- Email: [email protected]
Peptide-based cancer vaccines
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Peptide-based cancer vaccines Cornelis JM Melief* , Rienk Offringa*, Rene EM Toes* and W Martin Kastt The application of molecularly defined vaccines composed of a limited number of tumor-specific T cell epitopes has resulted in protective antitumor T cell immunity in several mouse tumor models. The first encouraging results with such vaccines have been obtained in human beings. The development of the next generation of rationally designed vaccines that are both effective and safe for application in a clinical setting requires comparison of different modes of delivery of tumor-associated T cell epitopes in multiple epitope constructs.
Addresses *Department of Immunohaematology, University Hospital of Leiden, Rijnsburgerweg 10, Leiden, The Netherlands tCancer Immunology Program, Cardinal Bernadin Cancer Center, Loyola University of Chicago, 9160 South First Avenue, Maywood, IL 60153, USA Se-mail: [email protected] Current Opinion in Immunology 1996, 8:651-657 © Current Biology Ltd ISSN 0952-7915
Abbreviations Ad adenovirus CTL cytotoxic T lymphocyte DC dendriticcell EBNA Epstein-Barrnuclear antigen EBV Epstein-Barrvirus HBV hepatitisB virus HPV humanpapillomavirus IFA incompleteFreund's adjuvant LCMV lymphocytic choriomeningitis virus
Introduction Evidence is accumulating that the immune system usually does not react against all possible antigenic determinants, but only against the most immunodominant ones [1]. This is particularly true in the case of tumors, which often elicit no demonstrable responses at all. Mechanisms that contribute to this regrettable state of affairs are general disturbances in the immunocompetence of cancer patients [2], the poor immunogenicity of cancer cells [3,4], and the production of paracrine factors promoting tumor growth and angiogenesis by tumor cells [5",6°]. Antitumor vaccination can help to overcome many but not all of these problems. Tremendous progress has recently been made in the identification of both viral and nonviral tumor-associated antigens. This allows the development of molecularly defined vaccines, containing the minimal essential components, that can be used to elicit specific T cell responses against both dominant and subdominant T cell epitopes [7,8]. In addition, nonspecific components that orient the T cell response in the desired Th.(CD4÷) or cytotoxic T lymphocyte (CTL) (CD8 ÷) direction, such as
the interleukin (IL)-2, interferon (IFN)-7 or IL-12 genes or gene products, can now be incorporated into vaccines. T h e development of such rational vaccine design has only just started. Great diligence will be required to make a significant impact on the cancer problem by vaccination. Chances are that vaccination will succeed primarily as adjuvant therapy following debulking by conventional treatment. If vaccination fails, more drastic, laborious and expensive treatment by adoptive transfer of tumor-specific T cells, expanded in vitro, will be needed. Nature of tumor antigens expressed on virus-induced and nonvirus-induced tumor cells Most virus-induced tumor cells display a variety of MHCbound peptides of viral origin at their cell surface, although exceptions to this rule have been found (reviewed in [9"]). For instance, Burkitt's lymphoma cell lines, which retain the original tumor cell phenotype of fresh biopsies, express only a single virus-encoded protein, the Epstein-Barr virus (EBV)-encoded nuclear antigen (EBNA)-I [9"]. Moreover, the processing of this protein for presentation by H L A class I antigens is inhibited by the internal repeat region of EBNA-1 [10"°]. Other EBV-induced neoplastic cells and long-term B lymphoblastoid lines that express a variety of EBV antigens, however, do present H L A class I binding EBV peptides to the T cell immune system [9"]. One of the EBV gene products, latent membrane protein (LMP)-I, even appears to stimulate antigen processing and presentation in that it enhances the expression of both TAP (transporter associated with antigen processing) and H L A class I molecules [9"].
In the case of human papilloma virus (HPV)16, one of the major human papiilomavirus types associated with cervical cancer, a variety of immunogenic peptides encoded by the HPV16 E6 and E7 viral oncogenes were identified that could be useful in vaccines for the prevention and treatment of cervical carcinoma [11",12]. T h e natural C T L response to these HLA-A*0201-binding peptides was only demonstrable in a minority of patients with HPV16-associated cervical lesions [13"'], indicating that most patients might benefit from vaccination that would induce vigorous C T L responses to these viral peptides [13"1. Substantial progress has been made in the definition of melanoma-associated antigens recognized by C T L s (reviewed in [14°']). A detailed discussion of these antigens is beyond the scope of this review but we shall return to several of these antigens in our discussion of clinical vaccine trials. T h e fact that most of these
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antigens are encoded by nonmutated cellular genes, and can therefore be regarded as self antigens, has strongly promoted the concept that T cell immunity directed against such antigens can be employed to eradicate tumors and has encouraged the search for novel tumor-associated self antigens. Additional autologous antigens that are overexpressed or mutated in certain human tumors, and against which CD8 ÷ CTLs or CD4 ÷ T h cells have been raised, include carcinoembryonic antigen [15°°], mutant Ras [16",17°], HER-2/Neu [18], mutant and wild-type p53 [19,20] and Bcr/Abl breakpoint peptides [21,22°°]. In a mouse model, CTLs were raised against a self peptide derived from the p53-binding protein mdm2 and these C T L s were capable of lysing target cells that carry this epitope as a naturally processed peptide [23°°]. In HLA-transgenic mice, CTLs were raised against HLAA*0201-binding wild-type human p53-derived peptides that are not fully conserved between human and mouse p53. These CTLs, while not recognizing nontransformed human cells, were able to lyse human tumor cell lines [24°°]. Recently, we have been able to raise C T L s against a wild-type mouse p53 peptide by immunization of p53 knockout mice with congenic p53-overexpressing tumor cells. These CTLs, upon adoptive transfer into p53+/÷ mice bearing p53-overexpressing tumors, were able to eradicate these tumors without signs of autoimmunity to the host (M Vierboom et al., unpublished data). T h e prospect of utilizing p53 as a general target for CTL-mediated therapy of cancer is particularly attractive because p53 is overexpressed in more than 50% of all human cancers.
tumors will grow to cause considerable difficulties in the immunotherapy of many tumors.
Vaccination with MHC class I binding peptides, a delicate balance between immune activation and immune suppression Protective C T L responses induced by vaccination with MHC class I binding peptides were first reported by Schulz et al. [33] for lymphocytic choriomeningitis virus (LCMV) and independently by Kast et al. [34] for Sendai virus. In these studies a substantial antiviral effect was obtained by subcutaneous vaccination with a single MHC class I binding peptide in incomplete Freund's adjuvant (IFA). Subsequent studies showed that strict peptide length is not required for the induction of protective immunity in that a 16-mer Sendal peptide with additional flanking residues protected similarly to the minimal 9-met that shows optimal MHC binding [35]. Vaccination with this immunodominant peptide in IFA leads to activation of Sendai virus specific T cells with a wide variety o f T cell receptors, all recognizing the same Sendai epitope [36].
Factors influencing processing and presentation of tumor antigens
When applied to a murine tumor model, the principle of vaccination with MHC class I binding immunogenic peptides also resulted in the induction of protective immunity. This was demonstrated by prevention of outgrowth of an HPV16-induced tumor in C57BL/6 mice by vaccination with a peptide derived from the HPV16 E7 oncoprotein in IFA [37]. Similarly, vaccination with either of two tumor-associated mutant connexin peptides, emulsified in IFA or loaded onto processing-defective RMA-S cells, induced protection against metastatic tumor spread [38,39°°]. C T L s capable of lysing human HPV16 ÷, HLA-A*0201+ cervical carcinoma cells were raised by in vitro stimulation of peripheral blood mononuclear cell cultures of healthy HLA-A*0201+ donors with HLAA*0201-binding HPV16 E7-derived peptides [11"°]. In the first application of peptide vaccination in human beings, a vigorous C T L response was induced in hepatitis B virus (HBV)-negative human volunteers by vaccination with a lipopeptide consisting of an HLA-A*0201-binding HBV peptide, a pan HLA-DR-binding peptide not based on the HBV sequence and two palmitic acid tails. These C T L s were capable of lysing HLA-A*0201 + HBV-infected target cells in vitro [40°']. Previously, a similar lipopeptide vaccine had already been shown to induce functional T cell responses in a murine model [41].
T h e factors that determine successful recognition of tumor antigens by C T L s include proper proteolytic cleavage by the proteasome complex [28,29",30°,31°°], efficiency of peptide transport via the TAP system [31"',32 °] and surface display of peptide-MHC complexes [9",10°°,32°]. Presentation of immunogenic peptide epitopes to T cells might be avoided through epitope mutations affecting proteasome cleavage [31"°], MHC class I binding [32°] or blockage of processing by an unknown mechanism [9"]. Undoubtedly the list of evasion mechanisms employed by
Subcutaneous peptide vaccination at moderate doses in IFA very consistently causes protective immunity with the peptides mentioned above. Surprisingly, administration of some of these peptides at very high doses by the intraperitoneal route causes immunological tolerance to the peptide rather than protective immunity. This was first shown with an H-2Db binding, LCMV-derived peptide [42 °°] and this principle could be used to prevent autoimmune diabetes in a transgenic mouse model [43].
Minor histocompatibility antigens are a special category of tumor-associated antigens. Notably, those minor histocompatibility antigens with tissue-specific expression (e.g. in hematopoeitic tissue) are attractive target antigens for cancer immunotherapy [25]. Last year the molecular nature of two human minor histocompatibility antigens was unveiled [26°°,27°]. One of the therapeutic regimens that can be considered is adoptive transfer of donor-derived CTLs against host hemopoietic cell specific minor histocompatibility antigens expressed on a relapsed leukemia following allogeneic bone marrow transplantation.
Peptide-besed cancer vaccines Melief et aL
Certain other peptides, when delivered subcutaneously in IFA, consistently fail to induce protective immunity across a wide dose range. This phenomenon was observed in a murine tumor model for two adenovirus (Ad)-derived peptides that constitute the major T cell epitopes of Ad-induced tumors in C57BL/6 mice. C T L s against these peptides, raised by immunization with irradiated Ad-induced tumor cells, are capable of eradicating large established tumors following intravenous infusion into tumor-bearing T cell deficient nude mice [44,45]. In contrast, vaccination of immunocompetent mice with a single dose (0.1-10~tg]mouse) of Ad peptide caused enhanced outgrowth of subsequently transplanted Ad-induced tumors. This increased tumor growth was parallelled by specific tolerance of the mice for the injected peptide epitope [46°',47°']. T h e effect of peptide vaccination in these experiments was so striking because the tumor models chosen were such that either of the two Ad peptides could be the single dominant C T L epitope determining T cell protection against tumor growth [46°°,47°°]. In tumor-host relationships in which multiple T cell epitopes are potential targets, the consequences of downregulation of the response against a single epitope are likely to be less conspicuous. On the other hand, the affected T cell response may play a pivotal role in the resistance against a given tumor and, therefore, even deletion of this part of the antitumor T cell response should be avoided at all times. Although in the Ad tumor model, subcutaneous immunization with peptides in IFA led to specific tolerance at all doses tested, different modes of peptide delivery (including immunization with irradiated tumor cells or with replication-defective Ad that encodes the relevant epitopes) led to protective immunity associated with strong tumor-specific C T L activity [46"']. Furthermore, protective C T L immunity, rather than tolerance, can be obtained by immunization with activated dendritic cells (DCs) that have been loaded with synthetic Ad peptides (REM Toes, R Offringa, WM Kast, CJM Melief, unpublished data). The reasons why the Ad peptides injected at low doses subcutaneously in IFA caused specific C T L tolerance, while various other C T L epitope carrying peptides administered in exactly the same way consistently caused protection associated with C T L memory [33,34,36,38], remain unclear at this time. T h e contrasting effects of vaccination with either Ad peptides in IFA or Ad peptides loaded onto DCs suggest that not the peptides perse but rather the context in which they are presented to the T cell immune system determines the outcome of immunization with peptide-based vaccines. Inflammatory and costimulatory signals, collectively also named 'danger' signals [481, are likely to constitute important factors in creating the appropriate context for stimulation of the peptide-specific T cell response. Apparently, the balance between tolerance and immunity
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is more precarious for the Ad peptides than for other peptides tested. These observations imply that individual peptides to be used for antivirus or anticancer vaccines should at least be analyzed in HLA-transgenic animals for their immunogenic/tolerogenic properties in vivo [11°°], before they are used in a clinical setting. Probably even better, modes of peptide delivery must be considered that uniformly induce protective immunity associated with strong C T L responses. One such delivery mode may be peptide presentation on DCs. Indeed, activated DCs, obtained by culturing bone marrow cells with granulocyte macrophage-colony stimulating factor (GM-CSF) and IL-4, loaded with ovalbumin-derived MHC class I binding peptides induced protection in mice against the outgrowth of ovalbumin-transfected B16 melanoma cells [49"]. T h e same group of investigators in collaboration with our group has reported comparable results with tumor-derived peptides [50"']. In this study, immunization with peptide-pulsed DCs not only elicited protective immunity, but in addition exerted therapeutic effects on already established tumors [50"']. This approach was also effective with unfractionated acid-eluted tumor peptides loaded onto DCs. The effectiveness of immunization with peptide-pulsed DCs, with respect to antitumor effects in this study, depended on appropriate B7 costimulation and on DC-induced production of Thl-associated cytokines such as tumor necrosis factor (TNF)-~ and IFN-y [51"]. Effective vaccination was also achieved against chemically induced sarcomas in BALB/c mice with a mutant p53 peptide in IFA [52] or with either mutant or wild-type p53 peptides loaded onto activated DCs [53"']. In patients with cancer, peptide vaccination has only been applied on a very limited scale. Surprisingly, even immunization with free peptides corresponding to an epitope encoded by the melanoma-associated gene MAGE-3 induced complete regression of two metastatic melanomas [54"']. This peptide vaccination trial was conducted as a phase I toxicity study and tumor responses in the absence of adjuvant had not been anticipated. In another study [55], three patients with advanced metastatic melanoma were vaccinated with autologous plastic-adherent mononuclear blood cells that had been cultured in recombinant GM-CSF and pulsed with an HLA-A*0101-binding MAGE-1 peptide. MAGE-1 peptide specific C T L s were demonstrable after, but not prior to, vaccination and these CTLs were capable of lysing HLA-A*0101 ÷ MAGE-1 ÷ melanoma cells in vitro. No major therapeutic responses were noted, possibly because of the advanced stage of the disease.
Future of vaccination with minimal T cell epitopes T h e results just reviewed clearly show that vaccination with tumor-associated MHC-binding peptide epitopes is a powerful tool for the induction of tumor-specific T cell responses. Protective immunity has been achieved by immunization with peptides in IFA, peptides pulsed onto DCs and peptides encoded by replication-deficient Ads.
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On the other hand, peptides in IFA do not always protect. Depending on yet unknown properties of peptides, vaccination with certain peptides can lead to tolerance in regimens that are associated with protection in the case of other peptides [7,8,37,39",46"',47"]. Vaccination with peptide-loaded DCs may be superior to peptide in adjuvant, especially with respect to peptides that are prone to induce tolerance, but this approach is clearly more cumbersome in a clinical setting. It is our estimation that neither peptides in adjuvants, nor lipopeptides, nor peptides loaded onto D e s will ultimately prove to constitute effective, safe and versatile anticancer vaccines. Such vaccine formulations would be too restricted with respect to HLA type and antigen specificity and/or too laborious with respect to application on a large scale. Rather, we believe that the results recently obtained with experiments in mouse models and with several clinical trials should boost rapid progress in the direction of DNA-, virus- or protein-based vaccines that encode/harbor a multitude of epitopes. In our Ad-tumor model in mice, we found the most effective vaccination approach involved the injection of replication-deficient Ad [46"']. Others demonstrated that vaccination with a fowlpox virus, genetically engineered to encode the model tumor antigen 13-galactosidase, protected mice against challenge with tumor cells that express this antigen [56]. Similarly, vaccination with a recombinant vaccinia virus harboring a minigene encoding an H-2Kbrestricted ovalbumin epitope induced protection against ovalbumin-expressing EL4 tumor cells [57]. Addition to epitope-encoding minigenes of an amino-terminal endoplasmic reticulum insertion sequence in some cases greatly enhanced the CD8 ÷ C T L immune response [58"]. Furthermore, vaccinia viruses carrying string-of-beads minigenes that encode multiple C T L epitopes have been shown to confer protection from lethal LCMV challenge in mice of different H-2 types [59]. Such string-of-beads constructs consist of the genetic code for multiple C T L epitope containing peptides that are linked together with or without additional amino acids in between the epitopes serving as spacers. Similarly, protection against LCMV was obtained using recombinant gene products consisting of several M H C class I binding peptides inserted into various positions of self and nonself proteins [60"]. Polyepitope proteins or gene constructs have yet to be applied for vaccination against tumors, although development of such anticancer vaccines is well underway. This is illustrated, for instance, by a recent report in which infection of cells with a recombinant vaccinia virus encoding multiple known H L A class I binding EBV peptides was shown to sensitize these cells to recognition by C T L s against all relevant EBV epitopes [61"].
has been around for many years (reviewed in [62]). Indeed, inactivated influenza virus when presented by D e s stimulates human CD8 ÷ C T L responses [63] and protective CD8 ÷ C T L responses can be elicited by vaccination with nonliving bacteria [64]. We recently observed that completely allogeneic tumor cells induce very effective antitumor immunity based on induction of C T L s against epitopes processed and presented by host antigen-presenting cells [65"].
Conclusion Vaccination experiments with individual M H C class I binding peptides have now led the way to vaccine constructs that encode or harbor multiple epitopes. As for the single epitope vaccines, the mode of delivery of muhiepitope vaccines will prove equally important. To compare efficiently the various delivery systems (viral vectors, protein in adjuvant, DNA; reviewed in [66]) it is important to perform comparative vaccination in animal model systems, using the same epitopes in different delivery systems. In this respect, the most useful model systems are based on HLA-transgenic mice, as such models in essence allow evaluation of vaccines that are intended for clinical use [11"]. T h e most promising vaccines can subsequently be tested in patients. Vaccination is likely to be effective only in patients who have minimal residual disease following conventional treatment, or who exhibit a very early stage of the disease, because vaccination has been shown to be capable of eradication of only low numbers of tumor cells. For patients in more advanced stages, adoptive transfer of large numbers of tumor-specific T cells that have been expanded in vitro can be considered. Even for this obviously more laborious treatment, proper vaccination might be a first step toward expansion of low numbers of autologous T cell precursors.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • •.
of special interest of outstanding interest Sercarz EE, Lehmann PV, Ametani Benichou G, Miller A, Moudqil K: Dominance and crypticity of T cell antigenic determinants. Annu Rev /mmunol 1993, 11:729-766. Ochoa A, Longo DL: Alteration of signal transduction in T cells from cancer patients, tn Important Advances in Oncolog¥. Edited by De Vita VT, Hellman S, Rosenberg SA. Phildelphia: JB Lippincott Company; 1995:43-54. Melief CJM: Tumor eradication by adoptive transfer of cytotoxic
T lymphocytes. Adv Cancer Res 1992, 58:143-175. Nanda NK, Sercarz EE: Induction of anti-self immunity to cure cancer. Ceil 1995, 82:13-17.
T h e concept of using exogenous protein for priming of C T L responses is gradually gaining ground, although evidence for cross-priming of C T L s by exogenous protein
Seung LP, Rowley DA, Dubey P, Schreiber H: Synergy between T cell immunity and inhibition of paracrine stimulation causes tumor rejection. Proc Nat/Acad Sci USA 1995. 92:6254-6258.
Peptide-based cancer vaccines Melief et al.
Paracrine factor produced by tumor cells counteracts tumor eradication by T lymphocytes. The factor attracts granulocytes that make the tumor cells grow faster, outsmarting the T lymphocytes. 6.
Parangi S, O'Reilly M, Christophori G, Holmgren L, Grosfeld J, Folkman J, Hanahan D: Anti-angiogenic therapy of transgenic mice impairs de novo tumor growth. Proc Nat/Acad Sci USA 1996, 93:2002-2007. Antiangiogenic therapy may eventually be used synergistically with T cell therapy. Who wants to try it first? •
7.
Melief CJM, Kast WM: Prospects for T cell immunotherapy of tumors by vaccination with immunodominant and subdominant peptides. In Vaccines against Vira//y Induced Cancers (Ciba Symposium 189). Chichester: John Wiley & Sons; 1994:97-102.
8.
Melief CJM, Kast WM: T cell immunotherapy by adoptive transfer of cytotoxic T lymphocytes and by vaccination with minimal essential epitopes. Immunol Ray 1995, 145:167-177.
9. •
Rowe M, Khanna R, Jacob CA, Argaet V, Kelly A, Powis S, Belich M, Croom-Carter D, Lee S, Burrows SR eta/.: Restoration of endogenous antigen processing in Burkitt's lymphoma cells by Epstein-Barr virus latent membrane protein: coordinate up-regulation of peptide transporters and HLA class I antigen expression. Eur J Immunol 1995, 25:1374-1384. EBV LMP may restore some of the antigen processing and presentation defects in Burkitt's lymphoma cells. 10. ••
Levitskaya J, Coram M, Levitsky V, Imreh S, Steiger Wald-Mullen PM, Klein G, Kurilla MG, Masucci MG: Inhibition of antigen processing by the internal repeat region of the EBV EBNA-1 antigen. Nature 1995, 375:685-688. A startling strategy of the viral gane product. C/s-acting failure of MHC class I processing and presentation of EBNA-1 CTL epitopes by an EBNA-t internal repeat region. 11. ••
RessingME, Sette A, Brand RMP, Rappert J, Wentworth PA, HartmanM, Oseroff C, Grey HM, Melief CJM, Kast WM: Human CTL epitopes encoded by HPV16 E6 and E7 identified through in vivo and in vitro immunogenicity studies of HLA-A*0201 binding peptides. J Immunol 1995, 154:5934-5943. Demonstration that CTL epitopes of the HPV16 E6 and E? regions identified by MHC class I binding assays can be immunogenic in HLA-transganic mice. This immunogenicity correlates with in vitro immunogenicity for responding lymphocytes of HLA-A*020t + healthy donors. 12.
Kast WM, Brandt RPM, Sidney J, Drijfhout JW, Kubo RF, Grey HM, Melief CJM, Sette A: Role of HLA-A motifs in identification of potential CTL epitopes in human paillomavirus type 16 E6 and E7 proteins. J Immunol 1994, 152:3904-3912.
13. ••
Rassing ME, Van Driel W, Cells E, Sette A, Brandt RMP, Hartman M, Anholts JDH, Schreuder GMT, Ter Harmsel WB, Fleuren GJ et al.: Occasional memory CTL responses of patients with human papillomavirus type 16 positive cervical lesions against a human leukocyte antigen A*0201 restricted E7 encoded epitope. Cancer Res 1996, 56:582-588. Patients infected with HPV16 only occasionally show CTL memory responses against one of two tested HPV16 E7-derived encoded HLAA*0201 -binding peptides. This demonstrates that such patients do not react to all possible viral epitopes and that vaccination might be beneficial for the large majority of patients. Boon T, Van der Bruggen P: Human tumor antigens recognized by T lymphocytes. J Exp Mad 1995, 183:725-729. of the first clinical vaccination trials against a tumor associated self antigen shows that T cell tolerance can be broken by deliberate vaccination. No clinical benefit was observed in this study, however.
peptides that activate CD4+ and CD8+ T cell responses. Eur J /mmunol 1996, 26:435-443. Identification of overlapping epitopes in mutant Ras peptides that activate CD4 + and CD8 + responses. 18.
Disis ML, Smith HW, Murphy AE, Chen W, Cheever MA: In vitro generation of human CTLs specific for peptides derived from the HER-2/neu protooncogene protein. Cancer Res 1994, 54:1071-1078.
19.
Houbiers JGA, Nijman HW, Van der Burg SH, Drijfhout JW, Kenemans P, Van de Velde CJM, Brand A, Momberg F, Kast WM, Melief CJM: In vitro induction of human cytotoxic T lymphocyte responses against peptides of mutant and wild type p53. /mmunology 1993, 23:2072-2077.
20.
NijmanHW, Van der Burg SH, Vierboom MPM, Houbiers JGA, Kast WM, Melief CJM: p53, a potential target for tumor-directed T cells./mmunol Lett 1994, 40:171-178.
21.
Ten Bosch GJA, Toornvliet AC, Friede T, Melief CJM, Leeksma OC: Recognition of peptides corresponding to the joining region of p210 BCR-ABLprotein by human T calls. Leukemia 1995, 9:1344-1348.
22. •.
Ten Bosch GJA, Joostan AM, Kessler JH, Melief CJM, Leeksma OC: Recognition of BCR-ABL positive leukemic blasts by human CD4+ T cells elicited by primary in vitro immunization with a BCR-ABL breakpoint peptide. Blood 1996, in press. First demonstration that T cells directed against a chromosomal breakpoint peptide recognize human cancer cells. 23. ••
Dahl AM, Beverley P, Stauss HJ: A synthetic peptide derived from the tumor-associated protein mdm2 can stimulate autoreactive, high avidity CTLs that recognise naturally processed protein. J Immunol 1996, in press. Autoreactive CTLs raised in mice against an mdm2 peptide lyse cells bearing processed mdm2. 24. ••
Theobald M, Biggs J, Dittmer D, Levine AJ, Sherman LA: Targeting p53 as a general tumor antigen. Proc Natl Acad Sci USA 1995, 92:11993-11997. CTLs raised against in HLA-A2-transganic mice against a wild-type p53 peptide can lyse human tumor cells. 25.
TsangKY, Zaremba S, Nieroda CA, Zhu MZ, Hamilton JM, Schlom J : Generation of human cytotoxic T cells specific for human carcinoembryonic antigen epitopes from patients immunized with recombinant vaccinia CEA vaccine. J Nat/Cancer Inst 1995 87:982-993. One of the first clinical vaccination trials against a tumor-associated self antigen shows that T cell tolerance can be broken by deliberate vaccination. No clinical benefit was observed in this study, however. 16. •
Van Elsas A, Nijman HW, Van der Minne CE, Mourer JS, Kast WM, Melief CJM, Schrier PI: Induction and characterization of CTL recognizing a mutated p21 ras peptide presented by HLA-A'0201. Int J Cancer 1995, 61:389-396. CTL epitope identified mutant p21 Ras presented by HLA-A*020t. 17. •
Abrams SI, Stanziale SF, Lunin SD, Zaremba S, Schlom J: Identification of overlapping epitopes in mutant ras oncogene
Goulmy E: Human minor histocompatibility antigens. Curt Opin Immuno11996, 8:75-81.
26. ••
Den Haan JMM, Sherman NE, Blokland E, Huckzo E, Koning F, Drijfhout JW, Shabanowitz J, Hunt DF, Engelhard VH, Goulmy E: Identification of graft versus host disease associated human minor histocompatibility antigen. Science 1995, 268:1478-1480. First molecular identification of a human minor histocompatibility antigen by tandem mass spectrometry. 27. •
Wang W, Meadows LR, Den Haan JMM, Sherman NE, Chen Y, Blokland E, Shabanowitz J, Agulnik AI, Hendrikson RC, Bishop CE eta/.: Human H-Y: a male-specific histocompatibility antigen derived from the SMCY protein. Science 1995, 269:1588-1590. Second human minor histocompatibility antigen characterized molecularly: the long sought after 'male-antigen'. Human minor H antigens could serve as target antigens for cancer therapy under various circumstances. 28.
14. ~ne
15. ••
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Goldberg AL, Gaczynska M, Grant E, Michalik M, Rock KL: Functions of the proteasome in antigen presentation. Cold Spring Harb Syrup C)uant Biol 1995, 50:47-61.
29. •
NiedermannG, Butz S, Ihlenfeldt HG, Grimm R, Lucchiari M, HoschiJtzkiH, Jung G, Maler B, Eichmann K: Contribution of proteasome-mediated proteolysis to the hierarchy of epitopes presented by major histocompatibility complex class I molecules. Immunity 1995, 2:289-299. This paper, along with [30"], documents that CTL epitope hierarchy is strongly influenced by proteasome cleavage patterns. 30.
Eggers M, Boas-Fabian B, Ruppert 1", Kloetzel PM, Koszinowski UH: The cleavage preference of the proteasome governs the yield of antigenic peptides. J Exp Mad 1995, 182:1865-1870. See annotation [29"]. •
31. ••
Ossandorp F, Eggers M, Neisig A, Ruppert T, Groettrap M, Sijts A, Mengedb E, KIoetzai PM, Neefjes J, Koszinowski UH, Melief CJM: A single residue exchange within a viral CTL epitope alters proteasome-mediated degradation resulting in lack of antigen presentation. Immunity 1996, in press. Demonstration that a virus-encoded dominant CTL epitope in a tumor can be destroyed prematurely by mutation at a single amino acid position in the epitope that results in accelerated proteasomal degradation.
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32. •
Neisig A, Roelse J, Sijts AJAM, Ossendorp F, Eeltkamp MCW, Kast WM, Melief CJM, Neefjes JJ: Major differences in TAP dependent translocation of MHC presentable peptides and the effect of flanking sequences. J Immuno/1995, 154:1273-1276. Demonstration that TAP-dependent translocation can sometimes depend on amino acids flanking the actual MHC binding epitope. 33.
Schulz M, Zinkernagel RM, Hengartner H: Peptide-induced antiviral protection by cytotoxic T-cells. Proc Nat/Acad Sci USA 1991, 88:991.
34.
Kast WM, Roux L, Curren J, Blom HJJ, Voordouw AC, Meloen RH, Kolakovsky D, Melief CJM: Protection against lethal Sendal virus infection by in vivo priming of virus-specific cytotoxic T lymphocytes with an unbound peptide. Proc Nat/Acad Sci USA 1991, 88:2283-2287.
35.
Kast WM, Brandt RMP, Melief CJM: Strict peptide length is not required for the induction of cytotoxic T lymphocyte-mediated antiviral protection by peptide vaccination. Eur J Immunol 1993, 23:1189-1192.
36.
Cole GA, Hogg TL, Woodland DI: The MHC class I restricted T-cell response to Sendal virus in C57BL/6 mice: a single immunodominant epitope elicits an extremely diverse repertoire of T-cells. Int Immunol 1994, 6:1767-1775.
37.
Feltkamp MCW, Smits HL, Vierboom MPM, Minnaar RP, De Jongh BM, Drijfhout JW, Tar Schegget J, Melief CJM, Kast WM: Vaccination with cytotoxic T lymphocyte epitopecontaining peptide protects against a tumor induced by human papillomavirus type 16-transformed cells. Eur J Immunol 1993, 23:2242-2249.
38.
Mandalboim O, Berke G, Fridkin M, Feldman M, Eisenstein M, Eisenbach L: CTL induction by a tumour-associated antigen octapeptide derived from a mudne lung carcinoma. Nature 1994, 369:67-71.
CTL tolerance associated with enhanced tumor outgrowth can also be achieved by a single injection of low dose immunodominant peptide in IFA. Excellent protection is achieved if the same epitope is presented by Ads. 47. •*
Toes REM, Offringa R, Blom RJJ, Melief CJM, Kast WM: Peptide vaccination can lead to enhanced tumor growth through specific T-cell tolerance induction. Proc Nat/Acad Sci USA 1996, in press. Same results as in [49 °] but with a different CTL epitope. 48. 49. •
Celluzzi CM, Mayordomo JI, Storkus WJ, Lotze MT, Falo LD: Peptide pulsed dendritic cells induce antigen-specific CTLmediated protective tumor immunity. J Exp Med 1996, 183:283-287. Dendritic cells are a potent delivery system in CTL epitope vaccination. 50. ••
Mayordomo JI, Zorma T, Storkus WJ, Zitvogel L, Celluzzi C, Falo LD, Melief CJM, Ildstad ST, Kast WM, Deleo AB, Lotze MT: Bone marrow-derived dendritic cells pulsed with synthetic tumour peptides elicit protective and therapeutic anti-tumour immunity. Nat Mad 1995, 1:1297-1302. Peptide-pulsed DCs can even eradicate (small) established tumours.
51. •
Zitvogel L, Mayordomo JI, Tjandrawan T, Deleo AB, Clarke MR, Lotze MT, Storkus WJ : Therapy of murine tumours with peptide-pulsed dendritic cells: dependence on T-cells, B7 costimulation, and T helper cell 1-associated cytokines. J Exp Mad 1996, 183:87-97. Nonidentified peptides, eluted from MHC class I molecules on tumors at low pH, can serve as an efficient preventive vaccine, if delivered on activated DCs.
52.
39. •*
Mandalboim O, Vadal E, Fridkin M, Katz-Hillel A, Feldman M, Berke G, Eisenbach L: Regression of established murine carcinoma metastases following vaccination with tumour-associated antigen peptides. Nat Med 1995, 1:1179-1183. The peptide identified in [38] can be used as a peptide vaccine to cause regression of metastatic tumors.
53. ••
40. •*
54. •*
Vitiello A, Ishioka G, Grey HM, Rose R, Famess P, LaFond R, Yuan L, Chisari FV, Urze J, Bartholomeuz R, Chesnut RW: Development of a lipopeptide-based therapeutic vaccine to treat chronic HBV infection h induction of a primary cytotoxic T lymphocyte response in humans. J C/in Invest 1995, 95:341-345. First application of an MHC class I binding peptide vaccine in humans. An HBV-derived peptide induces a vigorous CTL response in healthy volunteers capable of lysing HBV-infected target cells. 41.
Aichele P, Brduscha-Riem, Zinkernagel RM, Hengartner H, Pitcher H: T cell priming versus T-call tolerance induced by synthetic peptides. J Exp Med 1995, 182:261-266. High dose peptide in IFA, repeatedly injected intraperitoneally in mice, causes tolerance rather than immunity. 43.
Aichele P, Kybarz D, Ohashi PS, Odermatt B, Zinkernagel RM, Hengartner H, Pircher H: Peptide-induced T-cell tolerance to prevent autoimmune diabetes in a transgenic mouse model. Proc Nat/Acad Sci USA 1994, 91:444-448.
44.
Kast WM, Offringa R, Peters PJ, Voordouw A, Meloen RH, Van der Eb AJ, Malief CJM: Eradication of adenovirus E1 induced tumors by E1A-specific cytotoxic T lymphocytes. Ceil 1989, 59:603-614.
45.
ToesREM, Offringa R, Blom RJJ, Brandt RMP, Van der Eb AJ, Melief CJM, Kast WM: An adenovirus type 5 early region 1 Bencoded CTL epitope mediating tumor eradication by CTL clones is down-modulated by an activated ras oncogene. J Immuno11995, 154:3396-3405.
46. ••
ToesREM, Blom RJJ, Offringa R, Kast WM, Melief CJM: Functional deletion of tumor-specific CTLs induced by peptide vaccination can lead to the inability to reject tumours. J /mmunol 1996, 156:3911-3918.
Noguchi Y, Chen Yl', Old I1: A mouse mutant p53 product recognized by CD4+ and CD8 + T-cells. Proc Nat/Acad Sci USA 1994, 91:3171-3175.
Mayordomo JI, Loftus DJ, Sakamoto H, De Cesare CM, Appasany PM, Lotze MT, Storkus WJ, Appella E, Deleo AB: Therapy of murine tumors with p53 wild-type and mutant sequence peptide-based vaccines. J Exp Med 1996, 183:1357-1365. Both mutant and wild-type p53 MHC class I binding peptides delivered on DCs protect against tumor outgrowth. Marchand M, Wegmants P, Rankin E, Arienti F, Belli F, Parmiani G, Cascinelli N, Bourlond A, VanWijck R, Humblet Y e t el.: Tumor regression responses in melanoma patients treated with a peptide encoded by gene MAGE-3. Int Cancer 1995, 63:883-885. Free peptides delivered without adjuvant to patients with metastatic melanoma cause complete remission in two patients. 55.
MukherjiB, Chakraborty NG, Yamasaki S, Okino T, Yamase H, Sporn JR, Kurtzman SK, Ergrin MT, Ozols J, Meehan J, Mauri F: Induction of antigen-specific cytolytic T cells in situ in human melanoma by immunization with synthetic peptide-pulsed autologous antigen presenting cells. Proc Nat/Acad Sci USA 1995, 92:8078-8082.
56.
Wang M, Bronte V, Chen PW, Gritz L, Panicali D, Rosenberg SA, Restifo NP:Active immunotherapy of cancer with a nonreplicating recombinant fowlpox virus encoding a model tumor associated antigen. J Immunol 1995, 154:4685-4692.
57.
McCabe BJ, Irvine KR, Nishimura MI, Yang JC, Spiass PJ, Shulman EP, Rosenberg SA, Restifo NP: Minimal determinant expressed by a recombinant vaccinia virus elicits therapeutic anti tumor cytolytic T lymphocyte responses. Cancer Res 1995, 55:1741-1747.
Schild H, Norda M, Deres K, Falk K, R6tschke O, WiesmOller KH, Jung G, Rammensee HG: Fine specificity of cytotoxic T lymphocytes pdmed in vivo either with virus or synthetic lipopeptide vaccine or primed in vitro with peptide. J Exp Med t991, 174:1665-1669.
42. *•
Matzinger P: Tolerance, danger, and the extended family. Annu Rev Immunol 1994, 12:991-1045.
58. •
Restifo NP, Bacik I, Irivine KR, Yewdell JW, McCabe BJ, Anderson RW, Eisenlohr LC, Rosenberg SA, Bennink JR: Antigen processing in vivo and the elicitstion of primary CTL responses. J Immunol 1995, 154:4414-4422. Addition of ER insertion sequence to epitope construct in vaccinia virus vector can greatly augment induction of CD9 + CTLs. 59.
Whitton JL, Sheng N, Oldstone MBA, McKee TA: A 'string of-beads' vaccine, comprising linked minigenes, confers protection from lethal-dose virus challenge. J Virol 1993, 67:348-352.
60. •
Weidt G, Deppert W, Buchhop S, Dralle H, LehmannGrube F: Anti-viral protective immunity induced by major histocompatibility complex class I molecule restricted viral
Peptide-based cancer vaccines Melief et al.
T-lymphocyte epitopes inserted in various positions in immunologically self and nonself proteins. J Virol 1995, 69:2654-2658. First demonstration of protective effect of CTL epitope string-of-beads protein construct. Thompson SA, Khanna R, Gardner J. Burrows SR, Coupar B, Moss DJ, Suhrbier A: Minimal epitopes expressed in a recombinant poly epitope protein are processed and presented to CD8 + cytotoxic T-cells: implications for vaccine design. Proc Natl Acad Sci USA 1995, 92:5845-5849. String-of-beads CTL epitope design for EBV vaccination purposes.
elicits human CO8 + cytolytic T cell responses. J Exp Med, 1995, 182:1663-1671. 64.
61. •
62.
Bevan MJ: Antigen presentation to cytotoxic T lymphocytes in vivo. J Exp Med 1995, 182:639-64t.
63.
Bender A, Kim Bui L, Feldman MAV, Larsson M, Bhardway N: Inactivated influenza virus when presented on dendritic cells
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Szalay G, Ladel CH, Kaufmann SHE: Stimulation of protective CD8 + T lymphocytes by vaccination with nonliving bacteria. Proc Natl Acad Sci USA 1995, 92:12389-12393.
65.
Toes REM, Biota RJJ, Van der Voort EIH, Offringa R, Melief CJM, Kast WM: Protective anti-tumor immunity induced by immunization with completely allogeneic tumor cells. Cancer Res 1996, in press. Protective vaccination induced by immunization with completely allogeneic tumor cells is associated with CTL memory to viral antigens processed by host APCs. •
66.
Pardoll DM, Beckerleg AML: Exposing the immunology of naked DNA vaccines. Immunity 1995, 3:165-169.
Peptide-based cancer vaccines Cornelis JM Melief* , Rienk Offringa*, Rene EM Toes* and W Martin Kastt The application of molecularly defined vaccines composed of a limited number of tumor-specific T cell epitopes has resulted in protective antitumor T cell immunity in several mouse tumor models. The first encouraging results with such vaccines have been obtained in human beings. The development of the next generation of rationally designed vaccines that are both effective and safe for application in a clinical setting requires comparison of different modes of delivery of tumor-associated T cell epitopes in multiple epitope constructs.
Addresses *Department of Immunohaematology, University Hospital of Leiden, Rijnsburgerweg 10, Leiden, The Netherlands tCancer Immunology Program, Cardinal Bernadin Cancer Center, Loyola University of Chicago, 9160 South First Avenue, Maywood, IL 60153, USA Se-mail: [email protected] Current Opinion in Immunology 1996, 8:651-657 © Current Biology Ltd ISSN 0952-7915
Abbreviations Ad adenovirus CTL cytotoxic T lymphocyte DC dendriticcell EBNA Epstein-Barrnuclear antigen EBV Epstein-Barrvirus HBV hepatitisB virus HPV humanpapillomavirus IFA incompleteFreund's adjuvant LCMV lymphocytic choriomeningitis virus
Introduction Evidence is accumulating that the immune system usually does not react against all possible antigenic determinants, but only against the most immunodominant ones [1]. This is particularly true in the case of tumors, which often elicit no demonstrable responses at all. Mechanisms that contribute to this regrettable state of affairs are general disturbances in the immunocompetence of cancer patients [2], the poor immunogenicity of cancer cells [3,4], and the production of paracrine factors promoting tumor growth and angiogenesis by tumor cells [5",6°]. Antitumor vaccination can help to overcome many but not all of these problems. Tremendous progress has recently been made in the identification of both viral and nonviral tumor-associated antigens. This allows the development of molecularly defined vaccines, containing the minimal essential components, that can be used to elicit specific T cell responses against both dominant and subdominant T cell epitopes [7,8]. In addition, nonspecific components that orient the T cell response in the desired Th.(CD4÷) or cytotoxic T lymphocyte (CTL) (CD8 ÷) direction, such as
the interleukin (IL)-2, interferon (IFN)-7 or IL-12 genes or gene products, can now be incorporated into vaccines. T h e development of such rational vaccine design has only just started. Great diligence will be required to make a significant impact on the cancer problem by vaccination. Chances are that vaccination will succeed primarily as adjuvant therapy following debulking by conventional treatment. If vaccination fails, more drastic, laborious and expensive treatment by adoptive transfer of tumor-specific T cells, expanded in vitro, will be needed. Nature of tumor antigens expressed on virus-induced and nonvirus-induced tumor cells Most virus-induced tumor cells display a variety of MHCbound peptides of viral origin at their cell surface, although exceptions to this rule have been found (reviewed in [9"]). For instance, Burkitt's lymphoma cell lines, which retain the original tumor cell phenotype of fresh biopsies, express only a single virus-encoded protein, the Epstein-Barr virus (EBV)-encoded nuclear antigen (EBNA)-I [9"]. Moreover, the processing of this protein for presentation by H L A class I antigens is inhibited by the internal repeat region of EBNA-1 [10"°]. Other EBV-induced neoplastic cells and long-term B lymphoblastoid lines that express a variety of EBV antigens, however, do present H L A class I binding EBV peptides to the T cell immune system [9"]. One of the EBV gene products, latent membrane protein (LMP)-I, even appears to stimulate antigen processing and presentation in that it enhances the expression of both TAP (transporter associated with antigen processing) and H L A class I molecules [9"].
In the case of human papilloma virus (HPV)16, one of the major human papiilomavirus types associated with cervical cancer, a variety of immunogenic peptides encoded by the HPV16 E6 and E7 viral oncogenes were identified that could be useful in vaccines for the prevention and treatment of cervical carcinoma [11",12]. T h e natural C T L response to these HLA-A*0201-binding peptides was only demonstrable in a minority of patients with HPV16-associated cervical lesions [13"'], indicating that most patients might benefit from vaccination that would induce vigorous C T L responses to these viral peptides [13"1. Substantial progress has been made in the definition of melanoma-associated antigens recognized by C T L s (reviewed in [14°']). A detailed discussion of these antigens is beyond the scope of this review but we shall return to several of these antigens in our discussion of clinical vaccine trials. T h e fact that most of these
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antigens are encoded by nonmutated cellular genes, and can therefore be regarded as self antigens, has strongly promoted the concept that T cell immunity directed against such antigens can be employed to eradicate tumors and has encouraged the search for novel tumor-associated self antigens. Additional autologous antigens that are overexpressed or mutated in certain human tumors, and against which CD8 ÷ CTLs or CD4 ÷ T h cells have been raised, include carcinoembryonic antigen [15°°], mutant Ras [16",17°], HER-2/Neu [18], mutant and wild-type p53 [19,20] and Bcr/Abl breakpoint peptides [21,22°°]. In a mouse model, CTLs were raised against a self peptide derived from the p53-binding protein mdm2 and these C T L s were capable of lysing target cells that carry this epitope as a naturally processed peptide [23°°]. In HLA-transgenic mice, CTLs were raised against HLAA*0201-binding wild-type human p53-derived peptides that are not fully conserved between human and mouse p53. These CTLs, while not recognizing nontransformed human cells, were able to lyse human tumor cell lines [24°°]. Recently, we have been able to raise C T L s against a wild-type mouse p53 peptide by immunization of p53 knockout mice with congenic p53-overexpressing tumor cells. These CTLs, upon adoptive transfer into p53+/÷ mice bearing p53-overexpressing tumors, were able to eradicate these tumors without signs of autoimmunity to the host (M Vierboom et al., unpublished data). T h e prospect of utilizing p53 as a general target for CTL-mediated therapy of cancer is particularly attractive because p53 is overexpressed in more than 50% of all human cancers.
tumors will grow to cause considerable difficulties in the immunotherapy of many tumors.
Vaccination with MHC class I binding peptides, a delicate balance between immune activation and immune suppression Protective C T L responses induced by vaccination with MHC class I binding peptides were first reported by Schulz et al. [33] for lymphocytic choriomeningitis virus (LCMV) and independently by Kast et al. [34] for Sendai virus. In these studies a substantial antiviral effect was obtained by subcutaneous vaccination with a single MHC class I binding peptide in incomplete Freund's adjuvant (IFA). Subsequent studies showed that strict peptide length is not required for the induction of protective immunity in that a 16-mer Sendal peptide with additional flanking residues protected similarly to the minimal 9-met that shows optimal MHC binding [35]. Vaccination with this immunodominant peptide in IFA leads to activation of Sendai virus specific T cells with a wide variety o f T cell receptors, all recognizing the same Sendai epitope [36].
Factors influencing processing and presentation of tumor antigens
When applied to a murine tumor model, the principle of vaccination with MHC class I binding immunogenic peptides also resulted in the induction of protective immunity. This was demonstrated by prevention of outgrowth of an HPV16-induced tumor in C57BL/6 mice by vaccination with a peptide derived from the HPV16 E7 oncoprotein in IFA [37]. Similarly, vaccination with either of two tumor-associated mutant connexin peptides, emulsified in IFA or loaded onto processing-defective RMA-S cells, induced protection against metastatic tumor spread [38,39°°]. C T L s capable of lysing human HPV16 ÷, HLA-A*0201+ cervical carcinoma cells were raised by in vitro stimulation of peripheral blood mononuclear cell cultures of healthy HLA-A*0201+ donors with HLAA*0201-binding HPV16 E7-derived peptides [11"°]. In the first application of peptide vaccination in human beings, a vigorous C T L response was induced in hepatitis B virus (HBV)-negative human volunteers by vaccination with a lipopeptide consisting of an HLA-A*0201-binding HBV peptide, a pan HLA-DR-binding peptide not based on the HBV sequence and two palmitic acid tails. These C T L s were capable of lysing HLA-A*0201 + HBV-infected target cells in vitro [40°']. Previously, a similar lipopeptide vaccine had already been shown to induce functional T cell responses in a murine model [41].
T h e factors that determine successful recognition of tumor antigens by C T L s include proper proteolytic cleavage by the proteasome complex [28,29",30°,31°°], efficiency of peptide transport via the TAP system [31"',32 °] and surface display of peptide-MHC complexes [9",10°°,32°]. Presentation of immunogenic peptide epitopes to T cells might be avoided through epitope mutations affecting proteasome cleavage [31"°], MHC class I binding [32°] or blockage of processing by an unknown mechanism [9"]. Undoubtedly the list of evasion mechanisms employed by
Subcutaneous peptide vaccination at moderate doses in IFA very consistently causes protective immunity with the peptides mentioned above. Surprisingly, administration of some of these peptides at very high doses by the intraperitoneal route causes immunological tolerance to the peptide rather than protective immunity. This was first shown with an H-2Db binding, LCMV-derived peptide [42 °°] and this principle could be used to prevent autoimmune diabetes in a transgenic mouse model [43].
Minor histocompatibility antigens are a special category of tumor-associated antigens. Notably, those minor histocompatibility antigens with tissue-specific expression (e.g. in hematopoeitic tissue) are attractive target antigens for cancer immunotherapy [25]. Last year the molecular nature of two human minor histocompatibility antigens was unveiled [26°°,27°]. One of the therapeutic regimens that can be considered is adoptive transfer of donor-derived CTLs against host hemopoietic cell specific minor histocompatibility antigens expressed on a relapsed leukemia following allogeneic bone marrow transplantation.
Peptide-besed cancer vaccines Melief et aL
Certain other peptides, when delivered subcutaneously in IFA, consistently fail to induce protective immunity across a wide dose range. This phenomenon was observed in a murine tumor model for two adenovirus (Ad)-derived peptides that constitute the major T cell epitopes of Ad-induced tumors in C57BL/6 mice. C T L s against these peptides, raised by immunization with irradiated Ad-induced tumor cells, are capable of eradicating large established tumors following intravenous infusion into tumor-bearing T cell deficient nude mice [44,45]. In contrast, vaccination of immunocompetent mice with a single dose (0.1-10~tg]mouse) of Ad peptide caused enhanced outgrowth of subsequently transplanted Ad-induced tumors. This increased tumor growth was parallelled by specific tolerance of the mice for the injected peptide epitope [46°',47°']. T h e effect of peptide vaccination in these experiments was so striking because the tumor models chosen were such that either of the two Ad peptides could be the single dominant C T L epitope determining T cell protection against tumor growth [46°°,47°°]. In tumor-host relationships in which multiple T cell epitopes are potential targets, the consequences of downregulation of the response against a single epitope are likely to be less conspicuous. On the other hand, the affected T cell response may play a pivotal role in the resistance against a given tumor and, therefore, even deletion of this part of the antitumor T cell response should be avoided at all times. Although in the Ad tumor model, subcutaneous immunization with peptides in IFA led to specific tolerance at all doses tested, different modes of peptide delivery (including immunization with irradiated tumor cells or with replication-defective Ad that encodes the relevant epitopes) led to protective immunity associated with strong tumor-specific C T L activity [46"']. Furthermore, protective C T L immunity, rather than tolerance, can be obtained by immunization with activated dendritic cells (DCs) that have been loaded with synthetic Ad peptides (REM Toes, R Offringa, WM Kast, CJM Melief, unpublished data). The reasons why the Ad peptides injected at low doses subcutaneously in IFA caused specific C T L tolerance, while various other C T L epitope carrying peptides administered in exactly the same way consistently caused protection associated with C T L memory [33,34,36,38], remain unclear at this time. T h e contrasting effects of vaccination with either Ad peptides in IFA or Ad peptides loaded onto DCs suggest that not the peptides perse but rather the context in which they are presented to the T cell immune system determines the outcome of immunization with peptide-based vaccines. Inflammatory and costimulatory signals, collectively also named 'danger' signals [481, are likely to constitute important factors in creating the appropriate context for stimulation of the peptide-specific T cell response. Apparently, the balance between tolerance and immunity
653
is more precarious for the Ad peptides than for other peptides tested. These observations imply that individual peptides to be used for antivirus or anticancer vaccines should at least be analyzed in HLA-transgenic animals for their immunogenic/tolerogenic properties in vivo [11°°], before they are used in a clinical setting. Probably even better, modes of peptide delivery must be considered that uniformly induce protective immunity associated with strong C T L responses. One such delivery mode may be peptide presentation on DCs. Indeed, activated DCs, obtained by culturing bone marrow cells with granulocyte macrophage-colony stimulating factor (GM-CSF) and IL-4, loaded with ovalbumin-derived MHC class I binding peptides induced protection in mice against the outgrowth of ovalbumin-transfected B16 melanoma cells [49"]. T h e same group of investigators in collaboration with our group has reported comparable results with tumor-derived peptides [50"']. In this study, immunization with peptide-pulsed DCs not only elicited protective immunity, but in addition exerted therapeutic effects on already established tumors [50"']. This approach was also effective with unfractionated acid-eluted tumor peptides loaded onto DCs. The effectiveness of immunization with peptide-pulsed DCs, with respect to antitumor effects in this study, depended on appropriate B7 costimulation and on DC-induced production of Thl-associated cytokines such as tumor necrosis factor (TNF)-~ and IFN-y [51"]. Effective vaccination was also achieved against chemically induced sarcomas in BALB/c mice with a mutant p53 peptide in IFA [52] or with either mutant or wild-type p53 peptides loaded onto activated DCs [53"']. In patients with cancer, peptide vaccination has only been applied on a very limited scale. Surprisingly, even immunization with free peptides corresponding to an epitope encoded by the melanoma-associated gene MAGE-3 induced complete regression of two metastatic melanomas [54"']. This peptide vaccination trial was conducted as a phase I toxicity study and tumor responses in the absence of adjuvant had not been anticipated. In another study [55], three patients with advanced metastatic melanoma were vaccinated with autologous plastic-adherent mononuclear blood cells that had been cultured in recombinant GM-CSF and pulsed with an HLA-A*0101-binding MAGE-1 peptide. MAGE-1 peptide specific C T L s were demonstrable after, but not prior to, vaccination and these CTLs were capable of lysing HLA-A*0101 ÷ MAGE-1 ÷ melanoma cells in vitro. No major therapeutic responses were noted, possibly because of the advanced stage of the disease.
Future of vaccination with minimal T cell epitopes T h e results just reviewed clearly show that vaccination with tumor-associated MHC-binding peptide epitopes is a powerful tool for the induction of tumor-specific T cell responses. Protective immunity has been achieved by immunization with peptides in IFA, peptides pulsed onto DCs and peptides encoded by replication-deficient Ads.
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On the other hand, peptides in IFA do not always protect. Depending on yet unknown properties of peptides, vaccination with certain peptides can lead to tolerance in regimens that are associated with protection in the case of other peptides [7,8,37,39",46"',47"]. Vaccination with peptide-loaded DCs may be superior to peptide in adjuvant, especially with respect to peptides that are prone to induce tolerance, but this approach is clearly more cumbersome in a clinical setting. It is our estimation that neither peptides in adjuvants, nor lipopeptides, nor peptides loaded onto D e s will ultimately prove to constitute effective, safe and versatile anticancer vaccines. Such vaccine formulations would be too restricted with respect to HLA type and antigen specificity and/or too laborious with respect to application on a large scale. Rather, we believe that the results recently obtained with experiments in mouse models and with several clinical trials should boost rapid progress in the direction of DNA-, virus- or protein-based vaccines that encode/harbor a multitude of epitopes. In our Ad-tumor model in mice, we found the most effective vaccination approach involved the injection of replication-deficient Ad [46"']. Others demonstrated that vaccination with a fowlpox virus, genetically engineered to encode the model tumor antigen 13-galactosidase, protected mice against challenge with tumor cells that express this antigen [56]. Similarly, vaccination with a recombinant vaccinia virus harboring a minigene encoding an H-2Kbrestricted ovalbumin epitope induced protection against ovalbumin-expressing EL4 tumor cells [57]. Addition to epitope-encoding minigenes of an amino-terminal endoplasmic reticulum insertion sequence in some cases greatly enhanced the CD8 ÷ C T L immune response [58"]. Furthermore, vaccinia viruses carrying string-of-beads minigenes that encode multiple C T L epitopes have been shown to confer protection from lethal LCMV challenge in mice of different H-2 types [59]. Such string-of-beads constructs consist of the genetic code for multiple C T L epitope containing peptides that are linked together with or without additional amino acids in between the epitopes serving as spacers. Similarly, protection against LCMV was obtained using recombinant gene products consisting of several M H C class I binding peptides inserted into various positions of self and nonself proteins [60"]. Polyepitope proteins or gene constructs have yet to be applied for vaccination against tumors, although development of such anticancer vaccines is well underway. This is illustrated, for instance, by a recent report in which infection of cells with a recombinant vaccinia virus encoding multiple known H L A class I binding EBV peptides was shown to sensitize these cells to recognition by C T L s against all relevant EBV epitopes [61"].
has been around for many years (reviewed in [62]). Indeed, inactivated influenza virus when presented by D e s stimulates human CD8 ÷ C T L responses [63] and protective CD8 ÷ C T L responses can be elicited by vaccination with nonliving bacteria [64]. We recently observed that completely allogeneic tumor cells induce very effective antitumor immunity based on induction of C T L s against epitopes processed and presented by host antigen-presenting cells [65"].
Conclusion Vaccination experiments with individual M H C class I binding peptides have now led the way to vaccine constructs that encode or harbor multiple epitopes. As for the single epitope vaccines, the mode of delivery of muhiepitope vaccines will prove equally important. To compare efficiently the various delivery systems (viral vectors, protein in adjuvant, DNA; reviewed in [66]) it is important to perform comparative vaccination in animal model systems, using the same epitopes in different delivery systems. In this respect, the most useful model systems are based on HLA-transgenic mice, as such models in essence allow evaluation of vaccines that are intended for clinical use [11"]. T h e most promising vaccines can subsequently be tested in patients. Vaccination is likely to be effective only in patients who have minimal residual disease following conventional treatment, or who exhibit a very early stage of the disease, because vaccination has been shown to be capable of eradication of only low numbers of tumor cells. For patients in more advanced stages, adoptive transfer of large numbers of tumor-specific T cells that have been expanded in vitro can be considered. Even for this obviously more laborious treatment, proper vaccination might be a first step toward expansion of low numbers of autologous T cell precursors.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • •.
of special interest of outstanding interest Sercarz EE, Lehmann PV, Ametani Benichou G, Miller A, Moudqil K: Dominance and crypticity of T cell antigenic determinants. Annu Rev /mmunol 1993, 11:729-766. Ochoa A, Longo DL: Alteration of signal transduction in T cells from cancer patients, tn Important Advances in Oncolog¥. Edited by De Vita VT, Hellman S, Rosenberg SA. Phildelphia: JB Lippincott Company; 1995:43-54. Melief CJM: Tumor eradication by adoptive transfer of cytotoxic
T lymphocytes. Adv Cancer Res 1992, 58:143-175. Nanda NK, Sercarz EE: Induction of anti-self immunity to cure cancer. Ceil 1995, 82:13-17.
T h e concept of using exogenous protein for priming of C T L responses is gradually gaining ground, although evidence for cross-priming of C T L s by exogenous protein
Seung LP, Rowley DA, Dubey P, Schreiber H: Synergy between T cell immunity and inhibition of paracrine stimulation causes tumor rejection. Proc Nat/Acad Sci USA 1995. 92:6254-6258.
Peptide-based cancer vaccines Melief et al.
Paracrine factor produced by tumor cells counteracts tumor eradication by T lymphocytes. The factor attracts granulocytes that make the tumor cells grow faster, outsmarting the T lymphocytes. 6.
Parangi S, O'Reilly M, Christophori G, Holmgren L, Grosfeld J, Folkman J, Hanahan D: Anti-angiogenic therapy of transgenic mice impairs de novo tumor growth. Proc Nat/Acad Sci USA 1996, 93:2002-2007. Antiangiogenic therapy may eventually be used synergistically with T cell therapy. Who wants to try it first? •
7.
Melief CJM, Kast WM: Prospects for T cell immunotherapy of tumors by vaccination with immunodominant and subdominant peptides. In Vaccines against Vira//y Induced Cancers (Ciba Symposium 189). Chichester: John Wiley & Sons; 1994:97-102.
8.
Melief CJM, Kast WM: T cell immunotherapy by adoptive transfer of cytotoxic T lymphocytes and by vaccination with minimal essential epitopes. Immunol Ray 1995, 145:167-177.
9. •
Rowe M, Khanna R, Jacob CA, Argaet V, Kelly A, Powis S, Belich M, Croom-Carter D, Lee S, Burrows SR eta/.: Restoration of endogenous antigen processing in Burkitt's lymphoma cells by Epstein-Barr virus latent membrane protein: coordinate up-regulation of peptide transporters and HLA class I antigen expression. Eur J Immunol 1995, 25:1374-1384. EBV LMP may restore some of the antigen processing and presentation defects in Burkitt's lymphoma cells. 10. ••
Levitskaya J, Coram M, Levitsky V, Imreh S, Steiger Wald-Mullen PM, Klein G, Kurilla MG, Masucci MG: Inhibition of antigen processing by the internal repeat region of the EBV EBNA-1 antigen. Nature 1995, 375:685-688. A startling strategy of the viral gane product. C/s-acting failure of MHC class I processing and presentation of EBNA-1 CTL epitopes by an EBNA-t internal repeat region. 11. ••
RessingME, Sette A, Brand RMP, Rappert J, Wentworth PA, HartmanM, Oseroff C, Grey HM, Melief CJM, Kast WM: Human CTL epitopes encoded by HPV16 E6 and E7 identified through in vivo and in vitro immunogenicity studies of HLA-A*0201 binding peptides. J Immunol 1995, 154:5934-5943. Demonstration that CTL epitopes of the HPV16 E6 and E? regions identified by MHC class I binding assays can be immunogenic in HLA-transganic mice. This immunogenicity correlates with in vitro immunogenicity for responding lymphocytes of HLA-A*020t + healthy donors. 12.
Kast WM, Brandt RPM, Sidney J, Drijfhout JW, Kubo RF, Grey HM, Melief CJM, Sette A: Role of HLA-A motifs in identification of potential CTL epitopes in human paillomavirus type 16 E6 and E7 proteins. J Immunol 1994, 152:3904-3912.
13. ••
Rassing ME, Van Driel W, Cells E, Sette A, Brandt RMP, Hartman M, Anholts JDH, Schreuder GMT, Ter Harmsel WB, Fleuren GJ et al.: Occasional memory CTL responses of patients with human papillomavirus type 16 positive cervical lesions against a human leukocyte antigen A*0201 restricted E7 encoded epitope. Cancer Res 1996, 56:582-588. Patients infected with HPV16 only occasionally show CTL memory responses against one of two tested HPV16 E7-derived encoded HLAA*0201 -binding peptides. This demonstrates that such patients do not react to all possible viral epitopes and that vaccination might be beneficial for the large majority of patients. Boon T, Van der Bruggen P: Human tumor antigens recognized by T lymphocytes. J Exp Mad 1995, 183:725-729. of the first clinical vaccination trials against a tumor associated self antigen shows that T cell tolerance can be broken by deliberate vaccination. No clinical benefit was observed in this study, however.
peptides that activate CD4+ and CD8+ T cell responses. Eur J /mmunol 1996, 26:435-443. Identification of overlapping epitopes in mutant Ras peptides that activate CD4 + and CD8 + responses. 18.
Disis ML, Smith HW, Murphy AE, Chen W, Cheever MA: In vitro generation of human CTLs specific for peptides derived from the HER-2/neu protooncogene protein. Cancer Res 1994, 54:1071-1078.
19.
Houbiers JGA, Nijman HW, Van der Burg SH, Drijfhout JW, Kenemans P, Van de Velde CJM, Brand A, Momberg F, Kast WM, Melief CJM: In vitro induction of human cytotoxic T lymphocyte responses against peptides of mutant and wild type p53. /mmunology 1993, 23:2072-2077.
20.
NijmanHW, Van der Burg SH, Vierboom MPM, Houbiers JGA, Kast WM, Melief CJM: p53, a potential target for tumor-directed T cells./mmunol Lett 1994, 40:171-178.
21.
Ten Bosch GJA, Toornvliet AC, Friede T, Melief CJM, Leeksma OC: Recognition of peptides corresponding to the joining region of p210 BCR-ABLprotein by human T calls. Leukemia 1995, 9:1344-1348.
22. •.
Ten Bosch GJA, Joostan AM, Kessler JH, Melief CJM, Leeksma OC: Recognition of BCR-ABL positive leukemic blasts by human CD4+ T cells elicited by primary in vitro immunization with a BCR-ABL breakpoint peptide. Blood 1996, in press. First demonstration that T cells directed against a chromosomal breakpoint peptide recognize human cancer cells. 23. ••
Dahl AM, Beverley P, Stauss HJ: A synthetic peptide derived from the tumor-associated protein mdm2 can stimulate autoreactive, high avidity CTLs that recognise naturally processed protein. J Immunol 1996, in press. Autoreactive CTLs raised in mice against an mdm2 peptide lyse cells bearing processed mdm2. 24. ••
Theobald M, Biggs J, Dittmer D, Levine AJ, Sherman LA: Targeting p53 as a general tumor antigen. Proc Natl Acad Sci USA 1995, 92:11993-11997. CTLs raised against in HLA-A2-transganic mice against a wild-type p53 peptide can lyse human tumor cells. 25.
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31. ••
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Toes REM, Offringa R, Blom RJJ, Melief CJM, Kast WM: Peptide vaccination can lead to enhanced tumor growth through specific T-cell tolerance induction. Proc Nat/Acad Sci USA 1996, in press. Same results as in [49 °] but with a different CTL epitope. 48. 49. •
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53. ••
40. •*
54. •*
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