Assessment of synthetic peptides of severe acute respiratory syndrome coronavirus recognized by long-lasting immunity.

In order to determine highly immunogenic severe acute respiratory syndrome coronavirus (SARS-CoV) epitope peptides capable of inducing long-lasting immunity, we first screened immunoglobulin-G (IgG) antibodies reactive to 197 different overlapping 15-mers from the SARS-CoV proteins in the sera of three infected patients. Forty-two peptides among them were reactive to the sera from all three patients. Consequently, we tested for the reactivity of these 42 peptides to patients' sera (n = 45) at 6-month post-infection. The significantly higher levels of IgG antibodies specific to three (S791, M207 and N161) of 42 peptides were detectable in the post-infection sera from 23 (51%), 27 (60%) and 19 (42%) of 45 patients, respectively. These three peptides, recognized by their long-lasting immunity, may provide a better understanding of the immunogenicity of SARS-CoV.

New multiplexed flow cytometric assay to measure anti-peptide antibody: a novel tool for monitoring immune responses to peptides used for immunization.

Peptide-based vaccination has a great potential for the prevention and treatment of various diseases. There is, however, no appropriate monitoring system to measure immune responses to vaccinated peptides, which hampers the development of therapeutically effective vaccine regimens to various diseases. In this study a new multiplexed flow cytometric assay using the Luminex system to monitor humoral immune responses to vaccinated peptides is described. Although the sensitivity is mostly equal to that of the traditional enzyme-linked immunosorbent assay (ELISA), the new assay has several advantages over ELISA in that it minimizes the amount of sera needed, running costs and working periods, and thus will be a novel tool for monitoring immune responses to vaccinated peptides.

Identification of epidermal growth factor receptor-derived peptides immunogenic for HLA-A2(+) cancer patients.

Epidermal growth factor receptor (EGFR) is one of the most appropriate target molecules for cancer therapy because of its relatively high expression in about one-third of all epithelial cancers in correlation with neoplasmic progression. With respect to EGFR-targeted therapies, antibodies and tyrosine-kinase inhibitors have been intensively studied, a novel EGFR-tyrosine-kinase inhibitor ZD1839 has been approved as an anticancer drug, and many other agents are now under clinical trial. In addition, cytotoxic T lymphocyte (CTL)-directed epitope peptides could be another class of compounds useful in EGFR-targeted therapies. However, there is presently no information on CTL-directed peptides of EGFR. Therefore, from the viewpoint of development of peptide-based cancer therapy, this study was intended to determine the EGFR-derived peptides recognised by both cellular and humoral immunities in HLA-A2(+) epithelial cancer patients. We herein report finding of two such types of EGFR-derived peptides at position 479-488 and 1138-1147, both of which were recognised by the majority of patients' sera (IgG), and also possessed the ability to induce HLA-A2-restricted peptide-specific CTLs against EGFR-positive tumour cells in peripheral blood mononuclear cells (PBMCs) of epithelial cancer patients. These results may provide a scientific basis for the development of EGFR-based immunotherapy for HLA-A2(+) cancer patients.

Analysis of cellular localization of SART3 tumor antigen by a newly established monoclonal antibody: heterotopic expression of SART3 on the surface of B-lineage leukemic cells.

SART3 has been identified as a tumor antigen recognized by cytotoxic T-lymphocytes. Subcellular localization of SART3 is still controversial, although it has been studied extensively. In this study, we established an anti-SART3 monoclonal antibody (mAb) to analyze the subcellular localization of SART3 in tumor specimens. Immunohistochemical analysis demonstrated that SART3 was preferentially expressed in the nucleus of cancer cells. Flow cytometric analysis indicated that SART3 was not expressed on the cell surface of tumor cells, except for B-lineage cells. SART3 was detected on the cell surface of several B-lineage leukemic cells with different maturation stages, but not on normal peripheral blood B cells. These results suggest that heterotopically expressed SART3 is a tumor-associated antigen rather than a differentiation antigen on B-lineage cells.

Heat shock cognate protein 70 encodes antigenic epitopes recognised by HLA-B4601-restricted cytotoxic T lymphocytes from cancer patients.

Heat shock cognate protein 70 (HSC70), a highly conserved protein and a member of the family of molecular chaperones, has the ability to induce cytotoxic T lymphocyte (CTL) responses through binding and carrying antigenic peptides. We demonstrated in this study that the HSC70 gene encodes two antigenic peptides recognised by HLA-B46-restricted and tumour-reactive CTLs established from tumour-infiltrating lymphocytes of a colon cancer. These HSC70-derived peptides, at amino-acid positions 106-114 and 233-241, had the ability to induce HLA-B46-restricted and peptide-specific CTLs, which are reactive to tumour cells, from peripheral blood mononuclear cells of the majority of epithelial cancer patients tested. These results, along with those from the previous studies, indicate the two ways of HSC70 involvement in the immune responses to tumours: chaperones and antigens, and thus may provide a new insight for the development of HSC70-directed cancer-specific immunotherapy.

IgG reactive to CTL-directed epitopes of self-antigens is either lacking or unbalanced in atopic dermatitis patients.

We previously demonstrated that CTL-directed epitopes derived from non-mutated self-antigens elicit a type-I allergy in the majority of healthy donors (HD) as did the presence of IgE and IgG reactive to these peptides in the sera of the donors. We investigated in this study whether Igs reactive to eight types of CTL-directed peptides were elevated in the sera of 40 patients with atopic dermatitis (AD). Total IgE levels in the sera of AD patients were significantly higher than those of HD, however, no significant differences between the AD patients and the HD were observed in either the serum levels or the positive rates of IgE reactive to seven of the eight peptides. Total IgG levels were not different from each other, however, IgG reactive to the two peptides with no sequence similarity to other species and one peptide that had similarity to DNA helicase II of enterobacteria were not detectable in the sera of the AD patients. Although IgG reactive to the remaining five peptides, which had sequence similarity to other species, were detectable in both the AD patients and the HD, ratios of peptide-specific IgG1/IgG2 were mostly lower in the AD patients than in the HD. These results indicate that IgG reactive to CTL-directed epitopes of self-antigens is either lacking or unbalanced in AD patients. This information may provide new insight into the immune-mechanisms of elevated auto-reactivity of AD patients.

Identification of ribosomal proteins S2 and L10a as tumor antigens recognized by HLA-A26-restricted CTL.

Recent identification of cytotoxic T lymphocyte (CTL)-directed peptides binding to the HLA-A2 and -A24 alleles has opened the door to peptide-based cancer immunotherapies. However, subsequent studies have succeeded in identifying no more than a few CTL-directed peptides that bind to alleles other than HLA-A2 and -A24, thus hampering development of immunotherapies directed at other alleles. We have shown in this study that two genes coding for ribosomal proteins (S2 and L10a) encoded tumor antigens recognized by HLA-A26-restricted CTLs. The S2 mRNA was expressed in all of the cancer cells and non-malignant cell lines tested, but was not expressed in normal tissues except for the testis, muscle, and peripheral mononuclear leukocyte cell (PBMC). In contrast, the L10a mRNA was expressed in all of these cancer and non-malignant cell lines, and also normal tissues, although the expression levels in normal tissues were mostly low. One S2-derived peptide and two L10a-derived peptides had the ability to induce HLA-A26-restricted and peptide-specific CTLs reactive to tumor cells in PBMCs of cancer patients, respectively. These ribosomal protein-derived peptides, and particularly the S2-derived peptide, could be suitable for use in peptide-based immunotherapy for HLA-A26+ cancer patients.

Recognition of ADP-ribosylation factor 4-like by HLA-A2-restricted and tumor-reactive cytotoxic T lymphocytes from patients with brain tumors.

Although specific immunotherapy is one candidate treatment of brain tumor, the molecular basis of T-cell-mediated recognition of brain tumors has not yet been elucidated. In this study, we tried to identify brain tumor antigens using HLA-A2-restricted and tumor-reactive cytotoxic T lymphocytes (CTLs). As an HLA-A2-restricted OK-CTL line contained CTLs capable of responding to HLA-A2+ malignant glioma cells, this cell line was used for identification of brain tumor antigens. After screening a cDNA library from brain tumor cells, this CTL line was found to produce interferon (IFN)-gamma when cultured with COS-7 cells, which were cotransfected with both a cDNA clone (clone 1) and HLA-A0207 cDNA. Data base searches indicated that the clone 1 was 98% identical to that of the human ADP-ribosylation factor 4-like (ARF4L). Two peptides, ARF4L 15-24 and ARF4L 69-77, possessed the ability to induce HLA-A2-restricted and tumor-reactive CTLs from peripheral blood mononuclear cells of patients with brain tumors. Although ARF4L seemed to be ubiquitously expressed at the mRNA level, ARF4L-reactive CTLs failed to exhibit cytotoxicity against normal lymphoid blasts. These results indicate that these two ARF4L peptides could be targets for immunotherapy of HLA-A2+ patients with brain tumors.

UDP-Gal: betaGlcNAc beta1, 3-galactosyltransferase, polypeptide 3 (GALT3) is a tumour antigen recognised by HLA-A2-restricted cytotoxic T lymphocytes from patients with brain tumour.

Patient prognosis in the case of malignant brain tumours is generally poor, despite significant improvements in the early detection of the tumours, and thus the development of new treatment modalities is needed. One of the most prominent modalities is specific immunotherapy, for which the elucidation of antigenic molecules of malignant brain tumours recognized by T cells is essential. We report here a gene, UDP-Gal: betaGlcNAc beta1, 3-galactosyltransferase, polypeptide 3, encoding three epitope peptides recognised by tumor-reactive cytotoxic T lymphocytes in an HLA-A2-restricted manner. Two of the three peptides possessed an ability to induce HLA-A2-restricted and tumour-reactive cytotoxic T lymphocytes from peripheral blood mononuclear cells of patients with brain tumours. These peptides may be useful in the peptide-based specific immunotherapy for patients with malignant brain tumours.

Non-mutated tumor-rejection antigen peptides elicit type-I allergy in the majority of healthy individuals.

IgE-mediated type-I allergy is generally considered to be a hypersensitivity reaction to foreign antigens, and it is believed that self-antigens do not evoke this type of allergy. We report here, for the first time, that non-mutated self-antigen peptides identified as tumor-rejection antigen peptides recognized by HLA class I-restricted and tumor-specific cytotoxic T lymphocytes (CTLs) elicited a type-I allergy in the majority of healthy individuals. Peptide-specific IgE was detectable in sera from certain cases, although the levels did not always correlate with those of type-I allergy. Repeated vaccinations of nonallergic peptides derived from the same antigens possessing allergic peptides resulted in the suppression of both allergic peptide-specific IgE responses and type-I allergy, providing evidence for a new approach to the development of peptide-based desensitization.

Binding of a SART3 tumor-rejection antigen to a pre-mRNA splicing factor RNPS1: a possible regulation of splicing by a complex formation.

We recently reported the identification of a human SART3 gene that encodes a tumor-rejection antigen recognized by cytotoxic T lymphocytes (CTLs). The squamous-cell carcinoma antigen recognized by T cells-3 (SART3) is an RNA-binding protein expressed in the nucleus of the majority of proliferating cells, including normal cells and malignant cells, but not in normal tissues except for the testes and fetal liver. To determine its biologic function, we employed a 2-hybrid screening in yeast for proteins interacting with SART3, and this method yielded a pre-mRNA splicing factor (RNA-binding protein prevalent during the S phase or RNA-binding protein with a serine-rich domain [RNPS1]) that activated both constitutive and alternative splicing of pre-mRNA in vitro. Interaction of SART3 with RNPS1 through the physical association of N-terminal domains of RNPS1 was confirmed by both in vitro pull-down assay and immunoprecipitation assay. Cotransfection of the 2 genes changed the distribution pattern of SART3 from diffuse nucleoplasmic spreading to nuclear speckled regions in which the RNPS1 was colocalized, suggesting a complex formation of the 2 proteins. In cooperation with RNPS1, SART3 stimulated the proximal alternative 3' splicing of a calcitonin-dihydrofolate reductase chimeric minigene pre-mRNA. These results suggest that SART3 is involved in the regulation of mRNA splicing probably via its complex formation with RNPS1.

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-B(129 - 138) or the Cyp-B(172 - 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-B(172 - 180 (V)), which is a 9-mer peptide with valine added at the C terminus, showed no clear superiority over the parental Cyp-B(172 - 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.

Expression of SART3 antigen and induction of CTLs by SART3-derived peptides in breast cancer patients.

We recently reported the SART3 tumour-rejection antigen as possessing tumour epitopes capable of inducing HLA-class I-restricted cytotoxic T lymphocytes (CTLs). This study investigated expression of the SART3 antigen in breast cancer to explore an appropriate molecule for use in specific immunotherapy of breast cancer patients. The SART3 antigen was detected in all of the breast cancer cell lines tested, 30 of 40 (75%) breast cancer tissue samples, and 0 of 3 non-tumourous breast tissue samples. SART3 derived peptides at positions 109-118 and 315-323 induced HLA-A24 restricted CTLs that reacted to breast cancer cells from the peripheral blood mononuclear cells (PBMCs) of breast cancer patients. Therefore, the SART3 antigen and its peptides could be an appropriate molecule for use in specific immunotherapy of the majority of HLA-A24-positive breast cancer patients.

Molecular basis of T cell-mediated recognition of pancreatic cancer cells.

Pancreatic cancer continues to be a major unsolved health problem in the world. The prognosis of pancreatic cancer is extremely poor with a median survival of 3-4 months and the 5-year survival being 1-4%. This poor prognosis is primarily because of a lack of effective therapies, and thus development of new treatment modalities is needed. One of these treatments could involve specific immunotherapy, for which elucidation off the molecular basis of T cell-mediated recognition of cancer cells is required. We report here six different genes and 19 immunogenic epitopes from pancreatic adenocarcinoma cells and T-cell receptor beta usage of HLA-A2-restricted CTL clones reacting to some of these epitopes. Sixteen of 19 epitopes were found to possess the ability to induce HLA-A2-restricted CTL activity in the peripheral blood lymphocytes of patients with pancreatic and also colon adenocarcinomas. These results should provide a scientific basis for the development of specific immunotherapy for pancreatic and colon cancer patients.

Expression of the SART1 tumor-rejection antigen in hepatocellular carcinomas.

We previously reported a tumor-rejection antigen, SART1259, possessing tumor epitopes capable of inducing cytotoxic T lymphocytes (CTL) in epithelial cancer patients. The present study investigated the expression of the SART1259 antigen in hepatocellular carcinomas (HCC) in order to explore for a potential molecule for use in specific immunotherapy of HCC patients. Expression of the SART1 antigens in samples was analyzed by western blot analysis with anti-SART1259 and anti-SART1800 polyclonal antibodies. In addition HLA-A24- restricted CTLs were induced from peripheral blood mononuclear cells (PBMCs) of HLA-A24+ HCC patients by the SART1690-698 (EYRGFTQDF) peptide with an HLA-A24 binding motif. The SART1259 antigen was detected in the cytoplasmic fraction of 6 of 8 HCC cell lines and 12 of 23 (52%) HCC tissues, but in none of the normal liver tissues or those of chronic hepatitis or cirrhosis. The HLA-A24 restricted and SART1-specific CTLs recognized the HLA-A24+ and SART1259+ HCC cells. Further, in peripheral blood mononuclear cells of HCC patients, the SART1690-698 peptide induced CTLs that reacted to the HCC cells in an HLA-A24-restricted manner. These results suggest that the SART1259 antigen could be an appropriate target molecule for use in specific immunotherapy of HCC patients.

Recognition of the Lck tyrosine kinase as a tumor antigen by cytotoxic T lymphocytes of cancer patients with distant metastases.

The Lck protein (p56(lck)), a src family tyrosine kinase that is essential for T cell development and function, is aberrantly expressed in metastatic colon cancers. p56(lck) seems to facilitate the malignant transformation of epithelial cells through initiation of anchorage-independent proliferation. We demonstrate that the lck gene encodes antigenic epitopes recognized by the HLA class I-restricted and tumor-specific CTL of metastatic cancer patients. Lck peptides augmented CTL activity in peripheral blood mononuclear cells (PBMC) of colon and other epithelial cancer patients with distant metastases, but not those without distant metastases. CTL precursors recognizing the Lck peptide were identified in freshly prepared PBMC of patients with distant metastases, and their frequency was significantly augmented by stimulation with the peptide. Thus, Lck peptides could be useful in developing a specific immunotherapy for cancer patients with distant metastases.

Expression of tumor-rejection antigens in gynecologic cancers.

We recently reported the four tumor-rejection antigens (SART1(259), SART2, SART3, and ART4) that possess tumor epitopes capable of inducing HLA-A2402-restricted cytotoxic T lymphocytes (CTLs) in cancer patients. This study investigated the expression of these tumor antigens in gynecologic cancers, including 33 ovarian cancers, 38 cervical cancers, and 40 endometrial cancers. SART1(259) antigen was detected in 56%, 35%, and 30% of ovarian, cervical and endometrial cancers, while SART2 antigen was detected in 46%, 66%, and 30% of these cancers, respectively. Both SART3 and ART4 antigens were detectable in the majority of these gynecologic cancers tested. In contrast, none of these antigens was detectable in any of the normal ovarian and uterine tissues tested. Peripheral blood mononuclear cells (PBMCs) of HLA-A24(+) patients with gynecologic cancers were found to produce significant levels of interferon-gamma in response to HLA-A24(+) SART3(+) gynecologic cancer cells after having been stimulated three times in vitro with either SART3(109 - 118) or SART3(315 - 323) peptide. These PBMCs lysed HLA-A24(+) SART3(+) gynecologic cancer cells, but not HLA-A24(-) SART3(+) gynecologic cancer cells or HLA-A24(+) normal cells. Therefore, these four antigens and their peptides, including SART3 peptides, would be appropriate molecules for use in specific immunotherapy of HLA-A24(+) gynecologic cancer patients.

Identification of SART3-derived peptides capable of inducing HLA-A2-restricted and tumor-specific CTLs in cancer patients with different HLA-A2 subtypes.

We recently identified the SART3 antigen encoding shared tumor epitopes recognized by HLA-A2402-restricted and tumor-specific CTLs. Our study investigated whether the SART3 antigen encodes peptides recognized by the HLA-A2-restricted CTLs. The HLA-A2-restricted and tumor-specific CTL line recognized COS-7 cells co-transfected with the SART3 gene and either HLA-A0201, -A0206 or -A0207 cDNA but not those co-transfected with the SART3 gene and HLA-A2402 or -A2601 cDNA. The 2 SART3 peptides at positions 302 to 310 and 309 to 317 possessed the ability to induce HLA-A2-restricted and tumor-specific CTLs from peripheral blood mononuclear cells of cancer patients with various histological types and different HLA-A2 subtypes. Therefore, these 2 peptides could be useful for specific immunotherapy of a relatively large number of HLA-A2(+) cancer patients.

[Human tumor-rejection antigens and peptides from genes to clinical research].

One of the most significant advances in the field of modern tumor immunology is the identification of genes encoding tumor-rejection antigens that are recognized by human leukocyte antigen (HLA) class I-restricted and tumor-specific cytotoxic T lymphocytes (CTLs). Several peptides encoded by these genes are now under clinical trial as cancer vaccines, and major tumor regression has been observed in some melanoma patients. These results indicate that identification of the peptides capable of inducing CTLs may provide a new modality of cancer therapy. We investigated tumor-rejection antigens from epithelial cancers, and reported 7 genes encoding tumor-rejection antigens and peptides available for specific immunotherapy of HLA-A26 or -A24 patients with epithelial cancers. Furthermore, we identified more than 10 genes encoding tumor-rejection antigens and peptides available for specific immunotherapy of HLA-A2 patients with epithelial cancers. Therefore these new antigens and peptides could be applicable to the treatment of numerous epithelial cancer patients worldwide. Phase I clinical trials of cancer vaccine with these peptides for epithelial cancer patients are in progress at our university. Basic and clinical research will provide new insights for a better understanding of the molecular basis of T cell-mediated recognition of cancer cells and be important for the development of cancer vaccines.

A new tumor-rejection antigen recognized by cytotoxic T lymphocytes infiltrating into a lung adenocarcinoma.

Lung cancer is the most commonly occurring malignancy worldwide and one of the few that continues to show an increasing incidence. To understand the molecular basis of host immunity against lung cancer, we investigated tumor antigens recognized by HLA-A24-restricted CTLs established from T cells infiltrating into lung adenocarcinoma and report a new gene coding for antigens recognized by the CTLs. The mRNA of this gene was expressed at different levels in all of the malignant cells tested (high in adenocarcinomas and gliomas and low in esophageal cancers and malignant hematological disease). It was also expressed at the different levels in each of a panel of normal tissues (high in the thymus, low in peripheral blood mononuclear cells, and lowest in the stomach, small intestine, and skeletal muscle). This gene encodes a Mr 60,000 nuclear protein with 414 deduced amino acids. The three peptides at positions 158-165, 170-179, and 188-196 were recognized by the CTLs. One peptide at position 188-196 had the ability to induce HLA-A24-restricted and tumor-specific CTLs in peripheral blood mononuclear cells of lung cancer patients. These CTLs, however, did not lyse HLA-A24+ PHA-activated T cells in which the mRNA of this gene was highly expressed, even in the presence of excess amounts of a corresponding peptide in culture. These results suggest that this gene product and peptide could be applicable to specific immunotherapy of lung cancer patients.