Prognostic impact and immunogenicity of a novel osteosarcoma antigen, papillomavirus binding factor, in patients with osteosarcoma

To develop peptide-based immunotherapy for osteosarcoma, we previously identified papillomavirus binding factor (PBF) as a cytotoxic T lymphocytes (CTL)-defined osteosarcoma antigen in the context of human leukocyte antigen (HLA)-B55. In the present study, we analyzed the distribution profile of PBF in 83 biopsy specimens of osteosarcomas and also the prognostic impact of PBF expression in 78 patients with osteosarcoma who had completed the standard treatment protocols. Next, we determined the antigenic peptides from PBF that react with peripheral T lymphocytes of HLA-A24⁺ patients with osteosarcoma. Immunohistochemical analysis revealed that 92% of biopsy specimens of osteosarcoma expressed PBF. PBF-positive osteosarcoma conferred significantly poorer prognosis than those with negative expression of PBF ( P =  0.025). In accordance with the Bioinformatics and Molecular Analysis Section score, we synthesized 10 peptides from the PBF sequence. Subsequent screening with an HLA class I stabilization assay revealed that peptide PBF A24.2 had the highest affinity to HLA-A24. CD8⁺ T cells reacting with a PBF A24.2 peptide were detected in eight of nine HLA-A24-positive patients with osteosarcoma at the frequency from 5 × 10⁻⁷ to 7 × 10⁻⁶ using limiting dilution/mixed lymphocyte peptide culture followed by tetramer-based frequency analysis. PBF A24.2 peptide induced CTL lines from an HLA-A24-positive patient, which specifically killed an osteosarcoma cell line that expresses both PBF and HLA-A24. These findings suggested prognostic significance and immunodominancy of PBF in patients with osteosarcoma. PBF is the candidate target for immunotherapy in patients with osteosarcoma. ( Cancer Sci 2008; 99: 368–375)     

Identification of ribosomal protein L19 as a novel tumor antigen recognized by autologous cytotoxic T lymphocytes in lung adenocarcinoma

The purpose of the present study was to identify a novel tumor-specific antigen capable of inducing a specific cellular immune response in lung cancer patients. The co-culture of regional lymph node lymphocytes and the CD80-transfected autologous lung adenocarcinoma cell line H1224L resulted in a successful induction of bulk cytotoxic T lymphocytes (CTL). CTL clone L7/8 was established by the limiting dilution method from these bulk CTLs and lysed H1224L but not autologous Epstein–Barr virus-transformed B cells or K562. The CTL clone also recognized allogeneic lung cancer cell lines in an HLA-A*31012-restricted manner. Using the CTL clone, an antigen-coding gene was identified using the cDNA expression cloning technique, which encodes ribosomal protein L19 (RPL19). Finally, a 9 mer antigenic peptide was identified by means of construction of mini-genes. RPL19 was overexpressed in the lung cancer tissue from patient H1224. All of the normal tissues examined expressed lower levels of RPL19 mRNA than that of the lung cancer tissue. RPL19 was also found to be overexpressed in 12 of 30 (40%) non-small-cell lung cancer tissues by immunohistochemical staining. The expression level of RPL19 in tumor cell lines correlated positively with the production of interferon (IFN)-γby CTL clone L7/8 in response to such cell lines. In addition, the suppression of RPL19 expression by transfection with small interfering RNA resulted in the suppression of cyclinD1, D3 synthesis, and the growth inhibition of lung cancer cell lines overexpressing RPL19. Therefore, this growth suppression could be ascribed to the inhibition of the cell cycle. These results may indicate that RPL19 is a novel overexpressed antigen which may therefore be a useful candidate as a target for specific immunotherapy. ( Cancer Sci 2009)     

Identification of the HLA-Cw*0702-restricted tumor-associated antigen recognized by a CTL clone from a lung cancer patient.

PURPOSE: A large number of tumor-associated antigens have been used in vaccination trials for mainly melanomas. Our purpose of this study is to identify a novel tumor antigen useful for immunotherapy of lung cancer patients. EXPERIMENTAL DESIGN: Analysis of an autologous tumor-specific CTL clone F2a that was established from regional lymph node lymphocytes of a patient with lung cancer (A904) by a mixed lymphocyte-tumor cell culture. RESULTS: F2a recognized and killed autologous tumor cells (A904L), whereas it did not respond to autologous EBV-transformed B cells, phytohemagglutinin-blastoid T cells, and K562 cells. cDNA clone 31.2 was isolated by using cDNA expression cloning method as a gene encoding antigen. This gene was identical to the reported gene whose function was unknown. The antigen encoded by the cDNA was recognized by the CTL in a HLA-Cw*0702-restricted manner. Furthermore, a 9-mer peptide at positions 659 to 685 in cDNA clone 31.2 was identified as a novel epitope peptide. The CTL recognized some allogeneic cancer cell lines with HLA-Cw*0702 as well as some HLA-Cw*0702-negative cell lines when transfected with HLA-Cw*0702, thus indicating that the identified antigen was a cross-reactive antigen. CONCLUSIONS: Although exact mechanism to process the encoded protein and present the antigen in the context of HLA class I remains to be elucidated, the CTL recognized some of tumor cells in the context of HLA-Cw*0702 but did not recognize a variety of normal cells and also autologous EBV-transformed B cells. These results indicated that the antigen identified in this study may therefore be a possible target of tumor-specific immunotherapy for lung cancer patients.     

Autologous CTL response against cancer stem-like cells/cancer-initiating cells of bone malignant fibrous histiocytoma

Malignant fibrous histiocytoma (MFH) of the bone is an aggressive tumor with high rates of local recurrence and metastasis. The development of novel therapeutic approaches is critical to improve the prognosis of patients with MFH. We reported previously that the side population (SP) cells of the MFH2003 bone MFH cell line have the characteristics of cancer stem-like cells (CSC)/cancer-initiating cells. In the present study, to establish immunotherapy targeting CSC, we analyzed cell surface immune molecules on SP cells of the MHF2003 cell line, as well as autologous CTL responses against these SP cells in the tumor microenvironment and peripheral circulating lymphocytes, using autologous tumor-infiltrating lymphocytes and autologous CTL clones derived from peripheral blood, respectively. We found that the SP cells expressed human leukocyte antigen (HLA) Class I molecules on the cell surface. The autologous tumor-infiltrating lymphocyte line TIL2003 recognized both the SP and main population cells of the MFH2003 cell line. Next, we induced the CTL clone Tc4C-6 by mixed lymphocyte tumor cell culture using autologous peripheral blood mononuclear cells and freshly isolated SP cells, followed by a limiting dilution procedure. The Tc4C-6 clone showed specific cytotoxicity against the SP cells. Moreover, the cytotoxicity against SP cells was blocked by the anti-HLA Class I antibody W6/32. In conclusion, the findings of the present study support the idea that CSC of bone MFH are recognized by autologous CTL in the tumor microenvironment and peripheral circulating lymphocytes. Thus, CTL-based immunotherapy could target CSC of bone sarcoma to help prevent tumor recurrence.     

Interleukin-2-induced, melanoma-specific T cells recognize CAMEL, an unexpected translation product of LAGE-1.

Melanoma-specific cytotoxic T lymphocytes (CTLs) were induced by in vitro stimulation of peripheral blood mononuclear cells of a melanoma patient with autologous IL-2-producing melanoma 518/IL2.14 cells. CTL clone 1/29 recognized, in addition to autologous melanoma cell lines, a panel of HLA-A*0201-expressing allogeneic melanoma cell lines but was not reactive with normal melanocytes. Here, we report the full molecular characterization of the target structure for CTL 1/29, which was identified by cDNA expression cloning. The recognized antigen was named CAMEL (CTL-recognized antigen on melanoma). The CAMEL cDNA turned out to be derived from the LAGE-1 gene, a recently described tumor antigen that is strongly homologous to NY-ESO-1. CAMEL, however, is not encoded by the putative open reading frame (ORF) of LAGE-1 but by an alternative frame starting from the second ATG of the mRNA. The first 11 amino acids of the CAMEL protein, MLMAQEALAFL, constitute the epitope of CTL 1/29. This epitope is also encoded by a similar alternative ORF in NY-ESO-1. In summary, CTL induction with IL-2-transfected melanoma cells has revealed a new tumor antigen that may serve as a target for immunotherapy.     

Identification of HLA-A24 restricted shared antigen recognized by autologous cytotoxic T lymphocytes from a patient with large cell carcinoma of the lung

The aim of the present study was to elucidate the tumor-specific cellular immunological responses occurring in a patient with large cell carcinoma of the lung who had no evidence of recurrence following surgical resections of both a primary lung lesion and a metastatic adrenal lesion. We analyzed an autologous tumor-specific cytotoxic T lymphocytes (CTL clone F2b), which were HLA-A*2402 restricted from regional lymph node lymphocytes. The F2b possessed T cell receptor (TCR) using the Valpha5 and Vbeta7 gene segment. The existence of precursor CTL (pCTL) against autologous tumor cells (A904L) was analyzed using CTL clone-specific PCR. Lymphocytes with the same TCR as F2b were detected in the primary tumor tissue, regional lymph node and the peripheral blood collected from the patient 3 years after the operation. Using the F2b, we identified a cDNA clone encoding the tumor antigen using cDNA expression cloning method. The gene was found to encode splicing variant of the Tara gene. Finally, we identified the 9-mer Ag peptide, using constructions of mini-genes. The F2b recognized 3 out of 7 HLA-A24 positive allogeneic tumor cell lines and in 1 out of 7 HLA-A24 negative allogeneic tumor cell lines when transfected with HLA-A24. This peptide is therefore considered to be potentially useful for performing specific immunotherapy in a significant proportion of lung cancer patients bearing HLA-A24.     

Identification of a lung cancer antigen evading CTL attack due to loss of human leukocyte antigen (HLA) class I expression

The human lung cancer cell line, C831L, lost HLA class I expression due to a mutation of the β2‐microglobulin (β2m) gene, and it may have been the result of immunoediting by CTL cytotoxicity. By restoration of HLA class I expression, we could identify the antigen that may be associated with HLA downregulation. Such an antigen might be a promising target of immunotherapy because it potentially may induce a sufficient immune response to eradicate cancer cells. The CTL clone could be established from lymph node lymphocytes in patient C831 by stimulation with wild‐type β2m‐transduced C831L (C831L‐wβ2m). The CTL clone showed reactivity against C831L‐wβ2m in a HLA‐B*0702‐restricted manner, but not Parental‐C831L or autologous normal cells. The cDNA expression cloning method was used to identify the antigen coding gene recognized by the CTL clone. The cDNA clone exhibited a homology with a part of the mRNA that codes for leucine rich repeat containing eight family member A (LRRC8A). A transfection analysis of minigenes indicated that the antigen peptide was derived from protein translated from the downstream of the registered open reading frame in LRRC8A mRNA. The antigenic 9‐mer peptide (GPRESRPPA) was identified. The present methodology should be useful to find the crucial tumor antigens, which are potentially associated with loss of HLA expression. Furthermore, such an antigen may help in achieving a better understanding of the immunological escape mechanisms and it may also provide a favorable immune response in cancer immunotherapy. (Cancer Sci 2010; 00: 000–000)     

Cellular immune response to human renal-cell carcinomas: Definition of a common antigen recognized by HLA-A2-restricted cytotoxic T-Lymphocyte (CTL) clones

Cytotoxic T lymphocyte (CTL) clones directed against autologous renal-cell carcinoma (RCC) cell lines were generated by mixed lymphocyte/tumor-cell culture (MLTC) using peripheral blood lymphocytes (PBL). A CD8+, CD4- CTL clone MZ1257-CTL 5/30 with high cytolytic activity for the autologous tumor cell line MZ1257-RCC was established. No lysis of the autologous EBV-transformed B lymphocytes (EBV-B) or K562 cells was observed. A panel of HLA-A2-matched allogeneic RCC lines was recognized by CTL 5/30. Further specificity analysis showed a cross-reactivity with HLA-A2-matched allogeneic tumor cells of various origins, especially melanoma. CTL 5/30 was also cross-reactive with several HLA-A2-positive allogeneic normal kidney cells in culture. The restriction element identified for CTL 5/30 was HLA-A2, as shown by blocking of cytotoxicity using an anti-HLA-A2 monoclonal antibody (MAb) and by resistance of an HLA-A2-negative melanoma variant SK29-MEL. 1.22 against lysis by CTL 5/30. In this report we demonstrate HLA-A2-restricted recognition of a T-cell-defined antigen on autologous renal-cancer cells. This antigen is also expressed and recognized in association with HLA-A2 on normal kidney cells in culture and other HLA-A2-positive tumor cells. It may therefore be a normal differentiation antigen to which tolerance is incomplete in the renal-cell cancer system investigated.     

A survivin specific T-cell clone from a breast cancer patient display universal tumor cell lysis

Survivin is an attractive candidate for cancer immunotherapy since it is overexpressed in most common human cancers, poorly expressed in most normal adult tissues and is essential for cancer cell survival. Previously, we and others have demonstrated that survivin-specific immune responses are present in cancer patients. However, a significant limitation of these findings has been that antigen-specific lysis of tumors was achieved using polyclonal T-cell lines rather than a specific T-cell clone. In the present study we isolated and expanded a survivin specific cytotoxic T lymphocyte (CTL) clone from the peripheral blood of a cancer patient. The survivin specific CTL clone efficiently lysed a large panel of tumor cells of different origin, i.e., breast cancer, colon cancer and melanoma cells. The data support the notion that survivin may serve as a universal target antigen for anti-cancer immunotherapy.     

Identification of novel human leukocyte antigen‐A*11:01‐restricted cytotoxic T‐lymphocyte epitopes derived from osteosarcoma antigen papillomavirus binding factor

Osteosarcoma is the most common malignancy of bone that affects young people. Neoadjuvant chemotherapy and surgery have significantly improved the prognosis. However, the prognosis of non‐responders to chemotherapy is still poor. To develop peptide‐based immunotherapy for osteosarcoma, we previously identified CTL epitopes derived from papillomavirus binding factor (PBF) in the context of human leukocyte antigen (HLA)‐A2, HLA‐A24 and HLA‐B55. In the present study, we identified two novel CTL epitopes, QVT (QVTVWLLEQK) and LSA (LSALPPPLHK), in the context of HLA‐A11 using a sequence of screenings based on the predicted affinity of peptides, in vitro folding ability of peptide/HLA‐A11 complex, reactivity of peptide/HLA‐A11 tetramer and interferon (IFN)‐γ production of T cells that was induced by mixed lymphocyte peptide culture under a limiting dilution condition. CTL clones directed to QVT and LSA peptides showed specific cytotoxicity against HLA‐A11⁺PBF⁺ osteosarcoma (HOS‐A11) cells. In contrast, another epitope, ASV (ASVLSRRLGK), could highly induce cognate tetramer‐positive CTL. This might be because the ASV peptide mimics the peptide ASV (R6Q) (ASVLSQ RLGK) derived from bacterial polypeptides, ROK family proteins. However, ASV‐induced CTL did not show cytokine production against the cognate peptide. In conclusion, the CTL epitopes QVT and LSA peptides might be useful for the development of immunotherapy targeting PBF for patients with osteosarcoma.     

Proliferation potential-related protein, an ideal esophageal cancer antigen for immunotherapy, identified using complementary DNA microarray analysis.

PURPOSE: To establish effective antitumor immunotherapy for esophageal cancer, we tried to identify an useful target antigen of esophageal cancer. EXPERIMENTAL DESIGN: We did cDNA microarray analysis to find a novel candidate antigen, proliferation potential-related protein (PP-RP). We examined cytotoxicity against tumor cells in vitro and in vivo of CTLs specific to PP-RP established from esophageal cancer patients. RESULTS: In 26 esophageal cancer tissues, an average of relative ratio of the expression of the PP-RP mRNA in cancer cells versus adjacent normal esophageal tissues was 396.2. Immunohistochemical analysis revealed that, in 20 of the 22 esophageal cancer tissues, PP-RP protein was strongly expressed only in the cancer cells and not so in normal esophageal epithelial cells. PP-RP protein contains 10 epitopes recognized by HLA-A24-restricted CTLs. These CTLs, generated from HLA-A24-positive esophageal cancer patients, had cytotoxic activity against cancer cell lines positive for both PP-RP and HLA-A24. Furthermore, adoptive transfer of the PP-RP-specific CTL line inhibited the growth of a human esophageal cancer cell line engrafted in nude mice. CONCLUSIONS: The expression of PP-RP in esophageal cancer cells was significantly higher than in normal cells, and the CTLs recognizing PP-RP killed tumor cells in vitro and also showed tumor rejection effects in a xenograft model. Therefore, PP-RP may prove to be an ideal tumor antigen useful for diagnosis and immunotherapy for patients with esophageal cancer. cDNA microarray analysis is a useful method to identify ideal tumor-associated antigens.     

Identification of CLUAP1 as a human osteosarcoma tumor-associated antigen recognized by the humoral immune system

Since the prognosis of human osteosarcoma in advanced stage remains poor, the development of new and effective therapies including immunotherapy is required. To identify tumor-associated antigens of osteosarcoma applicable to the immunotherapy of this malignancy, we employed the serological analysis of recombinant cDNA expression library (SEREX) technique that defines tumor antigens recognized by the humoral immune system. Screening a cDNA library derived from an osteosarcoma cell line MG63 with sera from osteosarcoma patients identified 43 positive clones, representing 14 distinct antigens. Among them, CLUAP1 (clusterin-associated protein 1) was highly expressed in osteosarcoma tissue samples and cell lines. Overexpression of CLUAP1 was observed in other malignancies including ovarian, colon, and lung cancers. Our results suggest that CLUAP1 may be useful as a prognostic/diagnostic marker and/or for a target of immunotherapy of osteosarcoma.     

Serine proteinase inhibitor 9 can be recognized by cytotoxic T lymphocytes of epithelial cancer patients.

Serine proteinase inhibitor 9 (PI-9) inhibits granzyme B-mediated apoptosis and interleukin-1beta-converting enzyme activity. In this study, we report that the PI-9 gene encodes antigenic epitopes recognized by the HLA-A24-restricted and tumor-reactive cytotoxic T lymphocytes (CTLs) of epithelial cancer patients. Screening of an autologous cDNA library using a CTL line recognizing HLA-A24+ tumor cells resulted in the isolation of a cDNA, which had an identical coding region to the previously described PI-9 genes. PI-9 gene was expressed in approximately three-fourths of epithelial cancer cell lines and all leukemic cell lines tested. It was also expressed in normal peripheral blood mononuclear cells (PBMCs), but not in a normal fibroblast cell line. CTL sublines contained T cells capable of recognizing the PI-9(292-300) and PI-9(348-356) peptides among 13 different peptides having the HLA-A24 binding motifs. These two peptides were recognized by the CTL line in a dose-dependent and HLA class-I-restricted manner, and also possessed the ability to induce HLA class I-restricted and tumor-reactive CTLs in PBMCs from HLA-A24+ cancer patients. These results demonstrate that PI-9 is recognized by HLA class I-restricted and tumor-reactive CTLs of epithelial cancer patients.     

HER-2, gp100, and MAGE-1 are expressed in human glioblastoma and recognized by cytotoxic T cells

It has recently been demonstrated that malignant glioma cells express certain known tumor-associated antigens, such as HER-2, gp100, and MAGE-1. To further determine the possible utilization of these antigens for glioma immunotherapy and as surrogate markers for specific tumor antigen cytotoxicity, we characterized the presence of mRNA and protein expression in 43 primary glioblastoma multiforme (GBM) cell lines and 7 established human GBM cell lines. HER-2, gp100, and MAGE-1 mRNA expression was detected in 81.4%, 46.5%, and 39.5% of the GBM primary cell lines, respectively. Using immunoreactive staining analysis by flow cytometry, HER-2, gp100, and MAGE-1 protein expression was detected in 76%, 45%, and 38% of the GBM primary cell lines, respectively. HLA-A1-restricted epitope specific for MAGE-1 peptide (EADPTGHSY) CTL clone B07 and HLA-A2-restricted epitope specific for HER-2 peptide (KIFGSLAFL) CTL clone A05 and gp100 peptide (ITDQVPFSV) CTL clone CK3H6 were used in this study. The specificity of CTL clone was verified by HLA/peptide tetramer staining. Three CTL clones could efficiently recognize GBM tumor cells in an antigen-specific and MHC class I-restricted manner. IFN-gamma treatment can dramatically increase MHC class I expression of GBM tumor cells and significantly increase CTL recognition of tumor cells. Treatment with the DNA hypomethylating agent 5-aza-2'-deoxycytidine induced and up-regulated the mRNA expression of MAGE-1 and epitope presentation by autologous MHC. These data indicate that HER-2, gp100, and MAGE-1 could be used as tumor antigen targets for surrogate assays for antigen-specific CTLs or to develop antigen-specific active immunotherapy strategies for glioma patients.     

Screening of HLA-A24-restricted epitope peptides from prostate-specific membrane antigen that induce specific antitumor cytotoxic T lymphocytes.

PURPOSE: Prostate-specific membrane antigen (PSMA), which is a transmembrane glycoprotein predominantly expressed in prostate cancer, is an attractive target for tumor-specific immunotherapy. To identify human leukocyte antigen (HLA)-A24-restricted epitope peptides from PSMA for further application of the dendritic cell (DC)-based immunotherapy targeting prostate cancer, we have screened several PSMA-encoded HLA-A24-binding peptides for their capabilities to elicit specific antitumor CTL response in vitro. EXPERIMENTAL DESIGN: The amino acid sequence of PSMA was screened for peptides consisting of 9 or 10 amino acids, which possess the known HLA-A24-binding motif. Nine candidate peptides were screened for binding to HLA-A24 molecules. Then, each of these nine peptides was studied to determine whether CTL responses could be induced by primary in vitro immunization of CD8(+) T cells using peptide-pulsed autologous DCs derived from peripheral blood mononuclear cells of HLA-A24(+) healthy donor as antigen-presenting cells. The antigen specificity of the CTL lines was confirmed using several tumor cell lines as target cells, which were genetically modified to express both HLA-A24 and PSMA. RESULTS: Two peptides, LYSDPADYF and NYARTEDFF, were demonstrated to elicit CTL lines that lyse peptide-pulsed, HLA-A24(+) B-lymphoblastoid cells. Each of the CTL lines recognized their specific PSMA-expressing target cells in a HLA-A24-restricted manner. The capability to release IFN-gamma by the CTL lines was specifically inhibited by anti-MHC class I and anti-CD8 monoclonal antibodies but not by anti-MHC class II and anti-CD4 monoclonal antibodies. CONCLUSION: Two novel HLA-A24-restricted PSMA-derived epitopes were identified in this study. These epitopes can be used to further evaluate the clinical utility of DC-based immunotherapeutic strategies for treatment of hormone-refractory prostate cancers.