Mamoru Harada

Vaccination with predesignated or evidence-based peptides for patients with recurrent gynecologic cancers.

Two different trials of peptide vaccination were conducted for patients with recurrent gynecologic cancers. In the first regimen, four HLA-A24+ patients (two with cervical cancer and two with ovarian cancer) were vaccinated with peptides that were predesignated before vaccination. Three patients exhibited with a grade 1 adverse effect, and no clinical response was observed in any patients. In the second regimen, six HLA-A24+ and four HLA-A2+ patients (five with cervical cancer, one with endometrial cancer, one with uterine sarcoma, and three with ovarian cancer) were vaccinated with peptides (maximum four) to which preexisting cytotoxic T lymphocyte precursors in the periphery were confirmed before vaccination. With this regimen, grade 1 adverse effects were observed in eight patients, a grade 2 adverse effect in one patient, and a grade 3 adverse effect (ie, rectal bleeding) in one patient. However, this regimen was able to enhance peptide-specific cytotoxic T lymphocytes in seven of ten patients, and three of five cervical cancer patients showed objective tumor regression. Analysis of immunoglobulin G -reactive to administered peptides suggested that the induction of peptide-specific immunoglobulin G was correlated with clinical responses. Overall, these results suggest that peptide vaccination of patients showing evidence of preexisting peptide-specific cytotoxic T lymphocyte precursors could be superior to vaccination with predesignated peptides, and that the evidence-based regimen is applicable for clinical trials in treatment of patients with recurrent gynecologic cancers.

Peptide Vaccination for Patients With Melanoma and Other Types of Cancer Based on Pre-existing Peptide-Specific Cytotoxic T-Lymphocyte Precursors in the Periphery

Identification of antigenic peptides expressed on cancer cells enables us to treat cancer patients with peptide-based immunotherapy. Although optimal protocols for peptide-based vaccines have not yet been elucidated, boosting the immune system could be a better approach than priming the immune system to elicit prompt and potent peptide-specific T-cell responses in cancer patients. With this possibility in mind, the authors undertook a clinical trial in which cancer patients were vaccinated with peptides (maximum 4) after confirmation of pre-existing peptide-specific cytotoxic T-lymphocyte (CTL) precursors in the periphery. Fourteen patients (seven with melanoma and seven with other types of cancer) positive for either HLA-A24 or HLA-A2 were enrolled in this study. Fourteen and 16 peptides were used to screen for HLA-A24+ and HLA-A2+ patients, respectively. The vaccination was well tolerated, and the only adverse effects were local pain and fever. Kinetic analysis revealed that peptide-reactive CTLs increased after peptide vaccination in 7 of 14 patients. Immunoglobulin G (IgG) reactive to the administered peptides was detected in 2 patients before vaccination, although it became detectable in 8 of the other 12 patients after the peptide vaccination. Stable disease for more than 6 months was observed in five patients (one with melanoma and four with other types of cancer); all of these patients showed increased levels of peptide-specific IgG. These results indicate that peptide vaccination of patients showing evidence of pre-existing peptide-specific CTL precursors can be applied in further clinical trials aimed at the treatment of melanoma and other types of cancer.

Identification of new prostate stem cell antigen-derived peptides immunogenic in HLA-A2 + patients with hormone-refractory prostate cancer

PurposeProstate cancer refractory to hormonal manipulation requires new treatment modalities. In the present study we attempted to identify prostate stem cell antigen (PSCA)-derived peptides immunogenic in HLA-A2+ prostate cancer patients in order to develop peptide-based immunotherapy against hormone-refractory prostate cancer (HRPC).MethodsEleven different PSCA-derived peptides, which were prepared based on the HLA-A2 binding motif, were examined to determine whether they would be recognized by cellular and humoral immune responses in 12 HLA-A2+ patients (11 with HRPC and 1 with non-HRPC).ResultsAmong the PSCA-derived peptides, PSCA 7–15 and PSCA 21–30 peptides effectively induced HLA-A2-restricted peptide-specific and tumor-reactive cytotoxic T lymphocytes (CTLs) from peripheral blood mononuclear cells (PBMCs) of HLA-A2+ patients. The PSCA 21–30 peptide was capable of inducing peptide-specific CTLs in both cancer patients and healthy donors, whereas the PSCA 7–15 peptide was immunogenic in only cancer patients. Immunoglobulin G (IgG) reactive to the PSCA 21–30 peptide was detected in plasma of most patients and healthy donors, whereas IgG reactive to PSCA 7–15 was undetectable in all cases. These results indicate that the former peptide elicits both cellular and humoral immune responses in both patients and healthy donors, whereas the latter elicits only cellular responses in patients.ConclusionThese two PSCA peptides should be considered for use in clinical trials of immunotherapy for HLA-A2+ HRPC patients.

Identification of a prostate-specific membrane antigen-derived peptide capable of eliciting both cellular and humoral immune responses in HLA-A24+ prostate cancer patients

We tried to identify prostate‐specific membrane antigen (PSMA)‐derived peptides capable of eliciting both cellular and humoral immune responses in peripheral blood mononuclear cells (PBMCs) and plasma of HLA‐A24+ prostate cancer patients, respectively. For cellular response, peptide‐specific and prostate cancer‐reactive responses of in vitro‐stimulated PBMCs were examined with regard to interferon (IFN)‐γ production and cytotoxicity against both a parental HLA‐A24− prostate cancer cell line (PC‐93) and an HLA‐A24‐expressing transfectant cell line (PC93‐A24). For humoral response, patients’ plasma was tested for reactivity to the peptides by means of an enzyme‐linked immunosorbent assay (ELISA). Among 13 PSMA peptides, PSMA 624–632 peptide induced peptide‐specific and tumor‐reactive cytotoxic T lymphocytes (CTLs) most effectively. The PSMA 624–632 peptide‐stimulated PBMCs from either healthy donors or prostate cancer patients produced a significant level of IFN‐γ in response to prostate cancer cells in an HLA‐A24‐restricted manner, and also showed a higher level of cytotoxicity against PC93‐A24 than against PC93. Antibodies to the PSMA 624–632 peptide, but not to any others, were detected in prostate cancer patients. These results demonstrate that the PSMA 624–632 peptide could be an appropriate molecule for use in specific immunotherapy of HLA‐A24+ patients with prostate cancer.

Identification of Cyclophilin B-derived Peptides Capable of Inducing Histocompatibility Leukocyte Antigen-A2-restricted and Tumor-specific Cytotoxic T Lymphocytes

We recently suggested that cyclophilin B (Cyp‐B) is a tumor antigen recognized by histocompatibility leukocyte antigen (HLA)‐A24‐restricted and tumor‐specific cytotoxic T lymphocytes (CTLs). In this study, we tried to identify Cyp‐B‐derived epitopes, which can induce HLA‐A2‐restricted and tumor‐specific CTLs in cancer patients. The tumor‐infiltrating lymphocytes (TILs) from an HLA‐A0207 patient with colon cancer were found to respond to COS‐7 cells when co‐transfected with the Cyp‐B gene and either HLA‐A0201, ‐A0206, or ‐A0207 cDNA. These TILs contained CTLs capable of recognizing either the Cyp‐B129–138 or the Cyp‐B172–179 peptide among 28 different peptides, all of which were prepared based on the HLA‐A2 binding motif. Both Cyp‐B peptides possessed the ability to induce tumor‐specific CTLs in HLA‐A2+ cancer patients. Cyp‐B172–180 (v), which is a 9‐mer peptide with valine added at the C terminus, showed no clear superiority over the parental Cyp‐B172‐179 peptide in an in vitro sensitization experiment. In vitro ‐sensitized T cells with these peptides responded to cancer cells in an HLA‐A2‐restricted manner. These two Cyp‐B peptides could be useful for specific immunotherapy of HLA‐A2+ cancer patients.

Target molecules in specific immunotherapy against prostate cancer

Recent advances in molecular biology and tumor immunology have allowed us to identify genes encoding human cancer-related antigens and their peptides that are recognized by cytotoxic T lymphocytes (CTLs). Although these advances have been preceded by studies on melanoma antigens, prostate cancer is another target candidate for specific immunotherapy. Several prostate tissue-specific antigens can be target molecules in specific immunotherapy for prostate cancer. The distribution of prostate tissue-specific antigens is more localized than that of melanoma-related antigens. Prostate-specific antigen (PSA) is available as an evaluation indicator of clinical course. In addition, epithelial cancer-related antigens are also applicable for prostate cancer patients. These lines of evidence suggest that prostate cancer is the best candidate for specific immunotherapy among the various types of epithelial cancers. A number of epitope peptides which have the potential to generate prostate cancer-reactive CTLs have been identified to date, and clinical trials targeting these molecules have been conducted. In this article, we review prostate cancer-related antigens and their epitope peptides, which have potential for use in the immunotherapy of prostate cancer patients, and we introduce the current status of clinical trials of specific immunotherapy targeting these molecules.

Melanoma-Reactive CD8+ T cells recognize a novel tumor antigen expressed in a wide variety of tumor types.

An autologous melanoma cell line selected for loss of expression of the immunodominant MART-1 and gp100 antigens was initially used to carry out a mixed lymphocyte tumor culture (MLTC) in a patient who expressed the human leukocyte antigen (HLA)-A1 and HLA-A2 class I major histocompatibility complex alleles. Ten clones identified from this MLTC seemed to recognize melanoma in an HLA-A1–restricted manner but failed to recognize a panel of previously described melanoma antigens. The screening of an autologous melanoma cDNA library with one HLA-A1–restricted melanoma-reactive T-cell clone resulted in the isolation of a cDNA clone called AIM-2 (antigen isolated from immunoselected melanoma-2). The AIM-2 transcript seemed to have retained an intronic sequence based on its alignment with genomic sequences as well as expressed sequence tags. This transcript was not readily detected after Northern blot analysis of melanoma mRNA, indicating that only low levels of this product may be expressed in tumor cells. Quantitative reverse transcriptase–polymerase chain reaction analysis, however, demonstrated a correlation between T-cell recognition and expression in HLA-A1–expressing tumor cell lines. A peptide that was encoded within a short open reading frame of 23 amino acids and conformed to the HLA-A1 binding motif RSDSGQQARY was found to represent the T-cell epitope. The AIM-2–reactive T-cell clone recognized a number of neuroectodermal tumors as well as breast, ovarian, and colon carcinomas that expressed HLA-A1, indicating that this represents a widely expressed tumor antigen. Thus, AIM-2 may represent a potential target for the development of vaccines in patients bearing tumors of a variety of histologies.

Detection of peptide-specific CTL-precursors in peripheral blood lymphocytes of cancer patients.

Development of therapeutic vaccines is one of the major areas of tumour immunotherapy today. However, clinical trials of peptide-based cancer vaccines have rarely resulted in tumour regression. This failure might be due to an insufficient induction of cytotoxic T lymphocytes in the current regimes, in which cytotoxic T lymphocytes-precursors in pre-vaccination peripheral blood mononuclear cells are not measured. Initiation of immune-boosting through vaccination could be better than that of immune-priming with regard to induction of prompt and strong immunity. If this is also the case for therapeutic vaccines, pre-vaccination measurement of peptide-specific cytotoxic T lymphocytes-precursors will be important. In the present study, we investigated whether cytotoxic T lymphocytes-precursors reacting to 28 kinds of peptides of vaccine candidates (13 and 15 peptides for HLA-A24+ and HLA-A2+ patients, respectively) were detectable in pre-vaccination peripheral blood mononuclear cells of 80 cancer patients. Peptide-specific cytotoxic T lymphocytes-precursors were found to be detectable in peripheral blood mononuclear cells of the majority of cancer patients (57 out of 80 cases, 71%). The mean numbers of positive peptides were 2.0 peptides per positive case. Peripheral blood mononuclear cells incubated with positive peptides, not with negative peptides, showed significant levels of HLA-class-I-restricted cytotoxicity to cancer cells. The profiles of positive peptides entirely varied among patients, and were not influenced by the cancer origin. These results may provide a scientific basis for the development of a new approach to cancer immunotherapy, e.g.) cytotoxic T lymphocytes-precursor-oriented peptide vaccine.

In Vivo Evidence That Peptide Vaccination Can Induce HLA-DR-Restricted CD4+ T Cells Reactive to a Class I Tumor Peptide

Vaccination with class I tumor peptides has been performed to induce tumor-reactive CD8+ T cells in vivo. However, the kinds of immune responses that vaccination might elicit in patients are not fully understood. In this study we tried to elucidate the mechanisms by which vaccination of class I binding tumor peptides into an HLA-A2+ lung cancer patient elicited dramatic amounts of IgG1 and IgG2 specific to a nonamer peptide, ubiquitin-conjugated enzyme variant Kua (UBE2V)43–51. The UBE2V43–51 peptide contains cysteine at the sixth position. HLA-DR-restricted and UBE2V43–51 peptide-recognizing CD4+ T cells were induced from postvaccination, but not from prevaccination, PBMCs of the cancer patient. In addition, a CD4+ T cell line (UB-2) and its clone (UB-2.3), both of which recognize the UBE2V43–51 peptide in the context of HLA-DRB1*0403 molecules, were successfully established from postvaccination PBMCs. The peptide vaccination increased the frequency of peptide-specific T cells, especially CD4+ T cells. In contrast, mass spectrometric analysis revealed that the vaccinated UBE2V43–51 peptide contained both monomeric and dimeric forms. Both forms, fractionated by reverse phase HPLC, were recognized by UB-2 and UB-2.3 cells. Recognition by these CD4+ T cells was observed despite the addition of a reduction reagent or the fixation of APC. Overall, these results indicate that vaccination with class I tumor peptides can induce HLA-DR-restricted CD4+ T cells in vivo and elicit humoral immune responses, and that a cysteine-containing peptide can be recognized by CD4+ T cells not only as a monomer, but also as a dimer.

Elevation of serum MAGE-4 protein levels and prediction of hepatocellular carcinogenesis in patients with liver cirrhosis.

Early detection of hepatocellular carcinoma (HCC) is clinically important because advanced HCC limits treatment modalities for the cancer. We have previously reported that serum levels of MAGE‐4 protein are strongly associated with the development of HCC. The present study was designed to determine whether elevated serum MAGE‐4 protein levels can predict hepatocellular carcinogenesis in patients with liver cirrhosis before clinical diagnosis. Among 62 cirrhotic patients, 28 patients were diagnosed with HCC during the follow‐up period. The levels of MAGE‐4 protein and a‐fetoprotein (AFP) were significantly elevated in cirrhotic patients with HCC. Univariate and multivariate analyses suggest that elevated serum MAGE‐4 protein is more significant than AFP. Importantly, retrospective analysis of prefrozen sera of cirrhotic patients revealed a transient or continuous elevation of serum MAGE‐4 protein levels in 14 of 28 cirrhotic patients with HCC (50%) before clinical diagnosis. In contrast, elevated serum MAGE‐4 protein levels were observed in 3 of 33 cirrhotic patients without HCC (9%), and in 2 of 34 hepatitic patients (6%). These results indicate that elevated serum MAGE‐4 protein levels can be a predictive marker of hepatocellular carcinogenesis in cirrhotic patients, thereby enabling us to treat patients at an earlier stage.