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Breast cancer vaccines: a clinical reality or fairy tale?

2005; Elsevier BV; Volume: 17; Issue: 5 Linguagem: Inglês

10.1093/annonc/mdj083

1569-8041

Giuseppe Curigliano, Gianluca Spitaleri, Elisabetta Pietri, María Rescigno, Filippo de Braud, Anna Cardillo, Elisabetta Munzone, Andrea Rocca, Giuseppina Bonizzi, Vincent Brichard, Laura Orlando, Aron Goldhirsch,

Cancer Immunotherapy and Biomarkers

The characterization of tumor antigens recognized by immune effector cells has opened the perspective of developing therapeutic vaccines in the field of breast cancer. The potential advantages of the vaccines are: (i) the induction of a robust immune response against tumors that are spontaneously weekly immunogenic; (ii) the tumor specificity for some antigens; (iii) the good tolerance and safety profile and (iv) the long-term immune memory, critical to prevent efficiently tumor recurrence. Most trials evaluating breast cancer vaccines have been carried out in patients with extended metastatic breast cancer, characterized by aggressive tumors, resistant to standard cytotoxic treatments, so that clinical efficacy was difficult to achieve. However, some significant immune responses against tumor antigens induced upon vaccinations were recorded. The aim of this review is to analyze the activity of vaccination strategies in current clinical trials. Data of clinical activity have been observed by using vaccines targeting HER2/neu protein, human telomerase reverse transcriptase, carcinoembryonic antigen and carbohydrate antigen given after stem cell rescue. The review discusses possible future directions for vaccine development and applications in the adjuvant setting. The characterization of tumor antigens recognized by immune effector cells has opened the perspective of developing therapeutic vaccines in the field of breast cancer. The potential advantages of the vaccines are: (i) the induction of a robust immune response against tumors that are spontaneously weekly immunogenic; (ii) the tumor specificity for some antigens; (iii) the good tolerance and safety profile and (iv) the long-term immune memory, critical to prevent efficiently tumor recurrence. Most trials evaluating breast cancer vaccines have been carried out in patients with extended metastatic breast cancer, characterized by aggressive tumors, resistant to standard cytotoxic treatments, so that clinical efficacy was difficult to achieve. However, some significant immune responses against tumor antigens induced upon vaccinations were recorded. The aim of this review is to analyze the activity of vaccination strategies in current clinical trials. Data of clinical activity have been observed by using vaccines targeting HER2/neu protein, human telomerase reverse transcriptase, carcinoembryonic antigen and carbohydrate antigen given after stem cell rescue. The review discusses possible future directions for vaccine development and applications in the adjuvant setting. introductioTraditional cancer treatment regimes provide acceptable response rates and improve survival in patients with breast cancer, but they are generally not selective, inducing cytotoxicity in normal as well as in malignant cells and so they often are not well tolerated. Advances in the understanding of tumor biology have allowed targeted therapies against specific molecular targets to develop. The specificity is aimed at improving tumor targeting, thereby understanding cytotoxicity against normal cells. Recently, this approach has lead to the development of, for example, tyrosine kinase inhibitors or monoclonal antibodies (mAb). Recent results with mAb have validated the immunotherapy approach [1.Esteva F.J. Monoclonal antibodies, small molecules, and vaccines in the treatment of breast cancer.The Oncologist. 2004; 9: 4-9Crossref PubMed Scopus (62) Google Scholar]. An additional improvement may be brought by active immunization with the advantages of a non-toxic therapeutic modality potentially capable of inducing antitumor immune responses in patients with primary tumors and in those with metastases [2.Emens L.A. Jaffee E.M. Toward a breast cancer vaccine: work in progress.Oncology (Huntingt). 2003; 9: 1200-1211Google Scholar, 3.Nencioni A.F.F. Patrone G.F. Brossart P. Anticancer vaccination strategies.Ann Oncol. 2004; 15: 153-160Abstract Full Text PDF Scopus (26) Google Scholar, 4.Bernstein N. Overview of therapeutic vaccination approaches.Sem Oncol. 2003; 30: 1-8Crossref Scopus (20) Google Scholar, 5.Sung M.H. Simon R. Candidate epitope identification using peptide property models: application to cancer immunotherapy.Methods. 2004; 34: 460-467Crossref PubMed Scopus (16) Google Scholar]. Induction of strong immunity by cancer vaccines is expected to lead to the establishment of immune memory, thereby preventing tumor recurrence. A number of antigens recognized on tumor cells by T cells have been characterized.Human tumor antigens can be divided into (1) individual (e.g. incidental mutations that occur in CDK4 in melanoma cancer lines); (2) tumor-specific (e.g. common mutations or viral antigens such as HPV16 in cervix cancer or specifically activated in tumors such as MAGE) and tissue-restricted; or (3) differentiation antigens (i.e. PSA in prostatic cancer). Most of the cancer proteins are self-antigens. So the challenge within the field of cancer vaccines is to find the conditions to break a possible immune tolerance towards tumor antigens without inducing substantial autoimmune reactions that are harmful for healthy tissue [6.Huber C.H. Wolfel T. Immunotherapy of cancer: from vision to standard clinical practice.J Cancer Res Clin Oncol. 2004; 130: 367-374Crossref PubMed Google Scholar]. Vaccination in patients with breast cancer should induce an expansion of CD8+ cytotoxic T lymphocytes (CTLs) capable of rejecting tumor cells via recognition of tumor-associated antigenic (TAA) epitopes presented on the surface of cancer cells in association with human leukocyte antigen (HLA) class I molecules. However, optimal immunotherapeutic approaches should probably also prime CD4+ helper T cells, given the key role that these cells play in the control of immune responses and in the induction of cytotoxic responses.The antigens used in breast cancer vaccination strategies can be represented by whole tumor cells (either allogeneic or autologous) or of specific TAAs, which are delivered as DNA (naked or comprised in recombinant viruses), RNA, protein or HLA class I/II restricted peptide epitopes [3.Nencioni A.F.F. Patrone G.F. Brossart P. Anticancer vaccination strategies.Ann Oncol. 2004; 15: 153-160Abstract Full Text PDF Scopus (26) Google Scholar]. Antigenic materials can be injected directly, often coupled to immunostimulatory cytokines or adjuvants, or used for ex vivo loading of antigen presenting cells (APCs), usually dendritic cells (DCs). To generate a successful vaccine, this must have: a target antigen on tumor cells to direct the immune response; a platform to present the vaccine-derived antigen to immune system; an adjuvant to enhance immune stimulation, and appropriate monitoring techniques [4.Bernstein N. Overview of therapeutic vaccination approaches.Sem Oncol. 2003; 30: 1-8Crossref Scopus (20) Google Scholar].Table 1 summarizes different approaches used in monitoring vaccine therapies activities. There is a great difficulty in comparing the various approaches because monitoring assays have not yet been standardized. Identification of univocal surrogate immunological markers of activity should be a challenge for the future. Ideally, T-cell assays not only need to be sensitive, specific, reliable, reproducible, simple and quick to perform, but they should offer a good correlation with clinical outcome [7.Ko B.K. Kawano K. Murray J.L. et al.Clinical studies of vaccines targeting breast cancer.Clin Cancer Res. 2003; 9: 3222-3234PubMed Google Scholar]. Several immunological approaches in the treatment of breast cancer showed that it is possible to elicit an antitumor immune response that could potentially destroy tumor cells; however, the clinical activity is still discouraging. Several hypotheses can be postulated to explain these negative results including: the impact of previous oncolytic treatments (chemotherapy and radiotherapy) on immune system; the population of patients treated so far that is characterized by large tumor burden; the ability of large tumors to escape the immune system; and the difficulty to break immune tolerance [8.Lyerly H.K. Quantitating cellular immune response to cancer vaccines.Sem Oncol. 2003; 30: 9-16Crossref PubMed Scopus (35) Google Scholar]. Therapeutic cancer vaccines will probably be more active in minimal residual states, but most of the trials so far have been conducted on metastatic patients and limit the success of phase I/II trials [9.Emens L.A. Reilly R.T. Jafee E.M. Augmenting the potency of breast cancer vaccines: combined modality immunotherapy.Breast Dis. 2004; 20: 13-24Crossref PubMed Scopus (19) Google Scholar]. The aim of this review is to analyze the activity of vaccination strategies for patients with breast cancer in current clinical trials, focusing on possible future directions for vaccine development and applications in the adjuvant setting.Table 1Surrogate markers of immune response in vaccine therapy trialsTechniquesRationaleAdvantagesDisadvantagesCorrelation with clinical outcomeDTH testAgs are injected intradermally; evaluation of DTH responseSimple, low costNot standardized, subjective, not specificNoTetramer stainingFluorescent MHC/peptide Tetramers flow cytometryQuantitative cell number evaluation, specific, CD8+ restrictedText not evaluating activity of T cells, requires specific tetramers, limited to single epitopesNoLPAMeasurement of T-cell proliferation in response to Ag stimulation.SimpleInfluenced by non-specific immune response, not suitable for CD8+NoELISAMeasurement of cytokine secretionSensitiveNot specificNoSimpleNo enumeration of Ag-specific T cellsELIspotDirect enumeration of cytokine releasing Ag-specific T cellsQuantitative cell number evaluation, simple and reproducible, suitable for CD4/CD8+ cellsStandardization needs to be attemptedPossibleIntracellular cytokine measurement by flow cytometryFlow cytometry of stimulated cells after staining with a mixture of antibodiesQuantitative and functional assay, high specific rapid, simultaneous multiparameter valuationRequires incubation, unable to obtain live cells, technically complicatedYesqRT-PCRMolecular method for measuring amplified genesHigh accuracy, specific, indirect measurement of functionNo subset cells, high costsNoDirect cytotoxicity assaysIncubation of CTL with Ag-expressing cells labelled with cromium-51Suitable for CD8+Not sensitive, multiple stimulation are requestNoLimiting dilution analysisSerial dilutions of T-cells following in vitro stimulation and lysisQuantitative analysis, functional activityExpensive, operator-dependent and labor intensiveNoDTH, delayed-type hypersensitivity; tetramers, peptide MHC-teramers; LPA, lympoproliferation assays; ELISA, enzyme-linked immunosorbent assay; ELIspot, enzyme-linked immunospot assay; qRT-PCR, quantitative reverse transcriptase polymerase chain reaction; Ag(s), antigen(s); MHC, major histocompatibility complex. Open table in a new tab breast cancer vaccines based on characterized antigenMany tumor antigens used in breast-cancer immunotherapy are expressed on normal tissues but are overexpressed or mutated on tumor cells: MUC1, HER2, CEA, hTERT, p53 and carbohydrate antigens. Some of these antigens are universal tumor antigen (Ag) hTERT, as they are broadly expressed by most tumors. We will discuss all potential antigens that have been used to construct vaccines for the treatment of breast cancer.HER2/neHER2/neu is a 185-kDa protein receptor with tyrosine kinase activity and extensive homology to epidermal growth factor (EGF) receptor. HER2/neu is expressed in many epithelial tumors and over-expressed in approximately 30% of all primary breast cancer. Overexpression of HER2/neu is associated with a poor prognosis. HER2 is a suitable target because it involves an extracellular domain that can be targeted by antibodies produced by B cells. These antibodies could act either by a functional pathway (i.e. blocking the HER2 signalling pathway) or by an immune mechanism such as ADCC. Moreover, antigens derived from both the extra- or the intracellular domains can be presented by HLA class I or I to CD8+ or CD4+ T cells. Spontaneous T and B cells responses have been observed in patients with HER2/neu-positive tumors, confirming the immunogenicity of HER2/neu [10.Baxevanis C.N. Sotiropoulou P.A. Sotiriadou N.N. Papamichail M. Immunobiology of HER2/neu oncoprotein and its potential application in cancer immunotherapy.Cancer Immunol Immunother. 2004; 53: 166-175Crossref PubMed Scopus (63) Google Scholar].The use of HER2 peptides as a potential target for breast cancer immunotherapy arose from experimental evidence in the rat where it was shown that immunization with a mixture of peptides derived from the extracellular and intracellular domains of human HER2, but not the whole protein, elicited a delayed type hypersensitivity (DTH) [11.Disis M.L. Shiota F.M. Cheever M.A. Human H.E.R. 2/neu protein immunization circumvents tolerance to rat neu: a vaccine strategy for 'self' tumour antigens.J Immunol. 1998; 93: 192-199Crossref Scopus (62) Google Scholar]. Based on these findings, clinical trials with HER2 peptides have been conducted.Table 2 summarizes all clinical trials with HER2/neu peptides. Disis [12.Disis M.L. Grabstein K.H. Sleath P.R. et al.HER2/neu peptide vaccines elicit T cell immunity to the HER2/neu protein in patients with breast or ovarian cancer.Proc Am Soc Clin Oncol. 1998; 17: 97aGoogle Scholar] reported that nine of 17 patients with breast or ovarian cancer displayed a CD-8 immune response detected as IFN-γ production by positive enzyme-linked immunospot assay (ELIspot) after a vaccination with HER2 peptides mixed with G-CSF. This was associated with minimal toxicity, suggesting that it is possible to generate anti-HER2 responses also in humans without major signs of autoimmunity. In another pilot study, four HLA-A2+ patients with metastatic breast, ovarian or colorectal adenocarcinoma that overexpressed HER2/neu were immunized with the HLA-A2-binding epitope (p369–377) from HER2/neu in Freund's adjuvant. In three of four patients, peptide-specific CTLs were detected in the blood after one immunization. However, these CTLs failed to lyses HLA-A2+HER2+ tumors cells in vitro [13.Zaks T.Z. Rosenberg S.A. Immunization with a peptide epitope (p369–377) from HER2/neu leads to peptide specific cytotoxic T lymphocytes that fail to recognize HER2/neu+ tumors.Cancer Res. 1998; : 369-377Google Scholar]. In a following trial, the same HLA-restricted peptide-vaccine with HLA2 peptide and adjuvant GM-CSF has been tested in six patients with either stage III or IV breast or ovarian cancer. This vaccine resulted in the generation of low-level peptide-specific CD8 T-cell immunity that did not persist for an extended time after active immunization. Vaccination with HLA-class I peptides will probably require additional antigen specific or non-specific helper activity to generate long-lived immunity [14.Knutson K.L. Schiffman K. Cheever M.A. Disis M.L. Immunization of cancer patients with a HER2/neu, HLA-A2 peptide, p369–377, results in short-lived peptide-specific immunity.Clin Cancer Res. 2002; 8: 1014-1018PubMed Google Scholar]. The same group experimented the vaccine strategy with HLA-restricted helper HER2 peptides (p369–384, p688–703, p971–984) with encompassed HLA A2 epitopes (p369–377, p689–697, p971–979) in 19 patients with stage III or IV breast and ovarian cancer, producing a long lasting immune response (detected by ELISPOT for CD8+) (15). In another series of studies, 18 patients with breast cancer (four with stage III disease and 14 with stage IV disease) were given monthly vaccinations of three 15-amino acid HER2 peptides (369–384, 688–703 and 971–984) that contained the nested CTL epitopes 369–377, 688–696 and 971–979.Table 2Phase I trials with HER2 peptides-based vaccinesReferenceNo. and type of patientsVaccineGrade 3/4 toxicity rateNo. patients with ELIspot positiveDisis, 1999aThe trial reports 7/8 minimal response.,bNo-dose escalation trial.17 with advanced HER2-overexpressing breast or ovarian cancerHER2 peptides + GM-CSFNo9/17Zaks, 19994 advanced HER-overexpressing colorectal, breast or ovarian cancerHER2 peptides with Freund adjuvantNo3/4Knutson, 20026 advanced HER2-overexpressing breast or ovarian cancerHER2 helper peptides with GM-CSF adjuvantNRNR (low levels of CTL)Knutson, 200119 advanced HER-overexpressing breast or ovarian cancerHER2 peptides with HLA2 epitopesNR5/19Murray, 200018 advanced HER2-overexpressing breast cancerHER2 peptides with HLA2 epitopesNRNR (CD4+ CD8+ + long lasting responseMurray, 200214 advanced HER-overexpressing breast or ovarian cancerHER2 E75 plus GMCSFNo4/8Salazar, 200410 advanced HER-overexpressing breast or lung cancerFour HER2 peptides plus GMCSFNR25%Disis, 200429 with NED after surgery for HER2-overexpressing breast or ovarian cancerHER2 ICD peptideNR89%NR, not reported; NED, no evidence of disease; IDC, intracellular domain ECD, extracellular domain; GM-CSF, granulocyte-monocyte colony stimulating factor; ELIspot, enzyme-linked immunospot assay; ELISA, enzyme-linked immunosorbent assay; CTL, cytotoxic lymphocytes.a The trial reports 7/8 minimal response.b No-dose escalation trial. Open table in a new tab The goal of these studies was to overcome the problems associated with using class I epitopes alone, most of which are related to antigen instability or aggregation because of the short length of the peptide. The CD4+ and CD8+ cell responses were long lasting and remained detectable for more than 1 year after the final vaccination [16.Murray J.L. Przepiorka D. Ioannides C.G. Clinical trials of HER2/neu specific vaccines. Sem.Oncol. 2000; 27: 71-75Google Scholar]. Murray [17.Murray J.L. Gillogly M.E. Przepiorka D. et al.Toxicity, immunogenicity, and induction of E75-specific tumor-lytic CTLs by HER2 peptide E75 (369–377) combined with granulocyte macrophage colony-stimulating factor in HLA-A2+ patients with metastatic breast and ovarian cancer.Clin Cancer Res. 2002; 8: 3407-3418PubMed Google Scholar] used a vaccine containing E75 (p369–377) plus granulocyte-macrophage colony-stimulating factor (GM-CSF) for a phase I trial in 14 patients with metastatic breast (n = 13) or ovarian (n = 1) cancer. These patients were vaccinated with escalating doses (500–1000 μg) of E75 (p369–377) mixed with 250 μg of GM-CSF. No grade 3 toxic reactions to the vaccine were noted. Of eight patients tested for CTL induction, four had a CTL response after in vitro stimulation with autologous dendritic cells that had not been pulsed with peptide, consistent with the presence of activated/memory cells ex vivo. Four patients also had an E75-specific CTL response after in vitro stimulation with E75. In addition, CTLs from three patients specifically recognized E75 on indicator tumors, as demonstrated by cold target inhibition of tumor lysates. E75-specific tumor-lytic CTLs were present in some patients for more than 1 year after vaccination [17.Murray J.L. Gillogly M.E. Przepiorka D. et al.Toxicity, immunogenicity, and induction of E75-specific tumor-lytic CTLs by HER2 peptide E75 (369–377) combined with granulocyte macrophage colony-stimulating factor in HLA-A2+ patients with metastatic breast and ovarian cancer.Clin Cancer Res. 2002; 8: 3407-3418PubMed Google Scholar].Four putative class II HER2/neu peptides, which were found to generate detectable specific T-cell responses (stimulation index >2) in a majority of patients in a previous study, were used to formulate a single vaccine. The multipeptide vaccine was administered intradermally with GM-CSF as an adjuvant in 10 patients with HER2/neu overexpressing breast or lung cancer. Twenty-five percent of patients developed HER2/neu peptide-specific T-cell immunity and 50% developed HER2/neu peptide-specific antibody immunity. No patient developed HER2/neu entire protein-specific T-cell or antibody immunity. The majority of peptides exhibited high binding affinity, in vitro, to more than three of the 14 HLA-DR alleles analyzed. The group of peptides used in this study demonstrated high binding affinity to multiple DR alleles suggesting that in vitro binding affinity may be able to predict the in vivo immunogenicity of class II peptides. However, only a minority of patients immunized with the multipeptide vaccine developed HER2/neu peptide-specific T-cell or antibody immunity, and none developed HER2/neu protein-specific immunity [18.Salazar L.G. Fikes J. Southwood S. et al.Immunization of cancer patients with HER2/neu-derived peptides demonstrating high-affinity binding to multiple class II alleles.Clin Cancer Res. 2003; 9: 5559-5565PubMed Google Scholar].Interestingly, in a first study in the adjuvant setting, 29 patients with no evidence of disease after surgery for HER2/neu-overexpressing breast or ovarian cancer received three level doses (low 25 μg, intermediate 150 μg or high 900 μg) HER2/neu (intracellular domain) ICD protein vaccine. The vaccine was administered intradermally, monthly for 6 months, with GM-CSF as an adjuvant. The vaccine was well tolerated. The majority of patients (89%) developed HER2/neu ICD-specific T-cell immunity. The dose of vaccine did not predict the magnitude of the T-cell response. The majority of patients (82%) also developed HER2/neu-specific immunoglobulin G antibody immunity. Vaccine dose did not predict magnitude or avidity of the HER2/neu-specific humoral immune response. Time to development of detectable HER2/neu-specific immunity, however, was significantly earlier for the high- versus low-dose vaccine group (P = 0.003). Over half the patients retained HER2/neu-specific T-cell immunity 9–12 months after immunizations had ended. So, although the dose of vaccine did not impact on the magnitude of T cell or antibody immunity elicited, patients receiving the highest dose developed HER2/neu-specific immunity more rapidly than those who received the lowest dose. Clearly, we are waiting for the clinical outcome report, which needs a long-term follow-up [19.Disis M.L. Schiffman K. Guthrie K. et al.Effect of dose on immune response in patients vaccinated with an HER2/neu intracellular domain protein-based vaccine.J Clin Oncol. 2004; 22: 1916-1925Crossref PubMed Scopus (135) Google Scholar].Another study is ongoing in the adjuvant setting [20.Limentani S. Dorval T. White S. et al.Phase I dose-escalation trial of a recombinant HER2 vaccine in patients with Stage II/III HER2+ breast cancer.ASCO Proc. 2005; (Abstr 2520)Google Scholar]. The vaccine formulation contained a truncated recombinant HER2 protein (dHER2) combined with a new potent immunological adjuvant. dHER2 includes the extracellular domain (ECD) and a part of the ICD of the HER2 protein. The trial's objective was to evaluate safety and immunogenicity. Three dose levels of dHER2 protein were tested: 20, 100 and 500 μg. Three cohorts of 15 patients with stage II or III breast cancer were enrolled sequentially. Patients received six vaccinations over 14 weeks. All patients receiving the 20 and 100 μg doses have completed the course of six vaccinations, and those in the 500 μg cohort will have done so by March 2005. To date, the vaccine has been well tolerated overall and has shown minimal toxicity. Grade 3 fatigue and grade 3 neutropenia were recorded once each in different patients who continued treatment. No symptomatic cardiac dysfunction was observed. Ab responses against dHER2, ICD and ECD were elicited. After four vaccinations, two of 12 patients in the 20 μg and nine of 14 in the 100 μg cohort responded to the ECD. Results in the 500 μg cohort are pending. The dHER2 vaccine appears to be safe in the small number of patients tested. Ab to the ECD and the ICD are induced in a dose-dependent manner, suggesting that the higher-dose vaccine may be required for future phase II and III studies.Altogether, these results suggest that in order to have a maximal induction of the immune response against HER2 in terms of antibody and T cell responses, both the extracellular and intracellular domains of the protein should be included in the vaccine formulation. This should also guarantee the induction of a long-lasting immunological memory.MUC-MUC-1 is a membrane-associated glycoprotein expressed by many types of ductal epithelia, including pancreas, breast, lung and gastrointestinal tract. It is overexpressed and aberrantly glycosilated by malignant cells. It is a multifunctional protein involved in the protection of mucous membranes, signal transduction, and modulation of immune system. More than 70% of cancers overexpress MUC1, making this antigen a potential target for immunotherapy [21.Kohlgraf K.G. Gawron A.J. Higashi M. Tumor-specific immunity in MUC1. Tg mice induced by immunization with peptide vaccines from the cytoplasmatic tail of CD227 (MUC1).Cancer Immunol Immunother. 2004; 53: 1068-1084Crossref PubMed Scopus (29) Google Scholar, 22.Chen D. Xia J. Tanaka Y. et al.Immunotherapy of spontaneous mammary carcinoma with fusions of dendritic cells and mucin 1-positive carcinoma cells.Immunology. 2003; 109: 300-307Crossref PubMed Scopus (61) Google Scholar]. MUC1 is sufficiently immunogenic to elicit strong antitumor immunity as a tumor antigen [7.Ko B.K. Kawano K. Murray J.L. et al.Clinical studies of vaccines targeting breast cancer.Clin Cancer Res. 2003; 9: 3222-3234PubMed Google Scholar]. Preclinical studies, using tumor cells that expressed MUC-1 protein or peptide Ag, concluded that MUC-1 could induce humoral response without inducing cellular response [23.Ding L. Lalani E.N. Reddish M. et al.Immunogenicity of synthetic peptides related to the core peptide sequence encoded by the human MUC1 mucin gene: effect of immunization on the growth of murine mammary adenocarcinoma cells transfected with the human MUC1 gene.Cancer Immunol Immunother. 1993; 36: 9-17Crossref PubMed Scopus (101) Google Scholar, 24.Apostolopoulos V. Xing P.X. McKenzie I.F. Murine immune response to cells transfected with human MUC1: immunization with cellular and synthetic antigens.Cancer Res. 1994; 54: 5186-5193PubMed Google Scholar, 25.Zhang S. Graeber L.A. Helling F. et al.Augmenting the immunogenicity of synthetic MUC1 peptide vaccines in mice.Cancer Res. 1996; 56: 3315-3319PubMed Google Scholar, 26.Acres R.B. Hareuveni M. Balloul J.M. Kieny M.P. Vaccinia virus MUC1 immunization of mice: immune response and protection against the growth of murine tumors bearing the MUC1 antigen.J Immunother. 1993; 14: 136-143Crossref Scopus (82) Google Scholar].In a first phase I clinical trial, a 105-amino acid synthetic MUC-1 peptide with five repeated immunodominant epitopes mixed with Bacillus Calmette-Guerin (BCG) was tested in 63 patients with adenocarcinomas (including nine breast cancer patients) three times at 3-week intervals. The vaccine was well tolerated. Only three patients showed a strong immune response detected by DTH. In addition, the examination of 55 biopsies showed intense T-cell infiltration in 37 patients and lesser infiltration in seven patients, but only seven of 22 patients had a two- to four-fold increase in mucin-specific CTL precursors after vaccination. None of the breast cancer patients showed a disease control [27.Goydos J.S. Elder E. Whiteside T.L. Finn O.J. Lotze M.T. A phase I trial of a synthetic mucin peptide vaccine.Induction of specific immune reactivity in patients with adenocarcinoma. J Surg Res. 1996; 63: 298-304Google Scholar].An alternative approach was used in a following trial, where 16 patients with metastatic breast carcinoma were treated with a vaccine consisting of 5 μg of the 16-amino acid MUC-1 peptide (plus keyhole limpet hemocyanin and DETOX as adjuvants). All patients generated strong anti-keyhole limpet hemocyanin (KLH) IgG responses, but only three patients developed a weak anti-MUCIN IgG response. Evidence for class I-restricted killing of MUC1-expressing tumor cell lines was obtained in seven of the 11 tested patients. Five of these seven patients also had high anti-MUC-1 IgG titers. The clinical effects of inducing this level of MUC-1 immunity have not been reported.The apparent difficulty in generating CTL effectors in vivo may have been due to the induction of T-cell anergy by tumor-derived MUC-1 [28.Reddish M.A. MacLean G.D. Koganty R.R. et al.Anti-MUC1 class I restricted CTLs in metastatic breast cancer patients immunized with a synthetic MUC1 peptide.Int J Cancer. 1998; 76: 817-823Crossref PubMed Scopus (118) Google Scholar]. In an attempt to optimize MUC-1 presentation by APCs, several experimental studies in xenografts have targeted the mannose receptor on APCs, by using antigens that have been linked to mannose. Apostolopoulos et al. [29.Apostolopoulos V. Pietersz G.A. McKenzie I.F.C. Cell-mediated immune response to MUC-1 fusion protein coupled to mannan.Vaccine. 1996; 14: 930-938Crossref PubMed Scopus (137) Google Scholar, 30.Apostolopoulos V. Loveland B.E. Pietersz G.A. McKenzie IF. CTL in mice immunized with human mucin 1 are MHC-restricted.J Immunol. 1995; 155: 5089-5094PubMed Google Scholar, 31.Apostolopoulos V. Pietersz G.A. McKenzia I.F.C. Oxidative/reductive conjugation of mannan to antigen selects for T1 or T2 immune response.Proc Natl Acad Sci. 1995; 92: 10128-10132Crossref PubMed Scopus (191) Google Scholar] demonstrated that the use of the oxidized form of fusion mannan-protein typically produced high numbers of precursors. Based on these findings, 25 patients with advanced metastatic carcinoma (including eight breast cancer patients) were vaccinated with mannan-MUC1 in a phase I dose escalation study. High titers of high-affinity anti-MUC1 IgG Ab were produced in 13/25 patients, where the levels of Ab directly correlated with the amount of immunogen given [32.Karanikas V. Hwang L.A. Pearson J. Antibody and T-cell