Peptides derived from human insulin-like growth factor-II mRNA binding protein 3 can induce human leukocyte antigen-A2-restricted cytotoxic T lymphocytes reactive to cancer cells

Insulin-like growth factor-II mRNA binding protein 3 (IMP-3) is an oncofetal protein expressed in various malignancies including lung cancer. This study aimed to identify immunogenic peptides derived from IMP-3 that can induce tumor-reactive and human leukocyte antigen (HLA)-A2 (A*02:01)-restricted cytotoxic T lymphocytes (CTL) for lung cancer immunotherapy. Forty human IMP-3-derived peptides predicted to bind to HLA-A2 were analyzed to determine their capacity to induce HLA-A2-restricted T cells in HLA-A2.1 (HHD) transgenic mice (Tgm). We found that three IMP-3 peptides primed HLA-A2-restricted CTL in the HLA-A2.1 Tgm. Among them, human CTL lines reactive to IMP-3 (515) NLSSAEVVV(523) were reproducibly established from HLA-A2-positive healthy donors and lung cancer patients. On the other hand, IMP-3 (199) RLLVPTQFV(207) reproducibly induced IMP-3-specific and HLA-A2-restricted CTL from healthy donors, but did not sensitize CTL in the HLA-A2.1 Tgm. Importantly, these two IMP-3 peptide-specific CTL generated from healthy donors and cancer patients effectively killed the cancer cells naturally expressing both IMP-3 and HLA-A2. Cytotoxicity was significantly inhibited by anti-HLA class I and anti-HLA-A2 monoclonal antibodies, but not by the anti-HLA-class II monoclonal antibody. In addition, natural processing of these two epitopes derived from the IMP-3 protein was confirmed by specific killing of HLA-A2-positive IMP-3-transfectants but not the parental IMP-negative cell line by peptide-induced CTL. This suggests that these two IMP-3-derived peptides represent highly immunogenic CTL epitopes that may be attractive targets for lung cancer immunotherapy.     

Definition of an immunogenic region within the ovarian tumor antigen stratum corneum chymotryptic enzyme.

PURPOSE: The serine protease stratum corneum chymotryptic enzyme (SCCE) is overexpressed by ovarian tumor cells, but is not expressed by normal tissues, suggesting that SCCE may be an attractive target for immunotherapy. In this study, we tested the hypothesis that dendritic cells loaded with SCCE peptides will induce ovarian tumor antigen-specific CD8+ CTL responses and antigen-specific CD4+ helper T cell responses. EXPERIMENTAL DESIGN: Computer algorithms were used to identify candidate HLA-A2.1-restricted CD8+ CTL epitopes and HLA-DR-binding CD4+ helper T cell epitopes within SCCE. CD8+ CTL stimulated with peptide-loaded dendritic cells were tested against targets expressing endogenous SCCE, including HLA-A2.1-matched ovarian tumor cells. Dendritic cells were also loaded with an extended SCCE peptide, SCCE 110-139, which encompassed a defined CD8+ CTL epitope and multiple candidate CD4+ T helper cell epitopes. RESULTS: CD8+ CTL specific for SCCE 123-131 lysed autologous macrophages infected with an SCCE-expressing recombinant adenovirus, and also lysed HLA-A2.1-matched, SCCE-expressing ovarian tumor cells. Dendritic cells loaded with SCCE 5-13 peptide stimulated an HLA-A2.1-restricted CD8+ CTL response, but with a reduced level of lysis against ovarian tumor cells. Dendritic cells loaded with SCCE 110-139 induced antigen-specific CD4+ T cell and CD8+ T cell responses. Although SCCE 110-139-loaded dendritic cells processed and presented the 123-131 epitope, the dominant CD8+ CTL response was directed against alternative epitopes within SCCE 110-139. CONCLUSIONS: The 110-139 region of SCCE incorporates multiple CD8+ CTL and CD4+ helper T cell epitopes, and represents an attractive target antigen for immunotherapy of ovarian cancer.     

肿瘤相关抗原CT45的HLA-A2/A3限制性CTL表位的预测

目的:预测肿瘤相关抗原CT45的HLA-A2/A3限制性CTL表位,为CTL表位肽的合成和活性筛选提供依据。方法:基于新近发现的CT45抗原的氨基酸序列,用NetCTL 1.2 Server人工神经网络法远程预测系统进行HLA-A2/A3限制性CTL表位预测打分,挑选出分值较高的作为候选表位。结果:共预测出了5个潜在的HLA-A2限制性CTL表位九肽,15个潜在的HLA-A3限制性CTL表位九肽,所有这些表位均为首次报道。结论:NetCTL 1.2 Server人工神经网络法能高效的预测CTL表位肽,基于新近发现的CT45抗原,我们通过该系统成功预测出了5个潜在的HLA-A2限制性CTL表位,15个潜在的HLA-A3限制性CTL表位,可以对其进行进一步的研究。     

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.     

HLA-A2-Restricted Cytotoxic T Lymphocyte Epitopes from Human Heparanase as Novel Targets for Broad-Spectrum Tumor Immunotherapy

Peptide vaccination for cancer immunotherapy requires identification of peptide epitopes derived from antigenic proteins associated with tumors. Heparanase (Hpa) is broadly expressed in various advanced tumors and seems to be an attractive new tumor-associated antigen. The present study was designed to predict and identify HLA-A2-restricted cytotoxic T lymphocyte (CTL) epitopes in the protein of human Hpa. For this purpose, HLA-A2-restricted CTL epitopes were identified using the following four-step procedure: 1) a computer-based epitope prediction from the amino acid sequence of human Hpa, 2) a peptide-binding assay to determine the affinity of the predicted protein with the HLA-A2 molecule, 3) stimulation of the primary T-cell response against the predicted peptides in vitro, and 4) testing of the induced CTLs toward different kinds of carcinoma cells expressing Hpa antigens and/or HLA-A2. The results demonstrated that, of the tested peptides, effectors induced by peptides of human Hpa containing residues 525–533 (PAFSYSFFV, Hpa525), 277–285 (KMLKSFLKA, Hpa277), and 405–413 (WLSLLFKKL, Hpa405) could effectively lyse various tumor cell lines that were Hpa-positive and HLA-A2-matched. We also found that these peptide-specific CTLs could not lyse autologous lymphocytes with low Hpa activity. Further study revealed that Hpa525, Hpa277, and Hpa405 peptides increased the frequency of IFN-γ-producing T cells compared to a negative peptide. Our results suggest that Hpa525, Hpa277, and Hpa405 peptides are new HLA-A2-restricted CTL epitopes capable of inducing Hpa-specific CTLs in vitro. Because Hpa is expressed in most advanced malignant tumors, Hpa525, Hpa277, and Hpa405 peptide-based vaccines may be useful for the immunotherapy for patients with advanced tumors.     

Prediction and analysis of HLA-A2/A24-restricted cytotoxic T-lymphocyte epitopes of the tumor antigen MAGE-n using the artificial neural networks method on NetCTL1.2 Server

Cancer immunotherapy has become one of the most important therapeutic approaches to cancer in the past two decades. Tumor antigen-derived peptides have been widely used to elicit tumor-specific cytotoxic T lymphocytes (CTLs). Antigen-specific CTLs induced by MAGE-derived peptides have proven to be highly efficacious in the prevention and treatment of various types of tumor. MAGE-n is a new member of the MAGE gene family and has been shown to be closely associated with hepatocellular carcinoma. It is highly homologous to the MAGE-A gene subfamily, particularly to MAGE-3 (93%). MAGE-n-derived peptide QLVFGIEVV is a novel HLA-A2.1-restricted CTL epitope that induces MAGE-n-specific CTLs in vitro. Identification of these CTL epitopes may lead to clinical applications of these peptides as cancer vaccines for patients with MAGE-n(+)/HLA-A2(+) tumors. In the present study, HLA-A/A24-restricted CTL epitopes of antigen MAGE-n were predicted using the NetCTL1.2 Server on the web, COMB >0.85. The results showed that the NetCTL1.2 Server prediction method improved prediction efficacy and accuracy. Additionally, 8 HLA-A2- and 9 HLA-A24-restricted CTL epitope candidates (nonamers) derived from the tumor antigen MAGE-n were predicted. These nonamers, following identification via experimentation, may contribute to the development of potential antigen peptide tumor vaccines.     

Identification of HLA-A2-restricted CTL epitope encoded by the MAGE-n gene of human hepatocellular carcinoma

BACKGROUND: Identification of the cytotoxic T lymphocytes (CTL) restricted epitopes of tumor antigens opens up possibilities of developing a new cancer vaccine. For the MAGE-n has been demonstrated closely associated with hepatocellular carcinoma (HCC) and HLA-A2.1 is found in over 50% of HCC patients in China, we aim at identifying MAGE-n-encoded peptide presented by HLA-A2.1. MATERIALS: A HLA-A2.1-restricted CTL epitope was identified by using an improved "reverse immunology" strategy: (a) computer-based epitope prediction from the amino acid sequence of MAGE-n antigen; (b) peptide-binding assay to determine the affinity of the predicted peptide with HLA-A2.1 molecule; (c) stimulation of primary T-cell response against the predicted peptides in vitro; and (d) testing of the induced CTLs toward HCC cells expressing MAGE-n antigen and HLA-A2.1. RESULTS: Of the five tested peptides, effectors induced by a peptide of MAGE-n at residue position 159-167(QLVFGIEVV) lysed HCC cells expressing both MAGE-n and HLA-A2.1. Our results indicated that peptide QLVFGIEVV was a new HLA-A2.1-restricted CTL epitope capable of inducing MAGE-n specific CTLs in vitro. CONCLUSIONS: Identification of the MAGE-n /HLA-A2.1 peptide QLVFGIEVV may facilitate peptide-based specific immunotherapy for HCC. The combination of epitope prediction, epitope reconstruction method and immunological methods can improve the efficiency and accuracy of CTL epitope studies.     

Identification of HLA-A24-restricted CTL epitope from cancer-testis antigen, NY-ESO-1, and induction of a specific antitumor immune response.

PURPOSE: For the development of peptide-based, cancer-specific immunotherapy, the identification of CTL epitopes from additional tumor antigens is very important. NY-ESO-1, a cancer-testis antigen, is considered to be a promising target of tumor-specific immunotherapy. Because HLA-A24-expressing individuals cover >60% in the population of Japan, we aim at identifying NY-ESO-1-encoded peptide presented by HLA-A24. EXPERIMENTAL DESIGN: In our study, a HLA-A24-restricted CTL epitope was identified by using the following four-step procedure: (a) computer-based epitope prediction from the amino acid sequence of NY-ESO-1 antigen; (b) peptide-binding assay to determine the affinity of the predicted peptide with HLA-A24 molecule; (c) stimulation of primary T-cell response against the predicted peptides in vitro; and (d) testing of the induced CTLs toward various carcinoma cells expressing NY-ESO-1 antigen and HLA-A24. RESULTS: Of the tested peptides, effectors induced by a peptide of NY-ESO-1 at residue position 158-166 lysed three kinds of carcinoma cells expressing both NY-ESO-1 and HLA-A24. Our results indicate that peptide NY-ESO-1 (158-166) (LLMWITQCF) is a new HLA-A24-restricted CTL epitope capable of inducing NY-ESO-1-specific CTLs in vitro mediating HLA class I-restricted manner. CONCLUSIONS: We identified a novel HLA-A24-restricted NY-ESO-1-derived epitope peptide (LLMWITQCF) that could induce specific CTLs from the peripheral blood mononuclear cells of HLA-A24(+) healthy donors. This peptide would be useful in further evaluating the clinical utility of peptide-based, cancer-specific immunotherapy against various histological tumors.     

Identification of a new MAGE-A10 antigenic peptide presented by HLA-A*0201 on tumor cells

MAGE-A antigens belong to cancer/testis (CT) antigens that are expressed in tumors but not in normal tissues with the exception of testis and placenta. Among MAGE-A antigens, MAGE-A10 is extensively expressed in various histological types of tumors, representing an attractive target for tumor immunotherapy. Cytotoxic T lymphocytes (CTLs) play a key role in anti-tumor immune responses, so the identification of CTL epitopes derived from MAGE-A10 would contribute a lot to the design of epitope-based vaccines for tumor patients. In this study, we predicted HLA-A*0201-restricted CTL epitope peptides of MAGE-A10, followed by peptide/HLA-A*0201 binding affinity and complex stability assays, and induced peptide-specific CTL immune responses. Of the selected three peptides (designated P1, P2 and P3), P1 (MAGE-A10310-318, SLLKFLAKV) could elicit peptide-specific CTLs both in vitro from HLA-A*0201-positive PBMCs and in HLA-A*0201/Kb transgenic mice. And, the induced CTLs could lyse MAGE-A10-expressing tumor cells in a HLA-A*0201-restricted fashion but not MAGE-A10-negative tumor cells. Our results demonstrate that the peptide MAGE-A10310-318 is a HLA-A*0201-restricted CTL epitope of MAGE-A10 and could serve as a target for therapeutic antitumoral vaccination.     

HLA-A2-restricted CTL epitopes of a novel lung cancer-associated cancer testis antigen, cell division cycle associated 1, can induce tumor-reactive CTL

Toward the development of a novel cancer immunotherapy, we have previously identified several tumor-associated antigens (TAAs) and the epitopes recognized by human histocompatibility leukocyte (HLA)-A2/A24-restricted cytotoxic T lymphocyte (CTL). In this study, we tried to identify a TAA of lung cancer (LC) and its HLA-A2 restricted CTL epitopes to provide a target antigen useful for cancer immunotherapy of LC. We identified a novel cancer testis antigen, cell division cycle associated gene 1 (CDCA1), overexpressed in nonsmall cell LC using a cDNA microarray analysis. The expression levels of CDCA1 were also increased in the majority of small cell LC, cholangiocellular cancer, urinary bladder cancer and renal cell cancers. We used HLA-A2.1 transgenic mice to identify the HLA-A2 (A*0201)-restricted CDCA1 epitopes recognized by mouse CTL, and we investigated whether these peptides could induce CDCA1-reactive CTLs from the peripheral blood mononuclear cells (PBMCs) of HLA-A2-positive donors and a NSCLC patient. Consequently, we found that the CDCA1(65-73) (YMMPVNSEV) peptide and CDCA1(351-359) (KLATAQFKI) peptide could induce peptide-reactive CTLs in HLA-A2.1 transgenic mice. In HLA-A2(+) donors, in vitro stimulation of PBMC with these peptides could induce peptide-reactive CTLs which killed tumor cell lines endogenously expressing both HLA-A2 and CDCA1. As a result, CDCA1 is a novel cancer-testis antigen overexpressed in LC, cholangiocellular cancer, urinary bladder cancer and renal cell cancers, and CDCA1 may therefore be an ideal TAA useful for the diagnosis and immunotherapy of these cancers.     

Determination of cellularly processed HLA-A2402-restricted novel CTL epitopes derived from two cancer germ line genes, MAGE-A4 and SAGE.

PURPOSE: For identification of CTL epitopes useful for cancer vaccines, it is crucial to determine whether cognate epitopes are presented on the cell surface of target cancer cells through natural processing of endogenous proteins. For this purpose, we tried to use the cellular machinery of both mice and human to define naturally processed CTL epitopes derived from two "cancer germ line" genes, MAGE-A4 and SAGE. EXPERIMENTAL DESIGN: We vaccinated newly produced HLA-A2402 transgenic mice with DNA plasmids encoding target antigens. Following screening of synthesized peptides by splenic CD8(+) T cells of vaccinated mice, we selected candidate epitopes bound to HLA-A2402. We then examined whether human CD8(+) T cells sensitized with autologous CD4(+) PHA blasts transduced by mRNA for the cognate antigens could react with these selected peptides in an HLA-A2402-restricted manner. RESULTS: After DNA vaccination, murine CD8(+) T cells recognizing MAGE-A4(143-151) or SAGE(715-723) in an HLA-A2402-restricted manner became detectable. Human CTLs specific for these two peptides were generated after sensitization of HLA-A2402-positive CD8(+) T cells with autologous CD4(+) PHA blasts transduced with respective mRNA. CTL clones were cytotoxic toward tumor cell lines expressing HLA-A2402 and cognate genes. Taken together, these CTL epitopes defined in HLA-A24 transgenic mice are also processed and expressed with HLA-A2402 in human cells. The presence of SAGE(715-723)-specific precursors was observed in HLA-A2402-positive healthy individuals. CONCLUSIONS: Two novel HLA-A2402-restricted CTL epitopes, MAGE-A4(143-151) and SAGE(715-723), were identified. Our approach assisted by cellular machinery of both mice and human could be widely applicable to identify naturally processed CTL epitopes.     

Identification of HLA-A*0201-restricted cytotoxic T lymphocyte epitope from TRAG-3 antigen.

PURPOSE: Identification of tumor antigen and subsequent identification of T-cell epitope from these antigens make specific immunotherapy for malignant tumor applicable. Because TRAG-3 antigen is expressed in most melanomas and 54% of non-small cell lung carcinomas and HLA-A2.1-expressing individuals cover >50% in the population of China, we aim at identifying TRAG-3-encoded peptide presented by HLA-A2.1. EXPERIMENTAL DESIGN: In our study, a HLA-A2.1-restricted CTL epitope was identified by using the following four-step procedure: (a) computer-based epitope prediction from the amino acid sequence of TRAG-3 antigen; (b) peptide-binding assay to determine the affinity of the predicted peptide with HLA-A2.1 molecule; (c) stimulation of primary T-cell response against the predicted peptides in vitro; and (d) testing of the induced CTLs toward LB373-MEL cells expressing TRAG-3 antigen and HLA-A2.1. RESULTS: Of the four tested peptides, effectors induced by a peptide of TRAG-3 at residue position 58-66 lysed LB373-MEL cells expressing both TRAG-3 and HLA-A2.1. Our results indicate that peptide TRAG-3(58 approximately 66) (ILLRDAGLV) is a new HLA-A2.1-restricted CTL epitope capable of inducing TRAG-3 specific CTLs in vitro. CONCLUSIONS: Because TRAG-3 is a cancer/testis antigen expressed in most melanomas and half of non-small cell lung carcinomas, identification of the TRAG-3/HLA-A2.1 peptide ILLRDAGLV may facilitate peptide-based specific immunotherapy for various histological tumors.     

Multiple antigenic peptides of human heparanase elicit a much more potent immune response against tumors

Peptide vaccination for cancer immunotherapy requires an ideal immune response induced by epitope peptides derived from tumor-associated antigens (TAA). Heparanase is broadly expressed in various advanced tumors. Accumulating evidence suggests that heparanase can serve as a universal TAA for tumor immunotherapy. However, due to the low immunogenicity of peptide vaccines, an ideal immune response against tumors usually cannot be elicited in patients. To increase the immunogenicity of peptide vaccines, we designed three 4-branched multiple antigenic peptides (MAP) on the basis of the human leukocyte antigen (HLA)-A2-restricted cytotoxic T lymphocyte (CTL) epitopes of human heparanase that we identified previously as antigen carriers. Our results show that MAP vaccines based on the HLA-A2-restricted CLT epitopes of human heparanase were capable of inducing HLA-A2-restricted and heparanase-specific CTL in vitro and in mice. Moreover, compared with their corresponding linear peptides, heparanase MAP vaccines elicited much stronger lysis of tumor cells by activating CD8(+) T lymphocytes and increasing the releasing of IFN-γ. However, these heparanase-specific CTLs did not lyse heparanase-expressing autologous lymphocytes and dendritic cells, which confirm the safety of these MAP vaccines. Therefore, our findings indicate that MAP vaccines based on CTL epitopes of human heparanase can be used as potent immunogens for tumor immunotherapy because of advantages such as broad spectrum, high effectiveness, high specificity, and safety.     

Identification of an HLA-A*0201-restricted cytotoxic T lymphocyte epitope from the lung carcinoma antigen, Lengsin

Lengsin is an eye lens protein with a glutamine synthetase domain. We previously identified this protein as a lung carcinoma antigen through cDNA microarray analysis. Lengsin protein is overexpressed irrespective of the histological type of lung carcinoma, but not in normal tissues other than the lens. Therefore, to significantly extend the use of Lengsin-based T-cell immunotherapies for the treatment of patients with lung carcinoma, we searched for HLA-A*0201-restricted epitopes from this protein by screening predicted Lengsin-derived candidate peptides for the induction of tumor-reactive CTLs. Four Lengsin-derived peptides were selected by computerized algorithm based on a permissive HLA-A*0201 binding motif, and were used to immunize HLA-A*0201 transgenic (HHD) mice. Two of the immunizing peptides, Lengsin(206-215)(FIYDFCIFGV) and Lengsin(270-279)(FLPEFGISSA), induced peptide-specific cytotoxic T lymphocytes (CTLs) in HHD mice, and thus were used to stimulate human peripheral blood lymphocytes in?vitro. Lengsin(206-215) and Lengsin (270-279) also induced human peptide-specific CTLs, and we were able to generate Lengsin(206-215)- and Lengsin(270-279)-specific CTL clones. The Lengsin(270-279)-specific CTL clone specifically recognized peptide-pulsed T2 cells, COS-7 cells expressing HLA-A*0201 and Lengsin, and HLA-A*0201+/Lengsin+ lung carcinoma cells in an HLA-A*0201-restricted manner. On the other hand, the Lengsin(206-215)-specific CTL clone failed to recognize HLA-A*0201+/Lengsin+ target cells in the absence of cognate peptide. These results suggest that Lengsin(270-279) is naturally processed and presented by HLA-A*0201 molecules on the surface of lung carcinoma cells and may be a new target for antigen-specific T-cell immunotherapy against lung cancer.     

Cyclin D1-specific cytotoxic T lymphocytes are present in the repertoire of cancer patients: implications for cancer immunotherapy

PURPOSE: Cyclin D1, a key cell cycle regulator, is overexpressed in multiple types of cancer. Such tumor-associated genes may be useful targets for cancer immunotherapy. Nevertheless, it had previously been suggested that efficient T cells recognizing cyclin D1-derived epitopes are absent from the repertoire because of thymic deletion. We attempted to induce autologous CTL from healthy donors and patients with cyclin D1-overexpressing tumors using a highly efficient T-cell expansion system based on CD40-activated B cells as antigen-presenting cells. EXPERIMENTAL DESIGN: Cyclin D1-derived, HLA-A*0201-restricted epitopes were predicted by multiple computer algorithms, screened in HLA-A2-binding assays, and used for T-cell stimulation. The generated CTL lines and clones were analyzed by IFN-gamma enzyme-linked immunosorbent spot assay or cytolysis assay. RESULTS: After screening, at least two naturally processed and presented HLA-A*0201-binding cyclin D1 epitopes were identified. CTL specific for these epitopes could be successfully generated from HLA-A2(+) donors. T cells efficiently recognized target cells pulsed with the cognate peptide and cyclin D1-expressing tumor cell lines in an HLA-A*0201-restricted manner. More importantly, HLA-A*0201-matched, primary cyclin D1(+) tumor cells were efficiently recognized by cyclin D1-specific CTL. These CTL could be generated from patients with mantle cell lymphoma and cyclin D1(+) colon cancer. CONCLUSIONS: These results underscore that cyclin D1 needs to be considered as a target for broad-based antitumor immunotherapy.     

Novel breast-tumor-associated MUC1-derived peptides: characterization in Db-/- x beta2 microglobulin (beta2m) null mice transgenic for a chimeric HLA-A2.1/Db-beta2 microglobulin single chain

The MUC1 protein was found to be up-regulated in a spectrum of malignant tumors. T-cell responses to the MUC1 extracellular tandem repeat array (TRA) were observed in murine models as well as in breast-carcinoma patients. In the present study, we evaluated the anti-tumor potential of HLA-A2.1-motif-selected peptides from non-TRA domains of the molecule. Peptide immunogenicity was examined in the Db-/- x beta2 microglobulin (beta2m) null mice transgenic for a modified HLA-A2.1/Db-beta2 microglobulin single chain (HHD mice). Our results show the existence of 3 novel HLA-A2.1-restricted MUC1-derived cytotoxic T-lymphocyte (CTL) epitopes. These peptides are processed and presented by the HHD-transfected breast-tumor cell line MDA-MB-157. Moreover, CTL induced by these 3 peptides show higher lysis of target cells pulsed with breast-carcinoma-derived peptides than of targets pulsed with normal breast-tissue-derived peptides. These data suggest an important role for non-TRA MUC1-derived peptides as inducers of a MHC-restricted CTL reaction to a breast-carcinoma cell line and patient-derived tumor extracts.     

Identification of GP100-derived,melanoma-specific cytotoxic T-lymphocyte epitopes restricted by HLA-A3 supertype molecules by primary in vitro immunization with peptide-pulsed dendritic cells

The human melanocyte lineage-specific antigen gp100 contains several epitopes recognized by cytotoxic T lymphocytes (CTL). However, most of the epitopes reported to date are HLA-A2.1-restricted. Despite the high frequency of HLA-A2.1 in melanoma patients, effective population coverage requires the identification of epitopes restricted by other frequent HLA alleles. Herein, HLA-A3 binding, gp100-derived synthetic peptides were tested for their capacity to elicit anti-melanoma CTL in vitro using CD8⁺ T cells from healthy donors as responders and peptide-pulsed autologous dendritic cells as antigen-presenting cells. Of 7 peptides tested, 2 (gp100[9₈₇] and gp100[10₈₆] ) induced CTLs that killed melanoma cell lines expressing HLA-A3 and gp100. Additional MHC-binding studies to various HLA molecules belonging to the HLA-A3 superfamily (HLA-A*1101, -A*3101, -A*3301 and -A*6801) were performed to determine whether these CTL epitopes could further increase potential population coverage. Further experiments indicated that the peptide gp100\[9₈₇\], which bound to HLA-A11 with high affinity, was capable of inducing specific CTLs that killed melanoma cells expressing gp100 and HLA-A11 molecules. Our results indicate that the gp100\[9₈₇\] peptide corresponds to a CTL epitope which may be restricted by either the HLA-A3 or HLA-A11 allele, emphasizing its utility for the design and development of epitope-based therapies for melanoma. Int. J. Cancer, 78:518–524, 1998. © 1998 Wiley-Liss, Inc.     

In Vitro Generation of Cytotoxic T Lymphocytes Against HLA-A2.1 -Restricted Peptides Derived from Human Thymidylate Synthase

Abstract

5-Fluorouracil (5-FU) is a pyrimidine antimetabolite active against colorectal carcinoma and other malignancies of the digestive tract. Over-expression or mutation of thymidylate synthase (TS), the target enzyme of the 5-FU metabolite, 5-fluorodeoxyuridine monophosphate, is strictly correlated with cancer cell resistance to 5-FU. On this basis we investigated whether TS is a potential target for active specific immunotherapy of human colon carcinoma, which acquires resistance to 5-FU. Three TS-derived epitope peptides which fit defined amino acid consensus motifs for HLA-A2.1 binding were synthesized and investigated for their ability to induce human TS-specific cytotoxic T cell (CTL) responses In Vitro. CTL lines specific for each peptide were established by stimulating peripheral blood mononuclear cells (PBMC) from an HLA-A2.1 + healthy donor with autologous dendritic cells loaded with TS peptide. Specific CTL lines showed HLA-A2.1-restricted cytotoxicity In Vitro to HLA-A2.1+ target cells pulsed with the specific TS peptide and to HLA-class I matching colon carcinoma target cells over-expressing TS enzyme after exposure to 5-FU. Recognition by CTL lines suggests that these TS peptides may be potential candidates for use in a peptide-based vaccine against 5-FU resistant colon carcinoma.     

A novel tumor-associated antigen, cell division cycle 45-like can induce cytotoxic T-lymphocytes reactive to tumor cells

The present study attempted to identify a useful tumor-associated antigen (TAA) for lung cancer immunotherapy and potential immunogenic peptides derived from the TAA. We focused on cell division cycle 45-like (CDC45L), which has a critical role in the initiation and elongation steps of DNA replication, as a novel candidate TAA for immunotherapy based on a genome-wide cDNA microarray analysis of lung cancer. The CDC45L was overexpressed in the majority of lung cancer tissues, but not in the adjacent non-cancerous tissues or in many normal adult tissues. We examined the in vitro and in vivo anti-tumor effects of cytotoxic T-lymphocytes (CTL) specific to CDC45L-derived peptides induced from HLA-A24 (A*24:02)-positive donors. We identified three CDC45L-derived peptides that could reproducibly induce CDC45L-specific and HLA-A24-restricted CTL from both healthy donors and lung cancer patients. The CTL could effectively lyse lung cancer cells that endogenously expressed both CDC45L and HLA-A24. In addition, we found that CDC45L (556) KFLDALISL(564) was eminent in that it induced not only HLA-A24 but also HLA-A2 (A*02:01)-restricted antigen specific CTL. Furthermore, the adoptive transfer of the CDC45L-specific CTL inhibited the growth of human cancer cells engrafted into immunocompromised mice. These results suggest that these three CDC45L-derived peptides are highly immunogenic epitopes and CDC45L is a novel TAA that might be a useful target for lung cancer immunotherapy.     

In vitro generation of cytotoxic T lymphocytes against HLA-A2.1-restricted peptides derived from human thymidylate synthase.

5-Fluorouracil (5-FU) is a pyrimidine antimetabolite active against colorectal carcinoma and other malignancies of the digestive tract. Over-expression or mutation of thymidylate synthase (TS), the target enzyme of the 5-FU metabolite, 5-fluorodeoxyuridine monophosphate, is strictly correlated with cancer cell resistance to 5-FU. On this basis we investigated whether TS is a potential target for active specific immunotherapy of human colon carcinoma, which acquires resistance to 5-FU. Three TS-derived epitope peptides which fit defined amino acid consensus motifs for HLA-A2.1 binding were synthesized and investigated for their ability to induce human TS-specific cytotoxic T cell (CTL) responses in vitro. CTL lines specific for each peptide were established by stimulating peripheral blood mononuclear cells (PBMC) from an HLA-A2.1+ healthy donor with autologous dendritic cells loaded with TS peptide. Specific CTL lines showed HLA-A2.1-restricted cytotoxicity in vitro to HLA-A2.1+ target cells pulsed with the specific TS peptide and to HLA-class I matching colon carcinoma target cells over-expressing TS enzyme after exposure to 5-FU. Recognition by CTL lines suggests that these TS peptides may be potential candidates for use in a peptide-based vaccine against 5-FU resistant colon carcinoma.