Yuki Nabeta

Prognostic significance of HLA class I expression in osteosarcoma defined by anti‐pan HLA class I monoclonal antibody, EMR8‐5

With the goal of establishing efficacious peptide‐based immunotherapy for patients with bone and soft tissue sarcomas, we previously identified the cytotoxic T lymphocyte‐defined osteosarcoma antigenic gene Papillomavirus binding factor. The present study was designed to determine the status of HLA class I expression in osteosarcoma and other bone and soft tissue sarcomas. Seventy‐four formalin‐fixed paraffin‐embedded specimens of various bone and soft tissue sarcomas, including 33 osteosarcomas, were stained with the anti‐HLA class I monoclonal antibody EMR8‐5, which we recently generated. The expression of HLA class I was lost or downregulated in 46 of these specimens (62%). With respect to osteosarcoma, loss or downregulation of HLA class I expression was seen in 13 (52%) of 25 primary tumors and seven (88%) of eight metastatic tumors. In six of 11 HLA class I‐negative osteosarcoma specimens, the expression of β‐2 microglobulin was also lost. Subsequently the prognostic significance of HLA class I expression was analyzed in 21 patients with osteosarcoma who had completed multidrug neoadjuvant chemotherapy and undergone adequate surgery. Patients with osteosarcoma highly expressing HLA class I showed significantly better overall and event‐free survival than those with HLA class I‐negative osteosarcoma. In contrast, such prognostic significance of HLA class I expression was not found in 15 patients with malignant fibrous histiocytoma of soft tissue. These findings suggest that the class I‐restricted cytotoxic T lymphocyte pathway plays a major role in immune surveillance of patients with osteosarcoma. (Cancer Sci 2006; 97: 1374–1380)

Detection and Induction of CTLs Specific for SYT-SSX-Derived Peptides in HLA-A24+ Patients with Synovial Sarcoma

To investigate the immunogenic property of peptides derived from the synovial sarcoma-specific SYT-SSX fusion gene, we synthesized four peptides according to the binding motif for HLA-A24. The peptides, SS391 (PYGYDQIMPK) and SS393 (GYDQIMPKK), were derived from the breakpoint of SYT-SSX, and SS449a (AWTHRLRER) and SS449b (AWTHRLRERK) were from the SSX region. These peptides were tested for their reactivity with CTL precursors (CTLps) in 16 synovial sarcoma patients using HLA-A24/SYT-SSX peptide tetramers and also for induction of specific CTLs from four HLA-A24+ synovial sarcoma patients. Tetramer analysis indicated that the increased CTLp frequency to the SYT-SSX was associated with pulmonary metastasis in synovial sarcoma patients (p < 0.03). CTLs were induced from PBLs of two synovial sarcoma patients using the peptide mixture of SS391 and SS393, which lysed HLA-A24+ synovial sarcoma cells expressing SYT-SSX as well as the peptide-pulsed target cells in an HLA class I-restricted manner. These findings suggest that aberrantly expressed SYT-SSX gene products have primed SYT-SSX-specific CTLps in vivo and increased their frequency in synovial sarcoma patients. The identification of SYT-SSX peptides may offer an opportunity to design peptide-based immunotherapeutic approaches for HLA-A24+ patients with synovial sarcoma.

Early Active Motion and Weightbearing After Cross-Stitch Achilles Tendon Repair

Twenty-two closed Achilles tendon ruptures caused by sports injuries in 22 patients (average age, 37.6 years) were repaired with Kirschmayer core suture and cross-stitch epitenon suture, and early active ankle motion with weightbearing was implemented after surgery. This study was undertaken to evaluate the effectiveness of the repair technique and rehabilitation protocol by assessing clinical results and magnetic resonance imaging findings. The follow-up period averaged 24.6 months. Twenty of the tendons (91%) healed without rerupture, and two tendons (9%) suffered a partial rerupture at 23 and 56 days, respectively. Active ankle extension reached from the minus range to 0° in an average of 9.7 days, and ankle motion recovered to normal in an average of 6.0 weeks. Full weightbearing without heel raising became possible in an average of 16.4 days, and heel raising with both legs became possible in an average of 7.3 weeks. The patients returned to full sports activity in 13.1 weeks. The interval until the area of high-intensity signal at the tendon repair site on T2-weighted magnetic resonance imaging scans became intermediate-intensity signal averaged 6.9 weeks, and the tendon repair site became low-intensity signal in an average of 12.6 weeks, demonstrating excellent tendon healing. Treatment employing Kirschmayer core suture and cross-stitch epitenon suture may help athletes return to sports activity in a shorter period than that allowed by previous methods of repair for Achilles tendon ruptures.

Identification of Human Autologous Cytotoxic T-Lymphocyte-Defined Osteosarcoma Gene That Encodes a Transcriptional Regulator, Papillomavirus Binding Factor

The prognosis for patients with osteosarcoma who do not respond to current chemotherapy protocols still remains poor. Toward the goal of establishing efficacious peptide-based immunotherapy for those patients, we previously developed an autologous pair of CTLs and an osteosarcoma cell line. In the current study, we screened the cDNA library of this osteosarcoma cell line using an autologous CTL clone and identified cDNA encoding an antigen. The isolated cDNA was identical to papillomavirus binding factor (PBF), which was recently reported as a DNA binding transcription factor cooperating with RUNX1. Reverse transcription-PCR analysis revealed that PBF was expressed in 16 of 19 cases of bone and soft-tissue sarcoma cell lines (5 of 6 of osteosarcoma lines) and 57 of 76 sarcoma tissue samples (11 of 14 of osteosarcoma tissues). Also, PBF was expressed in 10 of 13 epithelial cancer cell lines and 20 of 34 of cancer tissues. In contrast, PBF was detected in some normal organs including ovary, pancreas, spleen, and liver by reverse transcription-PCR but was restricted in the cytoplasm by immunostaining and undetectable by Western blotting. Furthermore, a 12-mer peptide, CTACRWKKACQR, located at the COOH terminus of PBF, was found to be a minimum requirement for recognition by the CTL clone in the context of the HLA-B*5502 molecule. These findings suggest that PBF is a shared tumor-associated antigen, which may serve as a source of peptides applicable to peptide-based immunotherapy for osteosarcoma and other malignant tumors.

Identification of human antitumor cytotoxic T lymphocytes epitopes of recoverin, a cancer‐associated retinopathy antigen, possibly related with a better prognosis in a paraneoplastic syndrome

Cancer‐associated retinopathy (CAR) is a rare paraneoplastic syndrome, and the recoverin‐specific autoantibody is suggested to contribute to the pathogenesis of retinopathy, including apoptosis of retinal cells. Because it is known that CAR+ cancer patients have a preferable prognosis, we hypothesized that aberrantly expressed recoverin in cancer cells can become a target of cytotoxic T lymphocytes (CTL). Here we tested nine recoverin‐derived HLA‐A24‐binding peptides for their capacity to elicit antitumor CTL. We observed recoverin‐specific CTL responses in two HLA‐A24+ CAR+ cancer patients. In addition, the CTL responses were obtained from three of ten CAR– cancer patients and two of six healthy individuals. The CTL precursor frequency of CAR+ cancer patients and that of CAR– cancer patients was higher than that of healthy individuals. Of nine recoverin peptides, R49 (QFQSIYAKF), R49.2 (QFQSIYAKFF), and R64 (AYAQHVFRSF) were discovered to induce the peptide‐specific CTL. Taken together, our present data suggest that peripheral activation of recoverin‐specific antitumor CTL is likely to contribute to the preferable prognosis of CAR+ cancer patients. Moreover, in cases other than CAR+ cancer patients, recoverin may offer the opportunity to design epitope‐based immunotherapeutic approaches for treating HLA‐A24+ cancer patients with a recoverin‐expressing tumor.

Crisscross CTL Induction by SYT-SSX Junction Peptide and Its HLA-A*2402 Anchor Substitute

To investigate the effects of anchor substitutions in SYT-SSX junction peptide, an HLA-A24 anchor residue (position 9) of the SYT-SSX B peptide (GYDQIMPKK) was substituted to more favorable residues according to the HLA-A24-binding motif. Among four substitutes constructed, a substitute with isoleucine (termed K9I peptide) most apparently enhanced the affinity for HLA-A24 molecule. Subsequent in vitro CTL induction analysis using PBMCs of 15 HLA-A24+ synovial sarcoma patients revealed that the original B peptide allowed to induce synovial sarcoma-specific CTLs from 7 patients (47%), whereas such CTLs were inducible from 12 patients (80%) with K9I peptide. Moreover, the extent of cytotoxicity against HLA-A24+ synovial sarcoma cell lines was higher in K9I peptide-induced CTLs than B peptide-induced CTLs. Influence of anchor substitution on peptide/TCR interaction was evaluated by cytotoxicity assays against autologous cells and tetramer analysis. CTLs induced from a synovial sarcoma patient using K9I peptide did not lyse autologous PHA blasts or EBV-infected B cells. In vitro stimulations of PBMCs from 5 HLA-A24+ synovial sarcoma patients with K9I peptide increased the frequency of T cells reacting with both HLA-A24/K9I peptide tetramer and HLA-A24/B peptide tetramer. In contrast, the frequency of T cells reacting with HLA/HIV-derived peptide tetramer remained low. These findings support the validity in design of anchor residue substitution in SYT-SSX fusion gene-derived peptide, and provide a potential clue to the current stagnation in vaccination trials of fusion gene-derived natural junction peptides.

Human CD8 and CD4 T cell epitopes of epithelial cancer antigens

Abstract Recent human tumor immunology research has identified several genes coding immunogenic peptides recognized by CD8 cytotoxic T lymphocytes (CTLs) in melanoma tumors. Very recently, CD4 T cell antigenic epitopes were also determined in certain melanoma tumors. The use of these peptides in conjunction with human immunotherapy could prove to be of great benefit. However, such peptides in clinically common tumors of epithelial cell origin, such as of the stomach, colon, lung, etc., have not yet been determined extensively. We describe for the first time an HLA-A31 (A*31012)-restricted natural antigenic peptide recognized by the CD8 CTL TcHST-2 of gastric signet ring cell carcinoma cell line HST-2. We also identified the HLA-DRB1*08032-restricted peptide recognized by the CD4 T cell line TcOSC-20 of squamous cell carcinoma OSC-20 derived from the oral cavity. The antigenic peptide of HST-2, designated F4.2, is composed of 10 amino acid residues with two anchor motif residues necessary for binding to HLA-A31 molecules. The synthetic F4.2 peptide enhanced the reactivity of TcHST-2 against HST-2 cells. Furthermore, introduction of an expression minigene coding F4.2 peptide to HLA-A31(+) cells conferred cytotoxic susceptibility to TcHST-2 on the cells. Some stomach cancer lines into which the HLA-A31 gene had been introduced, such as MKN28-A31-2, were lysed by TcHST-2, suggesting the presence of F4.2 peptide in at least some HLA-A31(+) stomach cancers. Furthermore, F4.2 peptide induced an F4.2 peptide-specific CTL response in at least 30–40% of HLA-A31(+) peripheral blood lymphocytes from gastric cancer patients, suggesting that F4.2 peptide could be used as a cancer vaccine for gastric tumors. The natural antigenic peptide of OSC-20 was also determined using acid extraction and biochemical separation and by mass spectrometry. Consequently, OSC-20 peptide was designated as the 6-1-5 peptide, an HLA-DRB1*08032-restricted 16-mer peptide with two possible anchor motifs. It has an amino acid sequence identical to that of human α-enolase, suggesting that it was derived from the processed parental α-enolase protein. We are presently attempting to determine the genes that code tumor rejection antigens recognized by HLA-A24- and A26-restricted T cells, including those of pulmonary and pancreatic carcinomas. The search for these antigenic peptides may lead to the identification of immunogenic peptide antigens that would be suitable for clinical use in commonly occurring epithelial cancers.

Induction of cytotoxic T lymphocytes from peripheral blood of human histocompatibility antigen (HLA)-A31+ gastric cancer patients by in vitro stimulation with antigenic peptide of signet ring cell carcinoma

Antigenic peptides have been used as a cancer vaccine in melanoma patients and have led to a drastic regression of metastatic tumors. However, few antigens have been identified in non‐melanoma tumors. We recently purified a new natural antigenic peptide, designated F4.2, by biochemical elution from a human gastric signet cell carcinoma cell line and showed that it is recognized by an autologous human histocompatibility antigen (HLA)‐A31‐restricted cytotoxic T lymphocyte (CTL) clone. Here we describe in vitro induction of F4.2‐specific CTLs from peripheral blood T lymphocytes of HLA‐A31+ gastric cancer patients. The T cells of seven HLA‐A31+ patients with gastric cancers were stimulated in vitro by F4.2‐pulsed autologous dendritic cells which had been induced from peripheral blood of each patient by incubation in the presence of granulocyte macrophage colony‐stimulating factor (GM‐CSF) and IL‐4. We tested the cytotoxicity of the T cells against F4.2‐loaded C1R‐A *31012 by a 6‐h 51Cr release assay after 3 stimulations with F4.2‐pulsed dendritic cells. F4.2‐specific cytotoxicity was detectable in the stimulated T cells from two of the seven HLA‐A31+ patients. Further, both F4.2‐specific CTLs also lysed the gastric cancer cell line, HST‐2, from which F4.2 was derived. These results suggest that F4.2 peptide may be useful as an HLA‐A31‐restricted peptide vaccine in certain patients with gastric cancer.

Natural Antigenic Peptides from Squamous Cell Carcinoma Recognized by Autologous HLA-DR8-restricted CD4 + T Cells

A large number of human tumor antigens recognized by CD8+ cytotoxic T lymphocytes (CTL) have been identified. Some of them have been employed in clinical trials and have achieved some objective responses. However, little is known about those that are recognized by CD4+ T cells, except for a very few that were identified from melanomas. Previously, we reported that an oral squamous cell carcinoma (SCC) cell line, OSC–20, was effectively lysed by HLA‐DRB1·08032 (HLA‐DRS)‐restricted autologous CD4+ T cell line, TcOSC–20. In this study, we performed two steps of chromatographic purification of the tumor cell lysate in combination with mass spectrometry. We found one reverse‐phase high‐performance liquid chromatography (RP‐HPLC) fraction that was effectively recognized by the T cells. We analyzed the fraction by nano‐liquid chromatography/electrospray ionization ion trap mass spectrometry (LC/MS/MS) and found six representative ions. We could determine the primary amino acid sequence of each of the six ions. Three of them contained a potential HLA‐DR8 binding motif, and TcOSC–20 showed a rather strong cytotoxic response to one of the synthetic pep tides, namely, amino acid residues 321–336 of human a‐enolase. Thus, several gene products of squamous cancer cells are endogenously processed and may be presented on HLA class II molecules, so that they could constitute target molecules for autologous CD4+ T cells.

A gene encoding human gastric signet ring cell carcinoma antigen recognized by HLA-A31-restricted cytotoxic T lymphocytes.

We previously reported acid-extracted natural antigenic peptide (F4.2 [YSWMDISCWI]) of a gastric signet ring cell carcinoma HST-2 cells, recognized by HLA-A*31012-restricted autologous cytotoxic T lymphocytes, TcHST-2 line. In this study, the full-length cDNA (1101 bp), termed c98, predicting a protein composed of 170 amino acids was obtained. Because TcHST-2 cells could lyse the HLA-A31 antigen (+) allogeneic tumor cells that were introduced with c98 gene, this gene was suggested to possess antigenicity. Beginning at N-terminal 61 amino acid, the N-terminal six amino acid sequence that is completely identical to F4.2 was present in c98; however, a sequence of four amino acids in C-terminal was not found. Nevertheless, this peptide, c9861–70, seemed to be immunogenic, because cells pulsed with c9861–70 peptide were lysed in a dose-dependent manner by TcHST-2 cells. The c98 gene was expressed ubiquitously in tumor cells as well as in normal tissues. However, some tumor cells, including HST-2 cells, expressed this antigen in a high content, and such cells were lysed by TcHST-2 cells in the context of HLA-A31 antigen. However, TcHST-2 cells did not lyse cells that expressed lower amounts of c98 than HST-2 cells. These data suggested that c98-gene product and/or c9861–70 peptides could be used as a candidate for tumor vaccines in cancer immunotherapy.