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Keywords:

  • NY-ESO-1;
  • cancer vaccine;
  • cancer testis antigen;
  • heteroclitic antibody response

Abstract

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

NY-ESO-1 is a prototypic cancer/testis antigen. In a recent phase I clinical trial, we vaccinated 13 patients bearing NY-ESO-1-expressing tumors with a complex of cholesterol-bearing hydrophobized pullulan (CHP) and NY-ESO-1 protein (CHP-NY-ESO-1) and showed efficient induction of NY-ESO-1 antibody, and CD4 and CD8 T cell responses using peripheral blood from the patients. In our study, we analyzed heteroclitic serological responses in those patients after vaccination. Serological response against 11 tumor antigens including MAGE-A1, MAGE-A3, MAGE-A4, CT7/MAGEC1, CT10/MAGEC2, CT45, CT46/HORMAD1, SOX2, SSX2, XAGE1B and p53 was examined by enzyme-linked immunosorbent assay (ELISA) using sera from ten vaccinated patients. Expression of tumor antigens was determined by reverse transcription-polymerase chain reaction or immunohistochemistry. Eight of nine patients who showed antibody responses against NY-ESO-1 also showed an antibody response against at least 1 of these 11 tumor antigens after vaccination. In one patient, seven tumor antigens were recognized. Specificity analysis of the antibody response by ELISA using control recombinant proteins and synthetic peptides and by Western blot showed that the response was not against His6-tag and/or bacterial products included in a preparation of CHP-NY-ESO-1 used for vaccination. Thus, heteroclitic serological responses appear to be indicative of the overall immune response against the tumor, and their analysis could be useful for immune monitoring in cancer vaccine.

NY-ESO-1 antigen was originally identified in esophageal cancer by serological expression cloning (SEREX) using autologous patient serum1, 2 and has been shown to be strongly immunogenic. Patients bearing NY-ESO-1-expressing tumors often show antibody and CD4 and CD8 T cell responses spontaneously.3–5 Clinical trials using NY-ESO-1 peptide,6–9 protein10, 11 and viral constructs12 as cancer vaccine have been conducted. Those studies have demonstrated efficient induction of antibody, and CD4 and CD8 T cell responses, and have also shown in some sporadic cases efficacy of immunotherapy.2, 6–12

In a recent phase I clinical trial, we vaccinated 13 patients including eight Stage IV esophageal cancer patients, four Stage D3 prostate cancer patients and a Stage IV malignant melanoma patient with a complex of cholesterol-bearing hydrophobized pullulan (CHP) and NY-ESO-1 protein (CHP-NY-ESO-1).13–16 We showed that the vaccine was well tolerated and had a potent capacity to induce NY-ESO-1 antibody and CD4 and CD8 T cell responses using peripheral blood from patients. Beneficial effects such as tumor regression or no tumor growth for a prolonged period were observed in six of seven disease-evaluable patients. However, all patients died eventually. Immunohistochemical analysis of the tumors that grew after vaccination suggested involvement of different mechanisms resulting in immune impairment.15 NY-ESO-1-antigen loss was observed in a patient, disappearance of tumor-infiltrating CD4 and CD8 T cells was observed in two patients and an increase in the number of CD68+ macrophages was observed in another patient. Further study will be necessary to elucidate cellular mechanisms including regulatory T cells causing immune suppression at the local tumor site.

It has been shown that cytotoxic T cell responses to tumor antigens other than the antigen used for immunization occur after vaccination.17–26 This heteroclitic immune response following the initial response to a specific antigen was originally described in autoimmune disease and has been suggested to play a crucial role in clinical responses mediated by cancer vaccines.27, 28 Occurrence of CD8 T cell responses to unrelated tumor antigens was shown in studies of vaccination with MAGE-A1 and/or MAGE-A3,19, 20, 25 HER-2/neu,17, 18, 21, 22 MART-1/Melan-A23–25 and gp100.26

In our study, we analyzed the heteroclitic serological response against a panel of tumor antigens. Specificity analysis of antibody response was performed by enzyme-linked immunosorbent assay (ELISA) using control recombinant proteins and synthetic peptides, and by Western blot. The findings indicate frequent occurrence of heteroclitic serological responses in patients after CHP-NY-ESO-1 vaccination.

Material and Methods

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Patients and sera

Eight advanced esophageal cancer patients (E-1, E-2, E-3, E-4, E-5, E-6, E-7 and E-8) and two prostate cancer patients (P-2 and P-3) were enrolled in the clinical trial (protocol LUD 2002-005 of the Ludwig Institute for Cancer Research, New York, NY) (http://clinicaltrials.gov/ct2/show/NCT00106158?term=CHP+NY-ESO-1&rank=2).13–16 Peripheral blood was drawn from patients with written informed consent with the permission of the ethics committees of Osaka and Okayama Universities. Sera from MAGE-A4 seropositive esophageal cancer patients (EC-1, EC-2 and EC-3) and five healthy donors were drawn with written informed consent with the permission of the ethics committees of Osaka University. Sera were stored in −80°C freezer until use.

Preparation of a complex of cholesterol-bearing hydrophobized pullulan and NY-ESO-1 protein (CHP-NY-ESO-1)

Preparation of recombinant NY-ESO-1 protein for vaccine was described elsewhere.29 A complex of CHP and NY-ESO-1 protein (CHP-NY-ESO-1) and the schedule of vaccine were described previously.14 Briefly, patients with advanced cancers expressing NY-ESO-1 were injected 2–31 times subcutaneously at biweekly intervals with 100 μg of NY-ESO-1 recombinant protein formulated with 2 mg of CHP.

Recombinant protein and overlapping peptides

N-His6-tagged recombinant proteins, NY-ESO-1, LAGE-1, MAGE-A1, MAGE-A3, MAGE-A4, CT7/MAGEC1, CT10/MAGEC2, CT45, CT46/HORMAD1, p53, SOX2, SSX2, XAGE1B, dihydrofolate reductase (DHFR) and Akt,30–32 were produced by using pQE30 vector (QIAGEN, Hilden, Germany) and expressed in M15 E. coli cells. N-His6-tagged p53 produced in a Baculovirus system was purchased (Enzo Life Sciences, New York, NY). N-His6-tagged CCDC-62 protein was produced in a Baculovirus system at Okayama University.33

A series of 22 25-mer MAGE-A4 overlapping peptides (OLPs) were synthesized using standard solid-phase methods based on N-(9-fluorenyl)-methoxycarbonyl chemistry on an ABIMED Multiple Peptide Synthesizer (AMS422, ABIMED, Langenfeld, Germany) at Okayama University. MAGE-A4 OLPs: 1 (1–25), 2 (15–39), 3 (29–53), 4 (43–67), 5 (57–81), 6 (71–95), 7 (85–109), 8 (99–123), 9 (113–137), 10 (127–151), 11 (141–165), 12 (155–179), 13 (169–193), 14 (183–207), 15 (197–221), 16 (221–235), 17 (225–249), 18 (239–263), 19 (253–277), 20 (267–291), 21 (281–305), 22 (295–317).

MAGE-A4 transfectants

CMS5a, a murine fibrosarcoma cell line from a strain of BALB/c origin, was stably transfected with pcDNA3.1 MAGE-A4 plasmid as described previously.34 The plasmid was provided by Dr. A. Kuroda and Dr. M. Miyamoto (Hokkaido University, Sapporo, Japan) and purified using plasmid Mini kit.

ELISA

Serially diluted sera were added to 96-well plates coated overnight at 4°C with 1 μg/ml recombinant protein or 5 μg/ml peptide solution and blocked for 2 hr at room temperature. After overnight incubation, plates were extensively washed with PBS containing 0.2% Tween 20. For determining reciprocal titer by ELISA, shown in Figure 1, serum IgG bound to antigens was detected by alkaline phosphatase-conjugated specific monoclonal antibody (Southern Biotech, Birmingham, AL). After addition of ATTOPHOS substrate (Fisher Scientific, Waltham, MA), absorbance was measured using a Cytofluor Series 4000 fluorescence reader (PerSeptive Biosystems, Framingham, MA). A reciprocal titer was calculated for each sample as the maximal dilution still significantly reacting to a specific antigen. Specificity was determined by comparing seroreactivity among the various antigens tested.31 In each assay, positive and negative control sera were included. A positive result was defined as reciprocal titers >100. For conventional ELISA, peroxidase-conjugated goat anti-human IgG or IgM (Jackson Immuno Research Laboratory, West Grove, PA) was added to the wells for second antibody. After washing, signals were developed with o-phenylene diamine dihydrochloride, and absorbance at 490 nm was read using an ELISA reader (Benchmark Microplate Reader; Bio-Rad, Hercules, CA). Positivity was defined as sample optical density (OD) greater than three times that of the value for irrelevant control protein. Titers of Epstein–Barr virus (EBV) and Cytomegalovirus (CMV) antibody in sera were measured by EBV VCA kit (Denka Seiken, Tokyo, Japan) and CMV kit (Denka Seiken), respectively.

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Figure 1. Reciprocal antibody titer against 13 tumor antigens in sera from patients vaccinated with CHP-NY-ESO-1 by ELISA. Serially diluted sera obtained before and after vaccination were assayed against N-His6-tagged recombinant proteins NY-ESO-1, LAGE-1, MAGE-A1, MAGE-A3, MAGE-A4, CT7/MAGEC1, CT10/MAGEC2, CT45, CT46/HORMAD1, p53, SOX2, SSX2, XAGE1B and DHFR. The reciprocal titer was the maximal dilution showing significant reaction (open and closed circles). Closed circles indicate reciprocal titers exceeding 100 (positive reaction). In each assay, antibody positive and negative sera were included as controls. Positive (+) and negative (−) expression of tumor antigens indicated in boxes under each panel was analyzed by RT-PCR and/or IHC when sample was available (see Supporting Information Table). Titer of EBV and CMV antibody in sera were measured by EBV and CMV kits, respectively. Values exceeding 4.0 were positive by manufacturer's indication. +*; 57B and M3H67 mAbs generated against MAGE-A3 and MAGE-A4 recombinant proteins, respectively, were both shown to recognize multiple MAGE-A family molecules.

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Western blot

Recombinant protein (20 ng) or cell lysate (20 μg) in sample buffer (100 mM Tris-HCl, pH 8.8, 0.01% bromophenol blue, 36% glycerol, 4% SDS and 1 mM dithiothreitol) was boiled for 5 min and subjected to SDS-PAGE with 10–20% polyacrylamide BioRad Ready-Gels (Bio-Rad). After electrophoresis, the membrane (Hybond-P membrane, Amersham Pharmacia Biotech, Buckinghamshire, UK) was blocked with 5% FCS/PBS and then incubated with patients' sera diluted 1:1,000 for recombinant protein or 1:200 for cell lysate for 1 hr at room temperature. After washing, alkaline phosphatase-conjugated goat anti-human IgG (Jackson Immuno Research Laboratory) was added to the membrane. Signals were developed with a 5-bromo-4-chloro-3-indolylphosphate-nitroblue tetrazolium chromogenic substrate kit (Bio-Rad). Polyclonal rabbit anti-MAGE-A1 serum (Abcam, Cambridge, UK) and monoclonal anti-MAGE-A4 (clone 3D12; Abnova, Taipei, Taiwan), anti-p53 (clone PAb421; Enzo) and anti-His6-tag (clone OGHis; MBL, Nagoya, Japan) antibodies were used for positive controls at 1:1,000 dilution.

Immunohistochemistry

Immunohistochemistry (IHC) was performed using formalin-fixed paraffin-embedded specimens. Monoclonal antibodies used were anti-MAGE-A1 (clone MA454), anti-MAGE-A3 (clone M3H67), anti-MAGE-A4 (clone 57B), anti-CT7/MAGEC1 (clone CT7-33) and anti-CT10/MAGEC2 (clone LX-CT10.5). For cancer-testis (CT) antigens, only strong nuclear and/or cytoplasmic staining as observed in testicular tissue (positive control) in at least 5% of cells was scored as positive. 57B and M3H67 mAbs generated against MAGE-A3 and MAGE-A4 recombinant proteins, respectively, were both shown to recognize multiple MAGE-A family molecules.35, 36

Reverse transcription-polymerase chain reaction

Total cellular RNA was extracted from frozen tissue using TRIzol Reagent (Invitrogen, Carlsbad, CA). Conventional reverse transcription-polymerase chain reaction (RT-PCR) was performed against NY-ESO-1, LAGE-1, MAGE-A1, MAGE-A3, MAGE-A4, CT7/MAGEC1, CT10/MAGEC2, CT45, CT46/HORMAD1, SOX2, SSX2 and XAGE1B.30, 31

Results

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Antibody response against 13 tumor antigens in CHP-NY-ESO-1-vaccinated patients

We analyzed antibody responses against NY-ESO-1, NY-ESO-1-related antigen LAGE-1, other CT antigens MAGE-A1, MAGE-A3, MAGE-A4, CT7/MAGEC1, CT10/MAGEC2, CT45, CT46/HORMAD1, SSX2 and XAGE1B, SOX2 and p53 in esophageal cancer patients E-1, E-2, E-3, E-4, E-5, E-6, E-7 and E-8 and prostate cancer patients P-2 and P-3 before and after a cycle of CHP-NY-ESO-1 vaccination (Fig. 1 and Table 1). Before vaccination, strong antibody responses against NY-ESO-1 and/or LAGE-1 were observed in E-2 and P-3 and defined as baseline seropositive. Additionally, marginal antibody responses were observed in E-1, E-3 and E-7. In E-5, antibody against MAGE-A1, MAGE-A3, MAGE-A4 and SOX2 was observed. In E-7, antibody against MAGE-A4, CT7/MAGEC1, p53 and SOX2 was observed. In P-3, antibody against p53 was observed. After vaccination, in all patients except E-6, antibody response against NY-ESO-1 and LAGE-1 was increased or induced. In E-2, antibody responses against MAGE-A3 and MAGE-A4 were induced. In E-3, antibody response against SOX2 was induced. In E-4, antibody responses against MAGE-A3, MAGE-A4 and CT10/MAGEC2 were induced. In E-5, antibody responses against MAGE-A3 and MAGE-A4 were increased. In E-7, antibody responses against CT7/MAGEC1, p53 and SOX2 were increased and that against CT10/MAGEC2 was induced. In E-8, antibody responses against MAGE-A1, MAGE-A3, MAGE-A4, CT7/MAGEC1, CT45, CT46/HORMAD1 and p53 were induced. In P-2, antibody response against MAGE-A4 was induced. In P-3, antibody response against CT7/MAGEC1 was induced and that against p53 was increased. No antibody against DHFR included as a control was detected in any patient. Furthermore, no increase of antibody response was observed against EBV and CMV after CHP-NY-ESO-1 vaccination.

Table 1. Heteroclitic antibody response and clinical response after CHP-NY-ESO-1 vaccination
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Expression of 13 tumor antigens in tumor specimens

Expression of NY-ESO-1 was detected by RT-PCR and IHC in tumors from all patients before vaccination. Expression of other tumor antigens except p53 was analyzed by RT-PCR in E-1, E-5, E-6, E-7 and E-8, and expression of MAGE-A1, MAGE-A3, MAGE-A4, CT7/MAGEC1 and CT10/MAGEC2 was also analyzed by IHC in E-1, E-2, E-4, E-5, E-6, E-7 and E-8 (Fig. 1 and Supporting Information Table). Mutation of p53 was not determined in our study. Expression of corresponding antigen was confirmed with tumor specimens in patients who showed antibody against tumor antigens.

Antibody response against tumor antigens in CHP-NY-ESO-1-vaccinated patients: No involvement of antibody against His6-tag and the product of E. coli present in the vaccine

Antibody responses against selected tumor antigens were further confirmed in sera obtained at each time during multiple vaccinations. As shown in Figure 2, IgG antibody against MAGE-A4 was detected in sera from E-5 and E-7 before vaccination, and the response was increased or induced in E-2, E-4, E-5, E-8 and P-2 after vaccination. IgG antibody against p53 was detected in sera from E-7 and P-3 before vaccination, and the response was increased or induced in E-7, E-8 and P-3.

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Figure 2. IgG antibody response against NY-ESO-1, MAGE-A4 and p53 in sera from patients before and after CHP-NY-ESO-1 vaccination by ELISA. Sera diluted at 1:100 were assayed against N-His6-tagged recombinant proteins NY-ESO-1, MAGE-A4 and Akt produced in E. coli and recombinant proteins p53 and CCDC-62 produced in Baculovirus. Akt and CCDC-62 were included as negative control (dotted line). Positive reaction (closed circles) represented the OD values exceeding three times the control OD value.

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Induction of IgM antibody against MAGE-A4 was detected in sera from E-8 after vaccination (Supporting Information Fig. 1). IgM antibody against p53 was detected in sera from E-7 before vaccination. Increase or induction of IgM antibody against p53 was detected in E-7 and E-8 after vaccination. Interestingly, in E-8, transient IgM response against MAGE-A4 and p53 was followed by IgG response.

Recombinant NY-ESO-1 protein used for vaccination has His6-tag in the N-terminus and was produced in E. coli as the host cells. All antigens shown in Figure 1 also have His6-tag and were produced in E. coli. To exclude the possibility of detecting antibody against His6-tag and/or the product of E. coli in the assay that might be raised by vaccination, DHFR was tested as control. No antibody against DHFR was detected (see above). To further exclude the possibility, the antibody response against control antigens was examined by IgG ELISA using serum samples obtained in each time during multiple vaccinations. As shown in Figure 2, antibody against Akt protein with His6-tag and produced in E. coli was within a background level (<0.2 OD value). p53 used in the experiments shown in Figure 2 and Supporting Information Figure 1, but not in Figure 1, and CCDC-62 protein share His6-tag and were produced by Baculovirus. Antibody against CCDC-62 was undetectable in sera from any patients.

Next, we synthesized MAGE-A4 OLPs and investigated antibody response by ELISA. Antibody response against MAGE-A4 peptides 15 and 21 was frequently observed in patients showing antibody response against MAGE-A4 protein (Supporting Information Fig. 2). Serially diluted sera from patients E-4, E-5, E-7 and E-8 obtained before and after CHP-NY-ESO-1 vaccination were examined against MAGE-A4 peptides 15 and 21 by IgG ELISA (Fig. 3). Increase or induction of antibody response was observed in E-4, E-5 and E-8, but not E-7 after vaccination. No antibody response was detected in E-1 included as negative control. These results were consistent with those by ELISA using recombinant MAGE-A4 protein in Figure 2.

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Figure 3. Serially diluted sera from patients before (open squares) and after (closed squares) CHP-NY-ESO-1 vaccination were assayed against MAGE-A4 peptide 15 and peptide 21 by IgG ELISA.

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Western blot analysis

The specificity of antibody against MAGE-A1, MAGE-A4 and p53 in sera from all patients vaccinated was further analyzed by Western blot (Fig. 4). Each antibody as positive control showed the representative band for MAGE-A1 protein at 59 kDa, for MAGE-A4 protein at 49 kDa and for p53 protein at 55 kDa. Increase of reaction with the bands was observed with recombinant MAGE-A1 protein in sera from E-8, with recombinant MAGE-A4 protein in sera from E-2, E-4, E-5, E-8 and P-2 and with p53 in sera from E-7, E-8 and P-3 obtained after vaccination.

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Figure 4. Western blot analysis. Reaction of sera against MAGE-A1, MAGE-A4 and p53 was investigated. Recombinant proteins (20 ng) were run by SDS-PAGE and transferred to a membrane by electrophoresis. Sera (1:1,000) from all patients obtained before and after vaccination were examined. Marker and control bands of each protein detected by monoclonal or polyclonal antibody (1:1,000) are also shown.

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Specificity of the reaction was further confirmed using transfectants. As shown in Figure 5, sera from E-4 after vaccination and from E-5 before and after vaccination reacted to MAGE-A4 in lysate of MAGE-A4-transfected murine fibrosarcoma CMS5a cells. No reaction was observed with lysate of mock-transfected CMS5a cells.

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Figure 5. Western blot analysis of sera against MAGE-A4 in lysate of MAGE-A4-transfected CMS5a cells. Cell lysate (20 μg) was run by SDS-PAGE, transferred to a membrane by electrophoresis and sera (1:200) from E-4 and E-5 patients obtained before and after CHP-NY-ESO-1 vaccination were examined. Control band of the protein detected by monoclonal antibody (1:1,000) is shown.

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Discussion

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Efficient elicitation of host immune response is a prerequisite for successful immunotherapy using cancer vaccine, and immune monitoring of specific antibody, CD4 and CD8 T cell responses against tumor antigens after vaccination is crucial to evaluate the response. In our study, we investigated antibody response against 13 tumor antigens by ELISA using recombinant proteins to evaluate the immune response more precisely. Nine of ten patients analyzed except E-6 showed an increase or induction of antibody response against NY-ESO-1 and its related LAGE-1 antigen after CHP-NY-ESO-1 vaccination. Eight of these nine patients showed an increase or induction of antibody response to either of these antigens after vaccination. Previously, it was reported that sera from patients vaccinated with recombinant NY-ESO-1 protein and CpG in Montanide sometimes showed nonspecific production of antibody against other recombinant proteins used for control,11, 37 and some of these responses could be attributed to reactivity against bacterial components or His6-tag. To address this possibility, we performed specificity analysis of the antibody response using control recombinant proteins, synthetic peptides and by Western blot that showed heteroclitic responses were not against His6-tag and/or bacterial products included in a preparation of CHP-NY-ESO-1 used for vaccination.

We reported previously that those patients showed NY-ESO-1 specific antibody and CD4 and CD8 T cell responses during vaccination.14, 15 The findings suggest that increase or induction of antibody response against tumor antigens, e.g., MAGE-A3 and MAGE-A4, as well as NY-ESO-1 after CHP-NY-ESO-1 vaccination may be caused by their release from tumor cells damaged by NY-ESO-1-specific immunity. Therefore, antibody response to multiple tumor antigens may suggest an intensity of the overall host immune response against the tumor, and detection of multiple heteroclitic serological responses using a panel of recombinant proteins would be a new tool of immunological monitoring for antitumor responses. A clear correlation between heteroclitic antibody responses and clinical outcomes could not be established in the limited number of patients analyzed in our study (Table 1). However, antibody response as well as CD4 and/or CD8 T cell responses to heteroclitic tumor antigens would be useful for evaluating overall immune response to tumor.

A number of studies have shown the relationship between heteroclitic immune response and clinical response. Germeau et al.19 reported that the frequency of CTL precursor increased tenfold in some patients after vaccination using MAGE antigenic peptides, although they found no significant difference in the levels against immunizing antigens between the tumor-regressor and -progressor patients. They then analyzed CTL precursors against other tumor antigens than that utilized for vaccine and found that the immune responses elicited to those irrelevant antigens after vaccination might contribute to the whole immune response to a given tumor and was correlated to clinical responses. Similarly, Butterfield et al.23, 24 reported that peptide-specific T cell response was efficiently induced in most patients by immunization with MART-1/Melan-A peptide pulsed dendritic cells. However, cellular immune responses against not only MART-1/Melan-A but also gp100 and tyrosinase were detected only in a complete clinical responder. These findings suggest a relationship between heteroclitic CTL responses and clinical responses. Furthermore, Disis et al. reported induction of both cellular and humoral responses against other intramolecular determinants in patients immunized with HER-2/neu peptide vaccine, and of antibody response to p53 in patients immunized with HER-2/neu peptide vaccine.17, 22 They further studied the effect of HER-2/neu T-helper peptide-based vaccinated patients receiving trastuzumab therapy and observed prolonged immune responses against not only the vaccine antigen but also cryptic antigens.38 Collectively, the presence of either humoral or cellular immune response to multiple tumor antigens appears to be indicative of the strength of overall response against the tumor and predictive of clinical response. In our study, we used a panel of 13 tumor antigens for the detection of the humoral response. Serological detection of responses to multiple tumor antigens that were shown to be highly immunogenic in cancer patients would be convenient and could be included in routine immune monitoring.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The authors thank Ms. J. Mizuuchi for preparation of the manuscript, Dr. J. Wing for critical reading of this manuscript and Ms. Y. Tada for the excellent technical assistance.

References

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article

FilenameFormatSizeDescription
IJC_26074_sm_suppinfofig1.tif51KSupporting Information Figure 1.
IJC_26074_sm_suppinfofig2.tif68KSupporting Information Figure 2.

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