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Prophylactic cancer vaccines Olivera J Finn* and Guido Forni† Increasingly, data from distinct experimental systems show that immunity can be activated to prevent tumors. The rationale for prevention is strong because, in that setting, one deals with an immune system that is neither impaired by tumor- and treatment-induced suppression nor tolerant to tumorassociated antigens that have been encountered in the absence of correct presentation and costimulatory/danger signals. The use of overexpressed or mutated proteins, or mutated oncogenic growth factor receptors, as tumorassociated antigens yields rational targets for specific immunoprevention. Transgenic mouse models are providing encouraging indications of future usefulness of vaccines that are based on these molecules. Addresses *Department of Immunology, University of Pittsburgh School of Medicine and University of Pittsburgh Cancer Institute, W1142 Biomedical Science Tower, Pittsburgh, PA 15261, USA; e-mail: [email protected] † Department of Clinical and Biological Sciences, University of Torino, Ospedale San Luigi Gonzaga, 10043 Torino, Italy; e-mail: [email protected] Current Opinion in Immunology 2002, 14:172–177 0952-7915/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved. Abbreviations CEA carcinoembryonic antigen CTL cytotoxic T lymphocyte DTH delayed-type hypersensitivity Mab monoclonal antibody rrat TAA tumor-associated antigen
Introduction Tumor immunology has been advancing on two major fronts: first, the elucidation of the basic mechanisms of tumor recognition and rejection, and protection from recurrence; and, second, the application of that knowledge to cancer as a clinical problem. The basic science front has advanced rapidly and provided tools with which to explore two different approaches to disease — immunotherapy and cancer vaccines. Immunotherapy relies on administration of preformed effector mechanisms and it is ideally suited for application to the treatment of established tumors. Cancer vaccines, on the other hand, are being developed with an eye on prevention. Taking as an example the successes that vaccines have had in preventing infectious diseases, efforts are being made to define tumor targets and immunization protocols that would be equally effective in priming the immune system to eliminate cancer before it manifests itself clinically. Although the basic studies can be well designed and preclinical animal models continue to be developed and used to test the newly discovered scientific principles, there is hesitation in translating these findings to cancer prevention in humans.
With the exception of efforts to vaccinate against microorganisms known to cause human cancers, such as HPV (human papillomavirus) [1,2] and in that way prevent not only infection but also (indirectly) cancer, hundreds of clinical trials of cancer vaccines are currently being carried out in patients who already have cancer. These efforts have added a new term to the vaccine field — ‘therapeutic vaccines’. The efforts are driven more by the practicality of the approach than by strong supporting evidence from basic and preclinical research. Animal studies have shown that cancer vaccines are most effective in protection from tumor challenge or, in more recent studies that we review below, in prevention of tumor occurrence in genetically predisposed animals. Vaccines have shown limited potential in curing established tumors. Clinical trails of therapeutic cancer vaccines not surprisingly recapitulate the observations in animals. The trials show the ability of the vaccine to stimulate, to widely varied degrees, specific antibodies and T cells but, with few exceptions [3–6], rarely result in an objective clinical antitumor response. However, even though therapeutic vaccines are currently of marginal clinical utility, they will be ultimately useful for having paved the way for the use of cancer vaccines in people, in a setting that can be anticipated to have a much greater impact on human health — that of bona fide cancer prevention. In this review we will highlight studies published in the past year that are providing basic and preclinical support for preventive cancer vaccines.
Cancer vaccines protect from tumor challenge and prevent tumor occurrence Two schools of thought dominate research into tumor antigens and tumor vaccines. One believes that tumor rejection can be obtained only by immunizing with unique tumor-associated antigens (TAAs) [7,8], supporting the use of undefined tumor-derived materials — that would contain such antigens — as tumor vaccines. This is based on studies with carcinogen-induced tumors in rodents, where it was hard to demonstrate the presence of shared antigens (expressed on multiple tumors of the same tissue type) and cross-protective antigens (immunization against which could prevent occurrence of tumors in different individuals). In humans, shared antigens appear to be predominant targets of tumor-specific immunity [9]. Furthermore, as we will review below, animal models designed to test their tumor-rejection potential show that they can elicit tumorrejection responses. Other studies in animals showed that shared tumor antigens did exist. Carcinogen-induced fibrosarcomas in chickens not only cross-protected from tumor challenge [10], but also inhibited new carcinogenesis
Prophylactic cancer vaccines Finn and Forni
[11]. Vaccination with purified TAA was reported to suppress chemically induced mammary tumors in rats [12], and vaccination with p53 protein prevented chemically induced skin cancers in mice [13]. Most recently, TAAs — mostly self proteins — derived from spontaneously arising tumors in p53−/− mice were shown to cross-protect vaccinated mice against tumor challenge [14••]. Vaccination of C3H/HeOuj mice (which are known for their very high incidence of breast cancer) with a mouse breast cancer TAA — mouse MUC1 protein — significantly lowered the incidence of breast cancer in those mice [15]. A more important issue than unique versus shared antigens is the ability of a vaccine to appropriately prime immune effector mechanisms. TG.AC × C57BL/6 F1 mice carry in their germline a mutant ras oncogene that has an arginine at codon 12 instead of the glycine present in the wild-type. After physical promotion (by wounding) or chemical promotion, these mice develop papillomas that progress to cancer. Immunization with Arg(12)-mutant ras peptide induced T-cell reactivity and specific delayed-type hypersensitivity (DTH). However, when mice were painted with phorbol 12-myristate 13-acetate, tumors grew faster in the immunized mice. Immunization not only failed to protect against papillomas, but also induced a remarkable enhancement of their growth [16]. These findings are reminiscent of the inhibition of papilloma growth in mice with the TNF-α gene knocked out [17] and the enhancement of mammary carcinogenesis in mice transgenic for the PyMT oncogene and CSF-1 [18]. Specific immunity [19] and nonspecific inflammatory reactions [20,21] may in certain instances enhance tumor growth. The challenge will be to avoid stimulating the type of immunity that enhances carcinogenesis, by designing vaccines that promptly activate strong reactions at the site of the lesion. The very same immune mechanisms that have been seen to enhance tumor growth will lead to its rejection when fully activated [19].
Tumor prevention by stimulation of TAA-specific immunity: transgenic mouse models The use of overexpressed or mutated proteins, or mutated oncogenic growth-factor-receptors as TAAs has yielded rational targets for specific immunoprevention [22]. Transgenic mouse models are providing encouraging indications of future usefulness of vaccines based on these molecules and we describe four such models below.
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in virgin females or only after pregnancy and can involve one or all ten mammary glands [24]. Both the slow carcinogenesis driven by Her-2/neu protooncogene and the aggressive one due to the transforming r-Her-2/neu are inhibited by vaccination with proteins, peptides or DNA plasmids [25–28,29•,30–34,35••]. Inhibition appears to be mostly dependent on IFN-γ-based DTH and antibody, as suggested by pathological findings and in vitro tests [29•,32,35••]. In several lines of transgenic mice, anti-r-p185neu vaccination appears to be unable to elicit cytotoxic T lymphocyte (CTL)-mediated killing of p185neu+ cells [29•,32,35••,36], probably due to the difficulty of fully breaking their tolerance [37•]. Vaccination-induced antibodies appear to block carcinogenesis by inhibiting the ability of p185neu to transduce cell-activating signals and downregulating its membrane expression in preneoplastic cells [29•]. Their activity appears to be similar to that of passively administered anti-p185neu monoclonal antibody (Mab) [38]. By contrast, the role of antibodies in the rejection of r-p185neu+ transplantable tumors is controversial. In this setting, some vaccination protocols, anti-r-p185neu antibodies amplify the protection provided by T cells [36] whereas in others a strong CTL response leads to tumor rejection in the absence of antibodies [39•]. Thus, mechanisms other than CTL activity may inhibit carcinogenesis driven by the expression of oncogenic growth-factor-receptors on the cell membrane [22]. These mechanisms, interestingly, do not play a major role in the inhibition of growth of transplantable tumors [29•]. CEA-transgenic mice
Carcinoembryonic antigen (CEA) is a well-defined TAA that was first described in 1965 (see [40]). Human-CEAtransgenic mice have been generated that express the transgene under its own promoter [41,42]. They have been used to explore the immunogenicity and tumor-rejection potential of CEA in a setting in which, as in humans, it is also a self-antigen. The studies show that a certain degree of tolerance to this antigen exists in CEA-transgenic mice compared with wild-type mice, but this tolerance can be easily overcome with well-designed immunization protocols to generate immune responses that are protective against tumor challenge [43,44,45•]. Importantly, breaking tolerance to CEA does not result in any autoimmune reactions against normal tissues that express the transgene. MUC1-transgenic mice
HER-2/neu-transgenic mice
Thanks to the pioneering work of Muller (see [23]), transgenic mice overexpressing the rat (r-)Her-2/neu protooncogene or its mutated form constitute attractive models for testing immunoprevention of mammary carcinogenesis. These mice express r-p185neu protein at distinct stages of their life. Consequently, their tolerance levels to this protein and the aggressiveness of carcinogenesis are markedly different at various stages. Carcinogenesis takes place either
MUC1 was identified as a TAA by several groups simultaneously, using a tumor-specific mouse Mab [46], or tumor-specific T-cell lines from patients with epithelial adenocarcinomas [47,48]. In recent years, several lines of human MUC1-transgenic mice have been generated on the C57BL [49], BALB/c [50•] and DBA [51] backgrounds. These mice are transgenic for the entire human MUC1 gene driven by its own promoter. As such, this transgenic protein shows temporal and tissue expression typical of the
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endogenous antigen in humans. MUC1-transgenic mice showed peripheral tolerance to MUC1 in both T- and B-cell compartments, but this tolerance could be broken by increasing the immunogenicity of vaccine preparations [49,50•,51,52•,53,54•]. MUC1-specific immunity, primarily CD8+ T cells, which resulted in tumor rejection and longterm protection, did not result in autoimmunity. Several transgenic tumor models that express the SV-40 large T antigen under various tissue specific promoters and develop tumors in various tissue sites (such as breast, prostate, colon or pancreas), are being crossed with the MUC1-transgenic mice. One such double-transgenic model is the MET mouse that, because of elastase-driven T antigen, develops pancreatic tumors, which in the presence of the MUC1 transgene express MUC1. Concurrent with tumor development, these mice spontaneously develop MUC1-specific CTLs that do not stop growth of spontaneous tumors. However, when transferred to naïve mice, they can protect fully from MUC1+ tumor challenge [55••]. TRAMP transgenic mice
Prostate cancer is the most common malignancy in men. Its incidence is soaring due to the aging of the population and the introduction of diagnostic procedures that reveal its early stages. In TRAMP C57/BL transgenic mice, expression of SV40 T antigen is under the probasin promoter that is regulated by androgens and restricted to the prostate epithelial cells. Prostate tumor progression resembles the human disease [56]. A preventive immune response against primary tumors could be elicited in this model by using an irradiated-tumorcell vaccine and CTLA-4 blockade [57]. Furthermore, treatment of the prostate tumor cells with 15-deoxy-δ12,14prostaglandin J2 induced expression of heat-shock protein (Hsp) 70, and vaccination of TRAMP C57/BL transgenic mice with Hsp 70 from these tumors elicited tumor-specific CTLs and protected from tumor challenge [58].
to be expressed very early in the process of transformation and are frequently expressed in premalignant dysplasia and metaplasia. Several human tumors, such as colon, breast, prostate and lung, yield themselves to early detection and patients with familial history or other risk factors for these malignancies are under regular monitoring. Surgery is an option for removal of a small tumor but, with the exception of polyp removal for colon cancer prevention, there are no strategies for dealing with premalignant changes. A number of TAAs reviewed here would be candidate vaccines in the setting of premalignant disease [61−65]. Immune prevention could also be considered for individuals whose tumors have been eradicated by standard therapy. Clinical data on patients treated with Mab [66], and experimental data in mice immunized after tumor surgery [67,68] show that prevention of recurrences in conditions of minimal residual disease is a permissible immunological challenge. However, since the immune system has been imprinted by a previous encounter with a progressing tumor, tolerance to TAAs may need to be overcome in order to elicit an efficient response.
Acknowledgements This work was supported by grant 5R01 CA56103-10 from the National Cancer Institute and grants from the Italian Association for Cancer Research and the Italian Ministry for University and Scientific and Technological Research.
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Conclusions Different experimental systems show that immunity can be activated to prevent tumors. The rationale for prevention is strong because in that setting one deals with an immune system that is neither impaired by tumor- and treatment-induced suppression, nor tolerant to TAAs that have been encountered in the absence of correct presentation and costimulatory/danger signals. For a tumor to develop in healthy individuals with either a genetic risk of cancer or bearing preneoplastic lesions may take a considerable length of time. A certain length of time is also necessary for the induction of strong immunity to tumor TAAs. The elicited immune mechanisms are likely to have a greater impact on subclinical tumors than on those that have disseminated or become unresectable [59, 60•]. Although most antigen-discovery methods employed tumors as sources of antigen, many TAAs have been found
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35. Nanni P, Nicoletti G, De Giovanni C, Landuzzi L, Di Carlo E, Cavallo F, •• Pupa SM, Rossi I, Colombo MP, Ricci C et al.: Combined allogeneic tumor cell vaccination and systemic IL-12 prevents mammary carcinogenesis in HER-2/neu transgenic mice. J Exp Med 2001, 194:1195-1206. The very aggressive mammary carcinogenesis driven by the activated transforming Her-2/neu oncogene is fully inhibited for at least one year in transgenic BALB/c mice treated with systemic IL-12 and specifically immunized against the p185neu molecule presented by allogeneic cells. The protective efficacy of this combined treatment is markedly reduced when any one of its components (IL-12, p185neu or allogenic cells) is missing. Inhibition apparently does not rest on the activation of a CTL reactivity, but on both the production of anti-p185neu antibodies and an intratumor DTH in which IFN-γ plays a crucial role. 36. Reilly RT, Machiels JP, Emens LA, Ercolini AM, Okoye FI, Lei RY, Weintraub D, Jaffee EM: The collaboration of both humoral and cellular HER-2/neu-targeted immune responses is required for the complete eradication of HER-2/neu-expressing tumors. Cancer Res 2001, 61:880-883. 37. •
Reilly RT, Gottlieb MB, Ercolini AM, Machiels JP, Kane CE, Okoye FI, Muller WJ, Dixon KH, Jaffee EM: HER-2/neu is a tumor rejection target in tolerized HER-2/neu transgenic mice. Cancer Res 2000, 60:3569-3576. This paper examines the consequences of tolerance to Her-2/neu on the induction of an effective immune response. This is an issue of crucial importance, since it is expected that a similar tolerance is present in patients bearing incipient neoplastic lesions. The data show that, despite the evidence of significant tolerance to the transgene, both cellular and humoral neu-specific responses in transgenic mice can be boosted with neu-specific vaccination, although to a significantly lesser degree than in parental FVB/N mice; this indicates that the T cells involved are less responsive than in the nontolerant parental strain.
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MUC1 is broken and the tumors are rejected. Both T cells and antibodies are produced and the immune response is long-lasting. Mice that have rejected IL-12-transfected tumors are protected from subsequent tumor challenge.
39. Pilon SA, Piechocki MP, Wei WZ: Vaccination with cytoplasmic • Erbb-2 DNA protects mice from mammary tumor growth without anti-Erbb-2 antibody. J Immunol 2001, 167:3201-3206. The role of antibodies to p185neu in the inhibition of Her-2/neu carcinogenesis is still not well defined. The role of these antibodies in the rejection of transplantable p185neu cells is also not clear. In some instances, as in [38], anti-Her-2/neu antibodies appear to play an important role in enhancing the protective effects of cellular reactivity. In this paper, a DNA vaccine encoding a full-length r-Her-2/neu protein that is targeted to the cytosol and rapidly degraded there by the proteasomes was used to elicit a CTL response without an antibody response in normal parental mice. The feasibility of eliciting individual effector mechanisms by targeted DNA vaccine may open a new, interesting way of assessing the weight of cellular and antibody responses in the inhibition of carcinogenesis in transgenic mice.
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Barnd DL, Lan MS, Metzgar RS, Finn OJ: Specific, MHCunrestricted recognition of tumor-associated mucins by human cytotoxic T cells. Proc Natl Acad Sci USA 1989, 86:7159-7163.
48. Taylor-Papadimitriou J, Finn OJ: Biology, biochemistry and immunology of carcinoma-associated mucins. Immunol Today 1997, 18:105-107. 49. Rowse GJ, Tempero RM, VanLith ML, Hollingsworth MA, Gendler SJ: Tolerance and immunity to MUC1 in a human MUC1 transgenic murine model. Cancer Res 1998, 58:315-321. 50. Carr-Brandel V, Markovic D, Ferrer K, Smith M, Taylor-Papdimitriou J, • Cohen ER: Immunity to murine breast cancer cells modified to express MUC1, a human breast cancer antigen, in transgenic mice tolerant to human MUC1. Cancer Res 2000, 60:2435-2443. In this study, MUC1-transgenic mice are permissive of growth of a breast tumor line transfected with MUC1 and injected into the fat pad of the breast. However, when these cells are engeneered to express IL-12, tolerance to
52. Lees CJ, Apostolopulos V, Acres B, Ramshaw A, Ong CS, • McKenzie IF: Immunotherapy with mannan-MUC1 and IL-12 in MUC1 transgenic mice. Vaccine 2000, 19:158-162. This study provides support for importance of IL-12 in anti-MUC1 immunity. When double-transgenic MUC1/HLA-A2 mice are vaccinated with oxidized mannan−MUC1 fusion protein they generate MUC1-specific, HLA-A2restricted CTLs that are responsible for tumor protection. The frequency of these CTLs increases significantly and tumor protection is enhanced with the addition of IL-12. 53. Koido S, Kashiwaba M, Chen D, Gendler S, Kufe D, Gong J: Induction of antitumor immunity by vaccination of dendritic cells transfected with MUC1 RNA. J Immunol 2000, 165:5713-5719. 54. Soares MM, Mehta V, Finn OJ: Three different vaccines based on • the 140 amino acid MUC1 peptide with seven tandemly repeated tumor-specific epitopes elicit distinct immune effector mechanisms in wild-type versus MUC1 transgenic mice with different potential for tumor rejection. J Immunol 2001, 166:6555-6563. This study compares different adjuvants — GMCSF, SB-A2 and dendritic cells — for their effectiveness in increasing the immunogenicity of a MUC1 peptide in wild-type and MUC1-transgenic mice. Two important observations are made: first, peptide mixed with GMCSF and SB-A2 generates only antibody responses and no T-cell immunity, but anti-MUC1 antibodies do not elicit protection from tumor challenge; second, peptide loaded onto dendritic cells elicits CD4+ and CD8+ T cells in wild-type mice but only CD8+ T cells in MUC1-transgenic mice. MUC1-specific CD8+ T cells in the absence of MUC1-specific CD4+ T cells provide long-term protection against tumor challenge. 55. Mukherjee P, Ginardi AR, Madsen CS, Sterner CJ, Adriance MC, •• Tevethia MJ, Gendler SJ: Mice with spontaneous pancreatic cancer naturally develop MUC1-specific CTls that eradicate tumors when adoptively transferred. J Immunol 2000, 165:3451-3460. Patients with MUC1+ tumors have both anti-MUC1 antibodies and MUC1specific T cells whereas healthy individuals do not. It is not known when in the disease process the immune system responds to this antigen. This study, performed in MUC1-transgenic mice that spontaneously develop pancreatic cancer, addresses this problem. Tumors become detectable by immunohistology at 9 weeks of age and progressively grow through to 24 weeks of age. MUC1-specific CTLs appear at 12 weeks of age, increase through to 18 weeks and begin to disappear by 21 weeks. They are specific for a peptide from the tandem-repeat region previously shown to be a human class-Irestricted epitope. Although these CTLs did not appear to affect endogenous tumor progression, they provided long-term protection from tumor challenge when transferred to naïve mice. 56. Gingrich JR, Barrios RJ, Morton RA, Boyce BF, DeMayo FJ, Finegold MJ, Angelopoulou R, Rosen JM, Greenberg NM: Metastatic prostate cancer in a transgenic mouse. Cancer Res 1996, 56:4096-4102. 57.
Hurwitz AA, Foster BA, Kwon ED, Truong T, Choi EM, Greenberg NM, Burg MB, Allison JP: Combination immunotherapy of primary prostate cancer in a transgenic mouse model using CTLA-4 blockade. Cancer Res 2000, 60:2444-2448.
58. Vanaja DK, Grossmann ME, Celis E, Young CY: Tumor prevention and antitumor immunity with heat shock protein 70 induced by 15-deoxy-δδ12,14-prostaglandin J2 in transgenic adenocarcinoma of mouse prostate cells. Cancer Res 2000, 60:4714-4718. 59. Lollini P-L, Forni G: Specific and non-specific immunity in the prevention of spontaneous tumors. Immunol Today 1999, 20:343-347. 60. Forni G, Lollini P-L, Musiani P, Colombo MP: Immunoprevention of • cancer: is the time ripe? Cancer Res 2000, 60:2571-2575. This paper examines in depth the theoretical rationale and experimental findings that actively encourage the application of immunity to inhibit carcinogenesis and prevent tumors. The prospects of immune stimulation as a means of cancer prevention by inducing various forms of nonspecific and specific immunity are discussed. 61. Salem RR, Wolf BC, Sears HF, Lavin PT, Ravikumar TS, DeCoste D, D’Emilia JC, Herlyn M, Schlom J, Gottlieb LS et al.: Expression of
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colorectal carcinoma-associated antigens in colonic polyps. J Surg Res 1993, 55:249-255. 62. Reis CA, David L, Correa P, Carneiro F, deBolos C, Garcia E, Mandel U, Clausen H, Sobrinho-Simoes M: Intestinal metaplasia of human stomach displays distinct patterns of mucin (MUC1, MUC2, MUC5AC, and MUC6) expression. Cancer Res 1999, 59:1003-1007. 63. Nitta T, Sugihara K, Tsuyama S, Murata F: Immunohistochemical study of MUC1 mucin in premalignant oral lesions and oral squamous cell carcinoma: association with disease progression, mode of invasion and metastasis. Cancer 2000, 88:245-254. 64. Babino A, Opezzo P, Bianco S, Barrios E, Berois N, Navarette H, Osinaga E: Tn antigen is a pre-cancerous biomarker in breast tissue and serum in n-nitrosomethylurea-induced rat mammary carcinogenesis. Int J Cancer 2000, 86:753-759.
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65. Schmitt FC, Andrade L: Spectrum of carcinoembryonic antigen immunoreactivity from isolated ductal hyperplasias to atypical hyperplasias associated with infiltrating ductal breast cancer. J Clin Pathol 1995, 48:53-56. 66. Rietmuller G, Holz E, Schlimok G, Schmiegel W, Raab R, Hoffken K, Gruber R, Funke I, Pichlmaier H, Hirche K et al.: Monoclonal antibody therapy for resected Dukes’ C colorectel cancer: seven-year outcome of a multicenter randomized trial. J Clin Oncol 1998, 16:1788-1794. 67.
Porgador A, Tzehoval E, Vadai E, Feldman M, Eisenbach L: Combined vaccination with major histocompatibility class I and interleukin 2 gene-transduced melanoma cells synergizes the cure of postsurgical established lung metastases. Cancer Res 1995, 55:4941-4949.
68. Cavallo F, Di Carlo E, Butera M, Verrua R, Colombo MP, Musiani P, Forni G: Immune events associated with the cure of established tumors and spontaneous metastases by local and systemic IL-12. Cancer Res 1999, 59:414-421.
Prophylactic cancer vaccines Olivera J Finn* and Guido Forni† Increasingly, data from distinct experimental systems show that immunity can be activated to prevent tumors. The rationale for prevention is strong because, in that setting, one deals with an immune system that is neither impaired by tumor- and treatment-induced suppression nor tolerant to tumorassociated antigens that have been encountered in the absence of correct presentation and costimulatory/danger signals. The use of overexpressed or mutated proteins, or mutated oncogenic growth factor receptors, as tumorassociated antigens yields rational targets for specific immunoprevention. Transgenic mouse models are providing encouraging indications of future usefulness of vaccines that are based on these molecules. Addresses *Department of Immunology, University of Pittsburgh School of Medicine and University of Pittsburgh Cancer Institute, W1142 Biomedical Science Tower, Pittsburgh, PA 15261, USA; e-mail: [email protected] † Department of Clinical and Biological Sciences, University of Torino, Ospedale San Luigi Gonzaga, 10043 Torino, Italy; e-mail: [email protected] Current Opinion in Immunology 2002, 14:172–177 0952-7915/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved. Abbreviations CEA carcinoembryonic antigen CTL cytotoxic T lymphocyte DTH delayed-type hypersensitivity Mab monoclonal antibody rrat TAA tumor-associated antigen
Introduction Tumor immunology has been advancing on two major fronts: first, the elucidation of the basic mechanisms of tumor recognition and rejection, and protection from recurrence; and, second, the application of that knowledge to cancer as a clinical problem. The basic science front has advanced rapidly and provided tools with which to explore two different approaches to disease — immunotherapy and cancer vaccines. Immunotherapy relies on administration of preformed effector mechanisms and it is ideally suited for application to the treatment of established tumors. Cancer vaccines, on the other hand, are being developed with an eye on prevention. Taking as an example the successes that vaccines have had in preventing infectious diseases, efforts are being made to define tumor targets and immunization protocols that would be equally effective in priming the immune system to eliminate cancer before it manifests itself clinically. Although the basic studies can be well designed and preclinical animal models continue to be developed and used to test the newly discovered scientific principles, there is hesitation in translating these findings to cancer prevention in humans.
With the exception of efforts to vaccinate against microorganisms known to cause human cancers, such as HPV (human papillomavirus) [1,2] and in that way prevent not only infection but also (indirectly) cancer, hundreds of clinical trials of cancer vaccines are currently being carried out in patients who already have cancer. These efforts have added a new term to the vaccine field — ‘therapeutic vaccines’. The efforts are driven more by the practicality of the approach than by strong supporting evidence from basic and preclinical research. Animal studies have shown that cancer vaccines are most effective in protection from tumor challenge or, in more recent studies that we review below, in prevention of tumor occurrence in genetically predisposed animals. Vaccines have shown limited potential in curing established tumors. Clinical trails of therapeutic cancer vaccines not surprisingly recapitulate the observations in animals. The trials show the ability of the vaccine to stimulate, to widely varied degrees, specific antibodies and T cells but, with few exceptions [3–6], rarely result in an objective clinical antitumor response. However, even though therapeutic vaccines are currently of marginal clinical utility, they will be ultimately useful for having paved the way for the use of cancer vaccines in people, in a setting that can be anticipated to have a much greater impact on human health — that of bona fide cancer prevention. In this review we will highlight studies published in the past year that are providing basic and preclinical support for preventive cancer vaccines.
Cancer vaccines protect from tumor challenge and prevent tumor occurrence Two schools of thought dominate research into tumor antigens and tumor vaccines. One believes that tumor rejection can be obtained only by immunizing with unique tumor-associated antigens (TAAs) [7,8], supporting the use of undefined tumor-derived materials — that would contain such antigens — as tumor vaccines. This is based on studies with carcinogen-induced tumors in rodents, where it was hard to demonstrate the presence of shared antigens (expressed on multiple tumors of the same tissue type) and cross-protective antigens (immunization against which could prevent occurrence of tumors in different individuals). In humans, shared antigens appear to be predominant targets of tumor-specific immunity [9]. Furthermore, as we will review below, animal models designed to test their tumor-rejection potential show that they can elicit tumorrejection responses. Other studies in animals showed that shared tumor antigens did exist. Carcinogen-induced fibrosarcomas in chickens not only cross-protected from tumor challenge [10], but also inhibited new carcinogenesis
Prophylactic cancer vaccines Finn and Forni
[11]. Vaccination with purified TAA was reported to suppress chemically induced mammary tumors in rats [12], and vaccination with p53 protein prevented chemically induced skin cancers in mice [13]. Most recently, TAAs — mostly self proteins — derived from spontaneously arising tumors in p53−/− mice were shown to cross-protect vaccinated mice against tumor challenge [14••]. Vaccination of C3H/HeOuj mice (which are known for their very high incidence of breast cancer) with a mouse breast cancer TAA — mouse MUC1 protein — significantly lowered the incidence of breast cancer in those mice [15]. A more important issue than unique versus shared antigens is the ability of a vaccine to appropriately prime immune effector mechanisms. TG.AC × C57BL/6 F1 mice carry in their germline a mutant ras oncogene that has an arginine at codon 12 instead of the glycine present in the wild-type. After physical promotion (by wounding) or chemical promotion, these mice develop papillomas that progress to cancer. Immunization with Arg(12)-mutant ras peptide induced T-cell reactivity and specific delayed-type hypersensitivity (DTH). However, when mice were painted with phorbol 12-myristate 13-acetate, tumors grew faster in the immunized mice. Immunization not only failed to protect against papillomas, but also induced a remarkable enhancement of their growth [16]. These findings are reminiscent of the inhibition of papilloma growth in mice with the TNF-α gene knocked out [17] and the enhancement of mammary carcinogenesis in mice transgenic for the PyMT oncogene and CSF-1 [18]. Specific immunity [19] and nonspecific inflammatory reactions [20,21] may in certain instances enhance tumor growth. The challenge will be to avoid stimulating the type of immunity that enhances carcinogenesis, by designing vaccines that promptly activate strong reactions at the site of the lesion. The very same immune mechanisms that have been seen to enhance tumor growth will lead to its rejection when fully activated [19].
Tumor prevention by stimulation of TAA-specific immunity: transgenic mouse models The use of overexpressed or mutated proteins, or mutated oncogenic growth-factor-receptors as TAAs has yielded rational targets for specific immunoprevention [22]. Transgenic mouse models are providing encouraging indications of future usefulness of vaccines based on these molecules and we describe four such models below.
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in virgin females or only after pregnancy and can involve one or all ten mammary glands [24]. Both the slow carcinogenesis driven by Her-2/neu protooncogene and the aggressive one due to the transforming r-Her-2/neu are inhibited by vaccination with proteins, peptides or DNA plasmids [25–28,29•,30–34,35••]. Inhibition appears to be mostly dependent on IFN-γ-based DTH and antibody, as suggested by pathological findings and in vitro tests [29•,32,35••]. In several lines of transgenic mice, anti-r-p185neu vaccination appears to be unable to elicit cytotoxic T lymphocyte (CTL)-mediated killing of p185neu+ cells [29•,32,35••,36], probably due to the difficulty of fully breaking their tolerance [37•]. Vaccination-induced antibodies appear to block carcinogenesis by inhibiting the ability of p185neu to transduce cell-activating signals and downregulating its membrane expression in preneoplastic cells [29•]. Their activity appears to be similar to that of passively administered anti-p185neu monoclonal antibody (Mab) [38]. By contrast, the role of antibodies in the rejection of r-p185neu+ transplantable tumors is controversial. In this setting, some vaccination protocols, anti-r-p185neu antibodies amplify the protection provided by T cells [36] whereas in others a strong CTL response leads to tumor rejection in the absence of antibodies [39•]. Thus, mechanisms other than CTL activity may inhibit carcinogenesis driven by the expression of oncogenic growth-factor-receptors on the cell membrane [22]. These mechanisms, interestingly, do not play a major role in the inhibition of growth of transplantable tumors [29•]. CEA-transgenic mice
Carcinoembryonic antigen (CEA) is a well-defined TAA that was first described in 1965 (see [40]). Human-CEAtransgenic mice have been generated that express the transgene under its own promoter [41,42]. They have been used to explore the immunogenicity and tumor-rejection potential of CEA in a setting in which, as in humans, it is also a self-antigen. The studies show that a certain degree of tolerance to this antigen exists in CEA-transgenic mice compared with wild-type mice, but this tolerance can be easily overcome with well-designed immunization protocols to generate immune responses that are protective against tumor challenge [43,44,45•]. Importantly, breaking tolerance to CEA does not result in any autoimmune reactions against normal tissues that express the transgene. MUC1-transgenic mice
HER-2/neu-transgenic mice
Thanks to the pioneering work of Muller (see [23]), transgenic mice overexpressing the rat (r-)Her-2/neu protooncogene or its mutated form constitute attractive models for testing immunoprevention of mammary carcinogenesis. These mice express r-p185neu protein at distinct stages of their life. Consequently, their tolerance levels to this protein and the aggressiveness of carcinogenesis are markedly different at various stages. Carcinogenesis takes place either
MUC1 was identified as a TAA by several groups simultaneously, using a tumor-specific mouse Mab [46], or tumor-specific T-cell lines from patients with epithelial adenocarcinomas [47,48]. In recent years, several lines of human MUC1-transgenic mice have been generated on the C57BL [49], BALB/c [50•] and DBA [51] backgrounds. These mice are transgenic for the entire human MUC1 gene driven by its own promoter. As such, this transgenic protein shows temporal and tissue expression typical of the
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endogenous antigen in humans. MUC1-transgenic mice showed peripheral tolerance to MUC1 in both T- and B-cell compartments, but this tolerance could be broken by increasing the immunogenicity of vaccine preparations [49,50•,51,52•,53,54•]. MUC1-specific immunity, primarily CD8+ T cells, which resulted in tumor rejection and longterm protection, did not result in autoimmunity. Several transgenic tumor models that express the SV-40 large T antigen under various tissue specific promoters and develop tumors in various tissue sites (such as breast, prostate, colon or pancreas), are being crossed with the MUC1-transgenic mice. One such double-transgenic model is the MET mouse that, because of elastase-driven T antigen, develops pancreatic tumors, which in the presence of the MUC1 transgene express MUC1. Concurrent with tumor development, these mice spontaneously develop MUC1-specific CTLs that do not stop growth of spontaneous tumors. However, when transferred to naïve mice, they can protect fully from MUC1+ tumor challenge [55••]. TRAMP transgenic mice
Prostate cancer is the most common malignancy in men. Its incidence is soaring due to the aging of the population and the introduction of diagnostic procedures that reveal its early stages. In TRAMP C57/BL transgenic mice, expression of SV40 T antigen is under the probasin promoter that is regulated by androgens and restricted to the prostate epithelial cells. Prostate tumor progression resembles the human disease [56]. A preventive immune response against primary tumors could be elicited in this model by using an irradiated-tumorcell vaccine and CTLA-4 blockade [57]. Furthermore, treatment of the prostate tumor cells with 15-deoxy-δ12,14prostaglandin J2 induced expression of heat-shock protein (Hsp) 70, and vaccination of TRAMP C57/BL transgenic mice with Hsp 70 from these tumors elicited tumor-specific CTLs and protected from tumor challenge [58].
to be expressed very early in the process of transformation and are frequently expressed in premalignant dysplasia and metaplasia. Several human tumors, such as colon, breast, prostate and lung, yield themselves to early detection and patients with familial history or other risk factors for these malignancies are under regular monitoring. Surgery is an option for removal of a small tumor but, with the exception of polyp removal for colon cancer prevention, there are no strategies for dealing with premalignant changes. A number of TAAs reviewed here would be candidate vaccines in the setting of premalignant disease [61−65]. Immune prevention could also be considered for individuals whose tumors have been eradicated by standard therapy. Clinical data on patients treated with Mab [66], and experimental data in mice immunized after tumor surgery [67,68] show that prevention of recurrences in conditions of minimal residual disease is a permissible immunological challenge. However, since the immune system has been imprinted by a previous encounter with a progressing tumor, tolerance to TAAs may need to be overcome in order to elicit an efficient response.
Acknowledgements This work was supported by grant 5R01 CA56103-10 from the National Cancer Institute and grants from the Italian Association for Cancer Research and the Italian Ministry for University and Scientific and Technological Research.
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Conclusions Different experimental systems show that immunity can be activated to prevent tumors. The rationale for prevention is strong because in that setting one deals with an immune system that is neither impaired by tumor- and treatment-induced suppression, nor tolerant to TAAs that have been encountered in the absence of correct presentation and costimulatory/danger signals. For a tumor to develop in healthy individuals with either a genetic risk of cancer or bearing preneoplastic lesions may take a considerable length of time. A certain length of time is also necessary for the induction of strong immunity to tumor TAAs. The elicited immune mechanisms are likely to have a greater impact on subclinical tumors than on those that have disseminated or become unresectable [59, 60•]. Although most antigen-discovery methods employed tumors as sources of antigen, many TAAs have been found
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MUC1 is broken and the tumors are rejected. Both T cells and antibodies are produced and the immune response is long-lasting. Mice that have rejected IL-12-transfected tumors are protected from subsequent tumor challenge.
39. Pilon SA, Piechocki MP, Wei WZ: Vaccination with cytoplasmic • Erbb-2 DNA protects mice from mammary tumor growth without anti-Erbb-2 antibody. J Immunol 2001, 167:3201-3206. The role of antibodies to p185neu in the inhibition of Her-2/neu carcinogenesis is still not well defined. The role of these antibodies in the rejection of transplantable p185neu cells is also not clear. In some instances, as in [38], anti-Her-2/neu antibodies appear to play an important role in enhancing the protective effects of cellular reactivity. In this paper, a DNA vaccine encoding a full-length r-Her-2/neu protein that is targeted to the cytosol and rapidly degraded there by the proteasomes was used to elicit a CTL response without an antibody response in normal parental mice. The feasibility of eliciting individual effector mechanisms by targeted DNA vaccine may open a new, interesting way of assessing the weight of cellular and antibody responses in the inhibition of carcinogenesis in transgenic mice.
51. Acres B, Apostolopoulos V, Balloul J-M, Wreschner D, Xing P-X, Ali-Hadji D, Bizouarne N, Keiny MP, Mckenzie IFC: MUC1-specific immune responses in human MUC1 transgenic mice immunized with various human MUC1 vaccines. Cancer Immunol Immunother 2000, 48:588-594.
40. Hammarstrom S: The carcinoembryonic antigen (CEA) family: structures, suggested functions and expression in normal and malignant tissues. Semin Cancer Biol 1999, 2:67-81. 41. Eades-Perner A-M, van der Putten H, Hirth A, Thompson J, Neumaier M, von Kleist S, Zimmerman W: Mice transgenic for the human carcinoembryonic antigen gene maintain its spatiotemporal expression pattern. Cancer Res 1994, 54:4169-4176. 42. Clarke P, Mann J, Simpson JF, Rickard-Dickson K, Primus FJ: Mice transgenic for human carcinoembryonic antigen as a model for immunotherapy. Cancer Res 1998, 58:1469-1477. 43. Kass E, Schlom J, Thompson J, Guadagni F, Graziano P, Greiner JW: Induction of protective host immunity to carcinoembryonic antigen (CEA), self-antigen in CEA transgenic mice, by immunizing with a recombinant vaccinia-CEA virus. Cancer Res 1999, 59:676-683. 44. Mizobata S, Tomkins K, Simpson JF, Shyr Y, Primus FJ: Induction of cytotoxic T cells and their anti-tumor activity in mice transgenic for carcinoembryonic antigen. Cancer Immunol Immunother 2000, 49:285-295. 45. Xiang R, Siletti S, Lode HN, Dolman CS, Ruehlmann JM, • Niethammer AG, Pertl U, Gillies SD, Primus FJ, Reisfeld RA: Protective immunity against human carcinoembryonic antigen (CEA) induced by an oral DNA vaccine in CEA-transgenic mice. Clin Cancer Res Suppl 2001, 7:856-864. In this study, a CEA-based vaccine was delivered by an oral route in a live, attenuated AroA− strain of Salmonella typhimurium harboring CEA expression vectors that targeted CEA expression either to the cell surface or to the endoplasmic reticulum (ER). When mice were challenged with a lethal dose of tumor cells expressing CEA, mice immunized with Salmonella bearing the empty vector or the CEA targeted to the ER were not protected, whereas those immunized with the Salmonella bearing the vector that targeted CEA to the cell surface showed a dramatic decrease in tumor volume and showed complete protection in 50% of the animals. Furthermore, a fusion protein of an antibody against a second antigen on the tumor cells and IL-2, that had no antitumor effects when administered to naïve mice, increased antitumor effects in mice immunized with Salmonella−CEA. Antitumor effector cells were MHC-class-I-restricted CTLs. Targeting IL-2 to the tumor site via anti-CEA-antibody−IL-2 fusion protein increased release of proinflammatory cytokines from T cells of successfully vaccinated animals. 46. Gendler SJ, Spicer AP: Epithelial cell mucins. Annu Rev Physiol 1995, 57:607-634. 47.
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48. Taylor-Papadimitriou J, Finn OJ: Biology, biochemistry and immunology of carcinoma-associated mucins. Immunol Today 1997, 18:105-107. 49. Rowse GJ, Tempero RM, VanLith ML, Hollingsworth MA, Gendler SJ: Tolerance and immunity to MUC1 in a human MUC1 transgenic murine model. Cancer Res 1998, 58:315-321. 50. Carr-Brandel V, Markovic D, Ferrer K, Smith M, Taylor-Papdimitriou J, • Cohen ER: Immunity to murine breast cancer cells modified to express MUC1, a human breast cancer antigen, in transgenic mice tolerant to human MUC1. Cancer Res 2000, 60:2435-2443. In this study, MUC1-transgenic mice are permissive of growth of a breast tumor line transfected with MUC1 and injected into the fat pad of the breast. However, when these cells are engeneered to express IL-12, tolerance to
52. Lees CJ, Apostolopulos V, Acres B, Ramshaw A, Ong CS, • McKenzie IF: Immunotherapy with mannan-MUC1 and IL-12 in MUC1 transgenic mice. Vaccine 2000, 19:158-162. This study provides support for importance of IL-12 in anti-MUC1 immunity. When double-transgenic MUC1/HLA-A2 mice are vaccinated with oxidized mannan−MUC1 fusion protein they generate MUC1-specific, HLA-A2restricted CTLs that are responsible for tumor protection. The frequency of these CTLs increases significantly and tumor protection is enhanced with the addition of IL-12. 53. Koido S, Kashiwaba M, Chen D, Gendler S, Kufe D, Gong J: Induction of antitumor immunity by vaccination of dendritic cells transfected with MUC1 RNA. J Immunol 2000, 165:5713-5719. 54. Soares MM, Mehta V, Finn OJ: Three different vaccines based on • the 140 amino acid MUC1 peptide with seven tandemly repeated tumor-specific epitopes elicit distinct immune effector mechanisms in wild-type versus MUC1 transgenic mice with different potential for tumor rejection. J Immunol 2001, 166:6555-6563. This study compares different adjuvants — GMCSF, SB-A2 and dendritic cells — for their effectiveness in increasing the immunogenicity of a MUC1 peptide in wild-type and MUC1-transgenic mice. Two important observations are made: first, peptide mixed with GMCSF and SB-A2 generates only antibody responses and no T-cell immunity, but anti-MUC1 antibodies do not elicit protection from tumor challenge; second, peptide loaded onto dendritic cells elicits CD4+ and CD8+ T cells in wild-type mice but only CD8+ T cells in MUC1-transgenic mice. MUC1-specific CD8+ T cells in the absence of MUC1-specific CD4+ T cells provide long-term protection against tumor challenge. 55. Mukherjee P, Ginardi AR, Madsen CS, Sterner CJ, Adriance MC, •• Tevethia MJ, Gendler SJ: Mice with spontaneous pancreatic cancer naturally develop MUC1-specific CTls that eradicate tumors when adoptively transferred. J Immunol 2000, 165:3451-3460. Patients with MUC1+ tumors have both anti-MUC1 antibodies and MUC1specific T cells whereas healthy individuals do not. It is not known when in the disease process the immune system responds to this antigen. This study, performed in MUC1-transgenic mice that spontaneously develop pancreatic cancer, addresses this problem. Tumors become detectable by immunohistology at 9 weeks of age and progressively grow through to 24 weeks of age. MUC1-specific CTLs appear at 12 weeks of age, increase through to 18 weeks and begin to disappear by 21 weeks. They are specific for a peptide from the tandem-repeat region previously shown to be a human class-Irestricted epitope. Although these CTLs did not appear to affect endogenous tumor progression, they provided long-term protection from tumor challenge when transferred to naïve mice. 56. Gingrich JR, Barrios RJ, Morton RA, Boyce BF, DeMayo FJ, Finegold MJ, Angelopoulou R, Rosen JM, Greenberg NM: Metastatic prostate cancer in a transgenic mouse. Cancer Res 1996, 56:4096-4102. 57.
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