlAbstract/FREE Full Text
    6. ↵
      Knuth A, Wölfel T, Meyer zum Büschenfelde K-H. T cell responses to human malignant tumours. Cancer Surv 1992;13:39–52.
      OpenUrlPubMedWeb of Science
    7. ↵
      Van der Bruggen P, Traversari C, Chomez P, et al. A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma. Science 1991;254:1643–7.
      OpenUrlAbstract/FREE Full Text
    8. ↵
      Knuth A, Wölfel T, Meyer zum Büschenfelde K-H. Cellular and humoral responses against cancer: implications for cancer vaccines. Current Opin Immunol 1991;3:659–64.
      OpenUrlCrossRefPubMedWeb of Science
    9. ↵
      Van den Eynde B, Peeters O, De Backer O, et al. A new family of genes coding for an antigen recognized by autologous cytolytic T lymphocytes on a human melanoma. J Exp Med 1995;182:689–98.
      OpenUrlAbstract/FREE Full Text
    10. ↵
      Gaugler B, Van den Eynde B, van der Bruggen P, et al. Human gene MAGE-3 codes for an antigen recognized on a melanoma by autologous cytolytic T lymphocytes. J Exp Med 1994;179:921–30.
      OpenUrlAbstract/FREE Full Text
    11. Brasseur F, Rimoldi D, Lienard D, et al. Expression of MAGE genes in primary and metastatic cutaneous melanoma. Int J Cancer 1995;63:375–80.
      OpenUrlCrossRefPubMedWeb of Science
    12. ↵
      Traversari C, van der Bruggen P, Luescher IF, et al. A nonapeptide encoded by human gene MAGE-1 is recognized on HLA-A1 by cytolytic T-lymphocytes directed against tumor antigen MZ2-E. J Exp Med 1992;176:1453–7.
      OpenUrlAbstract/FREE Full Text
    13. ↵
      Chen Y-T, Scanlan MJ, Sahin U, et al. A testicular antigen aberrantly expressed in human cancers detected by autologous antibody screening. Proc Natl Acad Sci U S A 1997;94:1914–18.
      OpenUrlAbstract/FREE Full Text
    14. ↵
      Sahin U, Türeci Ö, Schmitt H, et al. Human neoplasms elicit multiple specific immune responses in the autologous host. Proc Natl Acad Sci U S A 1995;92:11810–13.
      OpenUrlAbstract/FREE Full Text
    15. ↵
      Jäger E, Chen Y-T, Drijfhout JW, et al. Simultaneous humoral and cellular immune response against cancer-testis antigen NY-ESO-1: definition of human histocompatibility leucocyte antigen (HLA)-A2-binding peptide epitopes. J Exp Med 1998;187:265–9.
      OpenUrlAbstract/FREE Full Text
    16. ↵
      Bakker AB, Schreurs MWJ, deBoer AJ, et al. Melanocyte lineage-specific antigen gp100 is recognized by melanoma-derived tumor-infiltrating lymphocytes. J Exp Med 1994;179:1005–9.
      OpenUrlAbstract/FREE Full Text
    17. Brichard V, Van Pel A, Wölfel T, et al. The tyrosinase gene codes for an antigen recognized by autologous cytolytic T lymphocytes on HLA-A2 melanomas. J Exp Med 1993;178:489–95.
      OpenUrlAbstract/FREE Full Text
    18. Coulie PG, Brichard V, Van Pel A, et al. A new gene coding for a differentiation antigen recognized by autologous cytolytic T lymphocytes on HLA-A2 melanomas. J Exp Med 1994;180:35–42.
      OpenUrlAbstract/FREE Full Text
    19. ↵
      Kawakami Y, Eliyahu S, Delgado CH, et al. Cloning of the gene coding for a shared human melanoma antigen recognized by autologous T cells infiltrating into tumor. Proc Natl Acad Sci U S A 1994;91:3515–19.
      OpenUrlAbstract/FREE Full Text
    20. ↵
      Kang XQ, Kawakami Y, El-Gamil M, et al. Identification of a tyrosinase epitope recognized by HLA-A24-restricted, tumor-infiltrating lymphocytes. J Immunol 1995;155:1343–8.
      OpenUrlAbstract/FREE Full Text
    21. Kawakami Y, Eliyahu S, Jennings C, et al. Recognition of multiple epitopes in the human melanoma antigen gp100 by tumor-infiltrating lymphocytes associated with in vivo tumor regression. J Immunol 1995;154:3961–8.
      OpenUrlAbstract
    22. ↵
      Wölfel T, Schneider J, Meyer zum Büschenfelde K-H, et al. Isolation of naturally processed peptides recognized by cytolytic T lymphocytes (CTL) on human melanoma cells in association with HLA-A2.1. Int J Cancer 1994;57:413–18.
      OpenUrlCrossRefPubMedWeb of Science
    23. ↵
      Jäger E, Bernhard H, Romero P, et al. Generation of cytotoxic T cell responses with synthetic melanoma associated peptides in vivo: implications for tumor vaccines with melanoma associated antigens. Int J Cancer 1996;66:162–9.
      OpenUrlCrossRefPubMedWeb of Science
    24. ↵
      Jäger E, Ringhoffer M, Dienes H-P, et al. Granulocyte macrophage colony-stimulating factor enhances immune responses to melanoma associated peptides in vivo. Int J Cancer 1996;67:54–62.
      OpenUrlCrossRefPubMedWeb of Science
    25. Marchand M, Weynants P, Rankin E, et al. Tumor regression responses in melanoma patients treated with a peptide encoded by gene MAGE-3. Int J Cancer 1995;63:883–5.
      OpenUrlCrossRefPubMedWeb of Science
    26. ↵
      Marchand M, van Baren N, Weynants P, et al. Tumor regressions observed in patients with metastatic melanoma treated with an antigenic peptide encoded by gene MAGE-3 and presented by HLA-A1. Int J Cancer 1998;80:219–30.
      OpenUrl
    27. ↵
      Coulie PG, Lehmann F, Lethe B, et al. A mutated intron sequence codes for an antigenic peptide recognized by cytolytic T lymphocytes on a human melanoma. Proc Natl Acad Sci U S A 1995;92:7976–80.
      OpenUrlAbstract/FREE Full Text
    28. Mandelboim O, Berke G, Fridkin M, et al. CTL induction by a tumour-associated antigen octapeptide derived from a murine lung carcinoma. Nature 1994;369:67–71.
      OpenUrlCrossRefPubMed
    29. ↵
      Wölfel T, Hauer M, Schneider J, et al. A p16INK4a-insensitive CDK4 mutant targeted by cytolytic T lymphocytes in a human melanoma. Science 1995;269:1281–4.
      OpenUrlAbstract/FREE Full Text
    30. ↵
      Disis ML, Cheever MA. Oncogenic proteins as tumor antigens. Current Opin Immunol 1996;8:637–42.
      OpenUrlCrossRefPubMed
    31. ↵
      Schlichtholz B, Legros Y, Gillet D, et al. Antibodies against p53 in breast cancer patients is directed against immunodominant epitopes unrelated to the mutational hot spot. Cancer Res 1992;52:6380–4.
      OpenUrlAbstract/FREE Full Text
    32. ↵
      Tilkin AF, Lubin R, Soussi T, et al. Primary proliferative T cell response to wild-type p53 protein in patients with breast cancer. Eur J Immunol 1995;25:1765–9.
      OpenUrlCrossRefPubMedWeb of Science
    33. ↵
      Noguchi Y, Richards EC, Chen YT, et al. Influence of interleukin-12 on p53 peptide vaccination against established Meth A sarcoma. Proc Natl Acad Sci U S A 1995;92:2219–23.
      OpenUrlAbstract/FREE Full Text
    34. ↵
      Fenton RG, Keller CJ, Hanna N, et al. Induction of T-cell immunity against Ras oncoprotein by soluble protein or Ras-expressing Escherichia coli. J Natl Cancer Inst 1995;87:1853–61.
      OpenUrlAbstract/FREE Full Text
    35. ↵
      Gjertsen MK, Bakka A, Breivik J, et al. Vaccination with mutant Ras peptides and induction of T-cell responsiveness in pancreatic carcinoma patients carrying the corresponding Ras mutation. Lancet 1995;346:1399–400.
      OpenUrlCrossRefPubMedWeb of Science
    36. ↵
      Murray RJ, Kurilla MG, Brooks JM, et al. Identification of target antigens for the human cytotoxic T cell response to Epstein-Barr virus (EBV): implications for the immune control of EBV-positive malignancies. J Exp Med 1992;176:157–68.
      OpenUrlAbstract/FREE Full Text
    37. ↵
      Koziel MJ, Dudley D, Afdhal N, et al. HLA-class I-restricted cytotoxic T lymphocytes specific for hepatitis C virus. Identification of multiple epitopes and characterization of patterns of cytokine release. J Clin Invest 1995;96:2311–21.
    38. ↵
      Rehermann B, Fowler P, Sidney J, et al. The cytotoxic T lymphocyte response to multiple hepatitis B virus polymerase epitopes during and after acute viral hepatitis. J Exp Med 1995;181:1047–58.
      OpenUrlAbstract/FREE Full Text
    39. ↵
      Feltkamp MC, Smits HL, Vierboom MP, et al. Vaccination with cytotoxic T lymphocyte epitope-containing peptide protects against a tumor induced by human papillomavirus type 16-transformed cells. Eur J Immunol 1993;23:2242–9.
      OpenUrlCrossRefPubMedWeb of Science
    40. ↵
      Koenig S, Woods RM, Brewar YA, et al. Characterization of MHC class I restricted cytotoxic T cell responses to tax in HTLV-1 infected patients with neurologic disease. J Immunol 1993;151:3874–83.
      OpenUrlAbstract
    41. ↵
      Boon T. Tumor antigens recognized by cytolytic T cells. Cancer Vaccine Week 1998;October 5–9:S01.
    42. ↵
      Marchand M, Weymants P, Rankin E, et al. Tumor regression responses in melanoma patients treated with a peptide encoded by gene MAGE-3. Int J Cancer 1995;63:883–5.
    43. ↵
      Herr W, Wölfel T, Heike M, et al. Frequency analysis of tumor-reactive cytotoxic T lymphocytes in peripheral blood of a melanoma patient vaccinated with autologous tumor cells. Cancer Immunol Immunother 1994;39:93–9.
      OpenUrlPubMedWeb of Science
    44. ↵
      Kawakami Y, Eliyahu S, Delgado CH, et al. Identification of a human melanoma antigen recognized by tumor-infiltrating lymphocytes associated with in vivo tumor rejection. Proc Natl Acad Sci U S A 1994;91:6458–62.
      OpenUrlAbstract/FREE Full Text
    45. ↵
      Robbins PF, El-Gamil M, Kawakami Y, et al. Recognition of tyrosinase by tumor-infiltrating lymphocytes from a patient responding to immunotherapy. Cancer Res 1994;54:3124–6.
      OpenUrlAbstract/FREE Full Text
    46. ↵
      Jäger E, Ringhoffer M, Arand M, et al Cytolytic T cell reactivity against melanoma associated differentiation antigens in peripheral blood of melanoma patients and healthy individuals. Melanoma Res 1996;6:419–25.
      OpenUrlCrossRefPubMedWeb of Science
    47. ↵
      Jäger E, Ringhoffer M, Karbach J, et al. Immunoselection in vivo: independent loss of MHC class I and melanocyte differentiation antigen expression in metastatic melanoma mediated by antigen-specific CTL. Int J Cancer 1997;71:142–7.
      OpenUrlCrossRefPubMedWeb of Science
    48. ↵
      Rivoltini L, Kawakami Y, Sakaguchi K, et al. Induction of tumor-reactive CTL from peripheral blood and tumor-infiltrating lymphocytes of melanoma patients by in vitro stimulation with an immunodominant peptide of the human melanoma antigen MART-1. J Immunol 1995;154:2257–65.
      OpenUrlAbstract
    49. ↵
      Visseren MJW, van Elsas A, van der Voort EIH, et al. CTL specific for the tyrosinase autoantigen can be induced from healthy donor blood to lyse melanoma cells. J Immunol 1995;154:3991–8.
      OpenUrlAbstract
    50. ↵
      Ogg GS, Dunbar PR, Romero P, et al. High frequency of skin-homing melanocyte-specific cytotoxic T lymphocytes in autoimmune vitiligo. J Exp Med 1998;188:1203–8.
      OpenUrlAbstract/FREE Full Text
    51. ↵
      Jäger E, Maeurer M, Höhn H, et al. Clonal expansion of Melan A-specific cytotoxic T lymphocytes (CTL) in a melanoma patient responding to continued immunization with melanoma-associated peptides. Int J Cancer 2000;86:538–47.
      OpenUrlCrossRefPubMedWeb of Science
    52. ↵
      Caux C, Dezutter-Dambuyant C, Schmitt D. GM-CSF and TNFa cooperate in the generation of dendritic Langerhans cells. Nature 1992;360:258–61.
      OpenUrlCrossRefPubMed
    53. ↵
      Kugler A, Stuhler G, Walden P, et al. Regression of human metastatic renal cell carcinoma after vaccination with tumor cell-dendritic cell hybrids. Nat Med 2000;6:332–6.
      OpenUrlCrossRefPubMedWeb of Science
    54. ↵
      Nestle FO, Alijagic S, Gilliet M, et al. Vaccination of melanoma patients with peptide- or tumor lysate-pulsed dendritic cells. Nat Med 1998;4:328–32.
      OpenUrlCrossRefPubMedWeb of Science
    55. ↵
      Chen Y-T, Stockert E, Jungbluth A, et al. Serological analysis of Melan A (MART-1), a melanocyte-specific protein homogenously expressed in human melanomas. Proc Natl Acad Sci U S A 1996;93:5915–19.
      OpenUrlAbstract/FREE Full Text
    56. ↵
      Chen YT, Stockert E, Tsang S, et al. Immunophenotyping of melanomas for tyrosinase: implications for vaccine development. Proc Natl Acad Sci U S A 1995;92:8125–9.
      OpenUrlAbstract/FREE Full Text
    57. ↵
      Stockert E, Jäger E, Chen Y-T, et al. A survey of the humoral immune response of cancer patients to a panel of human tumor antigens. J Exp Med 1998;187:1349–54.
      OpenUrlAbstract/FREE Full Text
    58. ↵
      Jäger E, Stockert E, Zidianakis Z, et al. Humoral immune responses of cancer patients against “cancer-testis” antigen NY-ESO-1: correlation with clinical events. Int J Cancer 1999;84:506–10.
      OpenUrlCrossRefPubMedWeb of Science
    59. ↵
      Jäger E, Jäger D, Karbach J, et al. Identification of NY-ESO-1 epitopes presented by HLA-DRB4 0101–0103 and recognized by CD4+ T lymphocytes of patients with NY-ESO-1 expressing melanoma. J Exp Med 2000;191:625–30.
      OpenUrlAbstract/FREE Full Text
    60. ↵
      Ressing ME, van Driel WJ, Brandt RMP, et al. Detection of T helper responses, but not of human papillomavirus specific CTL responses, after peptide vaccination of patients with cervical carcinoma. J Immunotherapy 2000;23:255–66.
    61. ↵
      Chang MH, Chen CJ, Lai MS. Universal hepatitis B vaccination in Taiwan and the incidence of hepatocellular carcinoma in children. N Engl J Med 1997;336:1855–9.
      OpenUrlCrossRefPubMedWeb of Science
    62. ↵
      Matzinger P. Tolerance, danger, and the extended family. Annu Rev Immunol 1994;12:991–1045.
      OpenUrlCrossRefPubMedWeb of Science
    63. ↵
      Alijagic S, Müller P, Artuc M, et al. Dendritic cells generated from peripheral blood transfected with human tyrosinase induce specific T cell activation. Eur J Immunol 1995;25:3100–7.
      OpenUrlCrossRefPubMedWeb of Science
    64. ↵
      Schirmbeck R, Melber K, Reimann J. Hepatitis B virus small surface antigen particles are processed in a novel endosomal pathway for major histocompatibility complex class I-restricted epitope presentation. Eur J Immunol 1995;25:1063–70.
      OpenUrlCrossRefPubMedWeb of Science
    65. ↵
      Dranoff G, Jaffee E, Lazenbry A, et al. Vaccination with irradiated tumor cells engineered to secrete murine granulocyte–macrophage colony-stimulating factor stimulates potent, specific, and long-lasting anti-tumor immunity. Proc Natl Acad Sci U S A 1993;90:3539–43.
      OpenUrlAbstract/FREE Full Text
    66. ↵
      Jäger E, Ringhoffer M, Karbach J, et al. Inverse relationship of melanocyte differentiation antigen expression in melanoma: evidence for immunoselection of antigen-loss variants in vivo. Int J Cancer 1996;66:470–6.
      OpenUrlCrossRefPubMedWeb of Science