Hideo Takasu

A simple culture protocol to detect peptide-specific cytotoxic T lymphocyte precursors in the circulation.

Abstract. The detection and monitoring of peptide-specific cytotoxic T lymphocyte (CTL) precursors is essential for successful peptide-based immunotherapy against cancers. In contrast to the development of effective methods of detecting antigen-specific CTL, such as ELISpot and HLA-class I tetramer assay, stimulation with peptide-pulsed antigen-presenting cells (APC) has for some time been conventionally employed to induce peptide-specific CTL from peripheral blood mononuclear cells (PBMC). This culture protocol, however, needs a substantial number of PBMC to test the reactivity against a panel of peptides. In the present study, we established a simple culture protocol which has no need of additional APC. Addition of a corresponding peptide every 3 days was found to induce not only Epstein–Barr virus (EBV)-specific CTL from healthy donors, but also tumor antigen-derived peptide-specific CTL from cancer patients. A 10-ml blood sample was almost sufficient to test the presence of CTL precursors against 20 different peptides in triplicate assays. Overall, this culture protocol can be useful in detecting and monitoring peptide-specific CTL precursors in the circulation in peptide-based immunotherapy against cancer.

Cep55/c10orf3, a tumor antigen derived from a centrosome residing protein in breast carcinoma.

Identification of tumor-associated antigens may facilitate vaccination strategies to treat patients with malignant diseases. We have found that the centrosomal protein, Cep55/c10orf3 acts as a novel breast carcinoma-associated tumor-associated antigen. Cep55/c10orf3 mRNA was detectable in a wide variety of tumor cell lines. Expression was barely detectable in normal tissues except for testis and thymus. Moreover, Cep55/c10orf3 protein could be detected by a monoclonal anti-Cep55/c10orf3 antibody (♯11-55) in 69.8% of breast carcinoma, 25% of colorectal carcinoma, and 57.8% of lung carcinoma tissues. The expression of Cep55/c10orf3 protein did not show any relationship with the hormone receptors such as estrogen receptor and progesterone receptor or expression patterns of p185HER2/neu. We designed 11 peptides which displayed a human leukocyte antigen-A24 binding motif. One Cep55/c10orf3-peptide, Cep55/c10orf3_193(10) (VYVKGLLAKI), induced cytotoxic T lymphocytes (CTLs) in 3 of 3 patients with Cep55/c10orf3 (♯11-55)-positive breast carcinoma. A Cep55/c10orf3_193(10)-specific CTL clone could also recognize Cep55/c10orf3 (+) displayed on human leukocyte antigen-A24 (+) cancer cell lines. These data indicate that Cep55/c10orf3 peptides were naturally presented by breast cancer cells and can cause CTL clonal expansion in vivo. Monoclonal antibody ♯11-55 and the Cep55/c10orf3_193(10) peptides may be useful as part of a therapeutic strategy for hormonal therapy or anti-p185HER2/neu monoclonal antibody therapy-resistant breast carcinoma patients.

Development of HLA-A2402/Kb transgenic mice

HLA‐transgenic mice have been developed to facilitate studies of HLA‐restricted cytotoxic responses, e.g., for the identification of immunodominant HLA‐restricted CTL epitopes and the optimization of peptide or DNA vaccine constructs for human use. We have developed HLA‐A2402/Kb‐transgenic mice expressing chimeric human (α1 and α2 domains of HLA‐A2402) and mouse (α3, transmembrane and cytoplasmic domains of H‐2Kb) class I molecules. Immunization of these HLA‐A2402/Kb‐transgenic mice with various known HLA‐A24‐restricted immunodominant cancer CTL epitope peptides derived from gp100, MAGE‐1, MAGE‐3, Her2/neu, CEA and TERT induced HLA‐A24‐restricted, peptide‐specific CTLs. Using these transgenic mice, we identified a novel HLA‐A24‐restricted CTL epitope, PSA152–160, encoded by human prostate‐specific antigen. Staining with HLA tetramers showed that the cytotoxic activity induced by immunizing with PSA152–160 in HLA‐A2402/Kb transgenic mice was HLA‐A2402‐restricted and CD8‐dependent. Therefore, PSA152–160 might be a candidate peptide for vaccination of HLA‐A24+ patients with prostate cancer. Our results suggest that HLA‐A2402/Kb transgenic mice might be useful in the search for HLA‐A24‐restricted CTL epitopes functioning as human cancer antigens and for the development of peptide‐based cancer immunotherapy.

Novel spliced form of a lens protein as a novel lung cancer antigen, Lengsin splicing variant 4.

A glutamine synthetase I family protein, Lengsin, was previously identified as a novel lens‐specific transcript in the vertebrate eye. In this report, we show for the first time that Lengsin is a novel tumor‐associated antigen expressed ectopically in lung cancer. Interestingly, a novel spliced form of human Lengsin termed ‘splicing variant 4’, gaining exon 3 that codes extra 63 amino acids, is the dominant transcript form in lung cancer cells. Lengsin mRNA could be detected in 7 of 12 (58%) lung cancer cell lines and 7 of 7 (100%) surgically resected lung cancer tissues. On the other hand, Lengsin transcripts could not be detected in normal major tissues or in other cancer cell lines, including melanoma, colorectal carcinoma, breast carcinoma and hepatocellular carcinoma. In addition, knockdown of Lengsin mRNA with RNAi caused cell death and a decrease of cell viability, suggesting that Lengsin has some essential role in cell survival. Since the lens is an immune‐privileged site, we regard Lengsin as a highly immunogenic cancer antigen. Anti‐Lengsin autoantibodies were detectable in sera of lung cancer patients, although these patients did not show any lens‐related disturbances. Hence, Lengsin splicing variant 4 might be an immunogenic lung cancer‐specific antigen that is suitable as a diagnostic marker and for molecular targeting therapy, including immunotherapy. (Cancer Sci 2009)

Identification of an Immunogenic CTL Epitope of HIFPH3 for Immunotherapy of Renal Cell Carcinoma

Purpose: CD8+ CTLs have an essential role in immune response against tumor. Although tumor-associated antigens have been identified in renal cell carcinoma (RCC), few of these are commonly shared and investigated as therapeutic targets in the clinical medicine. In this report, we show that HIFPH3, a member of prolyl hydroxylases that function as oxygen sensor, is a novel tumor antigen and HIFPH3-specific CTLs are induced from peripheral blood lymphocytes of RCC patients. Experimental Design: Expression of HIFPH3 was examined by reverse transcription-PCR and immunostaining with anti-HIFPH3 antibody. To identify HLA-A24-restricted T-cell epitopes of HIFPH3, eight peptides were selected from the amino acid sequence of this protein and screened for their binding affinity to HLA-A24. Peptide-specific CTLs were induced by stimulating peripheral blood lymphocytes of HLA-A24-positive RCC patients with these peptides in vitro. HLA-A24-restricted cytotoxicity of the CTLs against HIFPH3+ RCC lines was assessed by chromium release assay. Results: HIFPH3 was overexpressed in many RCC cell lines and primary RCC tissues, whereas it was not detectable in normal adult tissues by reverse transcription-PCR. Of the eight peptides that contained HLA-A24-binding motif, HIFPH3-8 peptide (amino acid sequence, RYAMTVWYF) could induce the peptide-specific CTLs from 3 of 6 patients with HIFPH3-positive RCC. Furthermore, HIFPH3-8 peptide-specific CTLs showed cytotoxicity against HIFPH3+ RCC cell lines in a HLA-A24-restricted manner. Conclusions: HIFPH3 may be a target antigen in immunotherapy for RCC and HIFPH3-8 peptide could be used as a peptide vaccine for HLA-A*2402+/HIFPH3+ RCC patients.

Identification of novel targets for antiangiogenic therapy by comparing the gene expressions of tumor and normal endothelial cells

Targeting tumor angiogenesis is an established strategy for cancer therapy. Because angiogenesis is not limited to pathological conditions such as cancer, molecular markers that can distinguish between physiological and pathological angiogenesis are required to develop more effective and safer approaches for cancer treatment. To identify such molecules, we determined the gene expression profiles of murine tumor endothelial cells (mTEC) and murine normal endothelial cells using DNA microarray analysis followed by quantitative reverse transcription–polymerase chain reaction analysis. We identified 131 genes that were differentially upregulated in mTEC. Functional analysis using siRNA‐mediated gene silencing revealed five novel tumor endothelial cell markers that were involved in the proliferation or migration of mTEC. The expression of DEF6 and TMEM176B was upregulated in tumor vessels of human renal cell carcinoma specimens, suggesting that they are potential targets for antiangiogenic intervention for renal cell carcinoma. Comparative gene expression analysis revealed molecular differences between tumor endothelial cells and normal endothelial cells and identified novel tumor endothelial cell markers that may be exploited to target tumor angiogenesis for cancer treatment.

Mouse homologue of the human SART3 gene encoding tumor-rejection antigen

We recently isolated a human SART3 (hSART3) gene encoding a tumor‐rejection antigen recognized by HLA‐A2402‐restricted cytotoxic T lymphocytes (CTLs). The hSART3 was also found to exist as an RNA‐binding nuclear protein of unknown biological function. In this study, we cloned and analyzed the homologous mouse SART3 (mSART3) gene in order to understand better the function of hSART3, and to aid in establishing animal models of specific immunotherapy. The cloned 3586‐bp cDNA encoded a 962‐amino acid polypeptide with high homology to hSART3 (80% or 86% identity at the nucleotide or protein level, respectively). Nonapeptides recognized by the HLA‐A2402‐restricted CTLs and all of the RNA‐binding motifs were conserved between hSART3 and mSART3. The mSART3 mRNA was ubiquitously expressed in normal tissues, with low level expression in the liver, heart, and skeletal muscle. It was widely expressed in various organs from as early as day 7 of gestation. mSART3 was mapped to chromosome 5, a syntenic region for human chromosome 12q23–24, and its genomic DNA extended over 28‐kb and consisted of 19 exons. This information should be important for studies of the biological functions of the SART3 protein and for the establishment of animal models of specific cancer immunotherapy.

Sequence Analysis of Genes Encoding Rodent Homologues of the Human Tumor‐rejection Antigen SART‐1

Human SART‐1 (hSART‐1) gene encodes a 125 kD protein with a leucine‐zipper motif expressed in the nucleus of all proliferating cells, and a 43 kD protein expressed in the cytosol of most epithelial cancers. In this study, two rodent genes (rSART‐1 and mSART‐1) homologous to hSART‐1 were cloned from cDNA libraries of murine brain and a rat tumor cell line, respectively. mSART‐1 and rSART‐1 were highly homologous to hSART‐1 with 86% and 84% identity at the nucleotide level, and 95% and 91% at the protein level, respectively. The leucine zipper domain and two basic amino acid portions that bind DNA, as well as peptide sequences recognized by human cyto‐toxic T lymphocytes (CTLs), were all conserved in these rodent genes. Nuclear protein homologous to the 125 kD hSART‐1800 protein, but not to the 43 kD cytosol SART‐1259 protein, was detectable with specific antibody in the nuclear fractions of rodent tumor cell lines, and normal rodent fetal liver and testis. These rodent genes should be a novel tool for studies on the biological roles of the SART‐1 gene, and also in the construction of animal models of specific immuno‐therapy using SART‐1 gene products.