EphA2 as target of anticancer immunotherapy: identification of HLA-A*0201-restricted epitopes

EphA2 (Eck) is a tyrosine kinase receptor that is overexpressed in several human cancers such as breast, colon, lung, prostate, gastric carcinoma, and metastatic melanoma but not in nonmalignant counterparts. To validate EphA2 as a tumor antigen recognized by CD8+ T lymphocytes, we used reverse immunology approach to identify HLA-A*0201-restricted epitopes. Peptides bearing the HLA-A*0201-specific anchor motifs were analyzed for their capacity to bind and stabilize the HLA-A*0201 molecules. Two peptides, EphA2(58) and EphA2(550), with a high affinity for HLA-A*0201 were selected. Both peptides were immunogenic in the HLA-A*0201-transgenic HHD mice. Interestingly, peptide-specific murine CTLs cell lines responded to COS-7 cells coexpressing HLA-A*0201 and EphA2 and to EphA2-positive human tumor cells of various origin (renal cell, lung, and colon carcinoma and sarcoma). This demonstrates that EphA2(58) and EphA2(550) are naturally processed from endogenous EphA2. In addition, EphA2(58) and EphA2(550) stimulated specific CD8(+) T cells from healthy donor peripheral blood mononuclear cells. These T cells recognized EphA2-positive human tumor cells in an HLA-A*0201-restricted manner. Interestingly, EphA2-specific CD8+ T cells were detected in the peripheral blood mononuclear cells of prostate cancer patients. These results show for the first time that EphA2 is a tumor rejection antigen and lead us to propose EphA2(58) and EphA2(550) peptides for a broad-spectrum-tumor immunotherapy.     

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.     

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.     

A polyepitope DNA vaccine targeted to Her-2/ErbB-2 elicits a broad range of human and murine CTL effectors to protect against tumor challenge

A cDNA vaccine (pVax1/pet-neu) was designed to encode 12 different Her-2/ErbB-2-derived, HLA-A*0201-restricted dominant and high-affinity heteroclitic cryptic epitopes. Vaccination with pVax1/pet-neu triggered multiple and ErbB-2-specific CTL responses in HLA-A*0201 transgenic HHD mice and in HLA-A*0201 healthy donors in vitro. Human and murine CTL specific for each one of the 12 ErbB-2 peptides recognized in vitro both human and murine tumor cells overexpressing endogenous ErbB-2. Furthermore, vaccination of HHD mice with pVax1/pet-neu significantly delayed the in vivo growth of challenged ErbB-2-expressing tumor (EL4/HHD/neu murine thymoma) more actively when compared with vaccination with the empty vector (pVax1) or vehicle alone. These data indicate that the pVax1/pet-neu cDNA vaccine coding for a poly-ErbB-2 epitope is able to generate simultaneous ErbB-2-specific antitumor responses against dominant and cryptic multiple epitopes.     

Inducible Hsp70 as target of anticancer immunotherapy: Identification of HLA-A*0201-restricted epitopes

The design of a broad application tumor vaccine requires the identification of tumor antigens expressed in a majority of tumors of various origins. We questioned whether the major stress-inducible heat shock protein Hsp70 (also known as Hsp72), a protein frequently overexpressed in human tumors of various histological origins, but not in most physiological normal tissues, constitutes a tumor antigen. We selected the p391 and p393 peptides from the sequence of the human inducible Hsp70 that had a high affinity for HLA-A*0201. These peptides were able to trigger a CTL response in vivo in HLA-A*0201-transgenic HHD mice and in vitro in HLA-A*0201+ healthy donors. p391- and p393-specific human and murine CTL recognized human tumor cells overexpressing Hsp70 in a HLA-A*0201-restricted manner. Tetramer analysis of TILs showed that these Hsp70 epitopes are targets of an immune response in many HLA-A*0201+ breast cancer patients. Hsp70 is a tumor antigen and the Hsp70-derived peptides p391 and p393 could be used to raise a cytotoxic response against tumors of various origins.     

A wild-type sequence p53 peptide presented by HLA-A24 induces cytotoxic T lymphocytes that recognize squamous cell carcinomas of the head and neck.

Evidence has accumulated indicating that HLA-A2-restricted CTLs specific for human wild-type sequence p53 epitopes lyse tumor cells expressing mutant p53. To explore the possibility that wild-type sequence p53 peptides could also be used in vaccines for patients expressing HLA-A24 antigen, another frequent HLA class I allele, we investigated the induction of HLA-A24-restricted p53-specific CTLs from the peripheral blood lymphocytes of normal donors. Of six p53-derived peptides possessing an HLA-A24 binding motif, the p53 peptide 125-134 (p53(125-134)) was found to have a high binding capacity and induced peptide-specific CTLs from peripheral blood mononuclear cells, using peptide-pulsed autologous dendritic cells and subsequent cultivation with cytokines interleukin 2 and interleukin 7. Bulk CTL populations lysed peptide-pulsed HLA-A24+ targets as well as HLA-A24+ squamous cell carcinoma of the head and neck (SCCHN) cell lines. However, IFN-gamma pretreatment of HLA-A24+ SCCHN cell lines was necessary for lysis, suggesting that a ligand density higher than that normally expressed by tumor cells is required for these CTLs to mediate lysis. Moreover, a cloned CTL, designated TH#99, isolated from the bulk population by limiting dilution, lysed HLA-A24+ SCCHN targets more efficiently than the bulk CTL population. Lysis was inhibited by anti-HLA class I monoclonal antibody but not by anti-HLA-DR monoclonal antibody. These results indicate that HLA-A24-restricted CTLs recognizing the wild-type sequence p53(125-134) can be generated using autologous dendritic cells from precursors present in peripheral blood lymphocytes obtained from normal HLA-A24+ donors. This finding suggests that vaccine strategies targeting wild-type sequence p53 epitopes can be extended to a wider range of cancer patients.     

Identification of HLA-A2- or HLA-A24-restricted CTL epitopes possibly useful for glypican-3-specific immunotherapy of hepatocellular carcinoma.

PURPOSE AND EXPERIMENTAL DESIGN: We previously reported that glypican-3 (GPC3) was overexpressed, specifically in hepatocellular carcinoma (HCC) and melanoma in humans, and it was useful as a novel tumor marker. We also reported that the preimmunization of BALB/c mice with dendritic cells pulsed with the H-2K(d)-restricted mouse GPC3(298-306) (EYILSLEEL) peptide prevented the growth of tumor-expressing mouse GPC3. Because of similarities in the peptide binding motifs between H-2K(d) and HLA-A24 (A*2402), the GPC3(298-306) peptide therefore seemed to be useful for the immunotherapy of HLA-A24+ patients with HCC and melanoma. In this report, we investigated whether the GPC3(298-306) peptide could induce GPC3-reactive CTLs from the peripheral blood mononuclear cells (PBMC) of HLA-A24 (A*2402)+ HCC patients. In addition, we used HLA-A2.1 (HHD) transgenic mice to identify the HLA-A2 (A*0201)-restricted GPC3 epitopes to expand the applications of GPC3-based immunotherapy to the HLA-A2+ HCC patients. RESULTS: We found that the GPC3(144-152) (FVGEFFTDV) peptide could induce peptide-reactive CTLs in HLA-A2.1 (HHD) transgenic mice without inducing autoimmunity. In five out of eight HLA-A2+ GPC3+ HCC patients, the GPC3(144-152) peptide-reactive CTLs were generated from PBMCs by in vitro stimulation with the peptide and the GPC3(298-306) peptide-reactive CTLs were also generated from PBMCs in four of six HLA-A24+ GPC3+ HCC patients. The inoculation of these CTLs reduced the human HCC tumor mass implanted into nonobese diabetic/severe combined immunodeficiency mice. CONCLUSION: Our study raises the possibility that these GPC3 peptides may therefore be applicable to cancer immunotherapy for a large number of HCC patients.     

Identification of a novel tumor-associated antigen, cadherin 3/P-cadherin, as a possible target for immunotherapy of pancreatic, gastric, and colorectal cancers

PURPOSE: To establish cancer immunotherapy, it is important to identify the tumor-associated antigens (TAA) that are strongly expressed in the tumor cells but not in the normal cells. In this study, to establish an effective anticancer immunotherapy, we tried to identify the useful TAA of pancreatic cancer. EXPERIMENTAL DESIGN: Based on a previous genome-wide cDNA microarray analysis of pancreatic cancer, we focused on cadherin 3 (CDH3)/P-cadherin as a novel candidate TAA for anticancer immunotherapy. To identify the HLA-A2 (A*0201)-restricted CTL epitopes of CDH3, we used HLA-A2.1 (HHD) transgenic mice (Tgm). Furthermore, we examined the cytotoxicity against the tumor cells in vitro and in vivo of CTLs specific to CDH3 induced from HLA-A2-positive healthy donors and cancer patients. RESULTS: CDH3 was overexpressed in the majority of pancreatic cancer and various other malignancies, including gastric and colorectal cancers, but not in their noncancerous counterparts or in many normal adult tissues. In the experiment using HLA-A2.1 Tgm, we found that the CDH3-4(655-663) (FILPVLGAV) and CDH3-7(757-765) (FIIENLKAA) peptides could induce HLA-A2-restricted CTLs in Tgm. In addition, peptides-reactive CTLs were successfully induced from peripheral blood mononuclear cells by in vitro stimulation with these two peptides in HLA-A2-positive healthy donors and cancer patients, and these CTLs exhibited cytotoxicity specific to cancer cells expressing both CDH3 and HLA-A2. Furthermore, the adoptive transfer of the CDH3-specific CTLs could inhibit the tumor growth of human cancer cells engrafted into nonobese diabetic/severe combined immunodeficiency mice. CONCLUSIONS: These results suggest that CDH3 is a novel TAA useful for immunotherapy against a broad spectrum of cancers, including pancreatic cancer.     

Generation of RAGE-1 and MAGE-9 peptide-specific cytotoxic T-lymphocyte lines for transfer in patients with renal cell carcinoma

Renal cell carcinomas (RCCs) are supposed to be immunogenic, and several clinical trials of immunotherapy using tumor lysate-pulsed dendritic cells (DCs) have been performed. We report on the generation of RAGE-1 and MAGE-9 peptide-specific CTL lines. RAGE-1 and MAGE-9 are expressed in 56% and 38% of RCCs. Seven MAGE-9- and 13 RAGE-1-derived peptides were found to be immunogenic in the context of the HLA-A*0201 MHC. CTLs were generated by coculture with peptide-pulsed, activated B cells, which were easily generated in great quantities and displayed functional activity for a prolonged period of time. MAGE-9 and RAGE-1 peptide-specific CTL lines were strictly peptide-specific and displayed high cytotoxic activity not only against peptide-loaded T2 cells but also against HLA-A*0201-positive RCC lines, which naturally express MAGE-9, RAGE-1 or both. Thus, B cells are well suited as APCs for the generation of large numbers of tumor peptide-specific CTLs for adoptive transfer. MAGE-9 as well as RAGE-1 may well provide suitable targets for immunotherapy of RCC.     

HLA-A2 and -A24-restricted glypican-3-derived peptide vaccine induces specific CTLs: preclinical study using mice

We previously reported that glypican-3 (GPC3) is uniquely overexpressed in human hepatocellular carcinoma and melanoma and that it is an ideal tumor antigen for immunotherapy in mouse models. We recently identified both HLA-A24 (A*2402) and H-2Kd-restricted GPC3298-306 (EYILSLEEL) and HLA-A2 (A*0201)-restricted GPC3144-152 (FVGEFFTDV), both of which can induce GPC3-reactive cytotoxic T cells (CTLs). The present study was a preclinical study in a mouse model that was conducted in order to design an optimal schedule for clinical trial of GPC3-derived peptide vaccine. When BALB/c mice were intradermally vaccinated at the base of the tail with Kd-restricted GPC3298-306 peptide mixed with incomplete Freund's adjuvant (IFA), the peptide-specific CTLs were induced. But the peptide alone could not induce peptide-specific CD8+ T cells. Furthermore, proteomic analyses showed that IFA protected the peptide against degradation in the human serum. Peptide-reactive CTLs were induced by peptide vaccine in a dose-dependent manner. In addition, at least two vaccinations with a single dose >10 microg were needed for the induction of GPC3298-306-specific CTLs. But repeated vaccination with a lower dose of GPC3298-306 did not induce peptide-specific CTLs. Similarly, induction of an Ag-specific immune response by HLA-A2 GPC3144-152 depended on the dose administered. The results of this study suggested that IFA is one of the indispensable adjuvants for peptide-based immunotherapy, and that the immunological effect of peptide vaccines depends on the dose of peptide injected.     

Mass-spectrometric evaluation of HLA-A*0201-associated peptides identifies dominant naturally processed forms of CTL epitopes from MART-1 and gp100.

Melanoma-reactive human cytotoxic T lymphocytes (CTLs) mediate tumor regression in vivo through specific recognition of MHC-associated peptide epitopes, many of which are encoded by the melanocytic tissue differentiation proteins gp100/Pme117 and MART-1/Melan-A. Vaccines using these peptides may induce protective or therapeutic immunity against melanoma. Rational design of such approaches is aided by a clear understanding of the identity of these antigenic peptides; however, most CTL epitopes described to date were identified indirectly. Especially where these peptides may be used in human clinical trials for the treatment or prevention of cancer, there is substantial need for direct evaluation of HLA-A*0201-associated peptides from MART-1 and gp100 that are naturally processed and presented. To that end, we have isolated peptides directly from HLA-A*0201 molecules of human melanoma cells and have determined that naturally processed epitopes for HLA-A*0201-restricted, melanoma-reactive CTLs include the nonamers MART-1(27-35) (AAGIGILTV), gp100(154-162) (KTWGQYWQV), gp100(209-217) (ITDQVPFSV) and gp100(280-288) (YLEPGPVTA) and the decamer gp100(476-485) (VLYRYGSFSV). Among these, the one that appears to be most abundant at the cell surface is gp100(154-162) (KTWGQYWQV). The others are among the less abundant peptides. HLA-A*0201-restricted CTLs from one melanoma patient who has survived metastatic disease recognized MART-1(27-35) (AAGIGILTV), gp100(280-288) (YLEPGPVTA) and gp100(154-162) (KTWGQYWQV) and were cross-reactive on longer peptides that contained these nonamer sequences. These peptides, identified by both an indirect genetic approach and by a direct peptide approach, can be used for tumor vaccine strategies with confidence that they are identical to the naturally processed peptide epitopes presented at the surface of melanoma cells in association with HLA-A*0201 molecules.     

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.     

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.     

Ring finger protein 43 as a new target for cancer immunotherapy.

We have performed genome-wide exploration by using cDNA microarray profiling, and successfully identified a new tumor-associated antigen (TAA) that can induce potent cytotoxic T lymphocytes (CTLs) specific to tumor cells. In our preceding study, we identified multiple new genes by using gene expression profiling with a genome-wide cDNA microarray containing 23,040 genes. Among them, we selected RNF43 (ring finger protein 43) as a promising candidate for a TAA expressed by colon cancer cells. In this study, we examined whether the RNF43 protein contains antigenic epitope peptides restricted to HLA-A*0201 or HLA-A*2402. The CTL clones were successfully induced with stimulation by using the peptides binding to HLA-A*0201 (ALWPWLLMA and ALWPWLLMAT) and HLA-A*2402 (NSQPVWLCL), and these CTL clones showed the cytotoxic activity specific to not only the peptide-pulsed targets but also the tumor cells expressing RNF43 and respective HLAs. Lytic activities mediated by two HLA-A2-restricted epitopes were marginal, whereas tumor lysis mediated by the HLA-A24 epitope was clearly better. These findings might be caused by the poor natural presentation of RNF43-11(IX) and RNF43-11(X) by tumors or poor T-cell receptor avidity for these specific epitopes. These results strongly suggest that RNF43 is a new TAA of colon cancer. Furthermore, these results also suggest that our strategy might be a promising one to efficiently discover clinically useful TAAs.     

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.     

A MAGE-A1 HLA-A A*0201 epitope identified by mass spectrometry

Peptides presented by HLA-A*0201 molecules on the surface of the human breast carcinoma cell line KS24.22 after IFN-gamma induction were analyzed by the "Predict-Calibrate-Detect" approach, which combines epitope prediction and high-performance liquid chromatography mass spectrometry. One of the predicted epitopes, MAGE-A1(278-286) (KVLEYVIKV), was found to be presented by HLA-A*0201, with an estimated copy number of 18 molecules/cell. HLA-A*0201 transgenic mice (HHD mice) were used to generate CTL lines that stained positive with an HLA-A*0201 tetramer folded around the KVLEYVIKV peptide and killed peptide-loaded mouse target cells expressing HLA-A*0201. IFN-gamma-treated or -nontreated HLA-A*0201 expressing HeLa cells transiently transfected with a plasmid expressing the MAGE-A1 gene stimulated in vitro cytokine production by the CTL lines. Moreover, IFN-gamma-treated KS24.22 cells, but not IFN-gamma-treated HLA-A*0201(+) MAGE-A1(-) cells or IFN-gamma-treated HLA-A*0201(-) MAGE-A1(+) cells, were killed by these CTLS: Thus, the combination of HLA epitope prediction, peptide analysis, and immunological methods is a powerful approach for the identification of tumor-associated epitopes.     

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.     

Identification of an HLA-A*0201 restricted Bcl2-derived epitope expressed on tumors

A large number of human tumor-associated antigen-derived peptides have been identified that are recognized by CTLs in a MHC-I restricted fashion. The apoptosis inhibitory protein Bcl2 is overexpressed in many human cancers as part of their neoplastic phenotype. Since inhibition or loss of Bcl2 expression might impair tumor growth and survival, this protein may serve as a rational target for vaccine-induced CTL responses. By Western blot technique, we screened a panel of established human tumor cell lines for proteins involved in the apoptotic process. Two of eight tumor cell lines, a B lymphoma (Loukes) and a colon carcinoma (CCL220) cell line showed increased Bcl2 protein expression whereas the majority of tumor cell lines expressed proapoptotic proteins. Neither fibroblasts nor peripheral blood mononuclear cells showed Bcl2 expression. An HLA-A*0201 restricted CTL epitope was deduced in silica from the amino acid sequence of the Bcl2 protein and its binding affinity for HLA-A*0201 was confirmed using a biochemical binding assay. We here demonstrate that the 9-mer peptide Bcl2(85-93) induces specific CTL reactivity in immunized C57-A2K(b) or -A2D(b) tg mice. These Bcl2(85-93) specific CTLs react with and lyse Bcl2-expressing human colon carcinoma CCL220 cells which have been transfected with a chimeric HLA-A*0201/H2-K(b) DNA construct similar to that expressed in the transgenic mice. Based on these observations, we suggest that Bcl2(85-93) may be a target for immune therapy.     

Human CD8 and CD4 T cell epitopes of epithelial cancer antigens

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 !-enolase, suggesting that it was derived from the processed parental !-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.     

Identification of C-met oncogene as a broadly expressed tumor-associated antigen recognized by cytotoxic T-lymphocytes.

PURPOSE: C-Met proto-oncogene is a receptor tyrosine kinase that mediates the oncogenic activities of the hepatocyte growth factor. Using a DNA chip analysis of tumor samples from patients with renal cell carcinoma and sequencing of peptides bound to the HLA-A*0201 molecules on tumor cells a peptide derived from the c-Met protein was identified recently. EXPERIMENTAL DESIGN: We used this novel HLA-A*0201 peptide for the induction of specific CTLs to analyze the presentation of this epitope by malignant cells. RESULTS: The induced CTL efficiently lysed target cells pulsed with the cognate peptide, as well as HLA-A*0201-matched tumor cell lines in an antigen-specific and HLA-restricted manner. Furthermore, the induced c-Met-specific CTLs recognized autologous dendritic cells (DCs) pulsed with the peptide or transfected with whole-tumor mRNA purified from c-Met-expressing cell lines. We next induced c-Met-specific CTLs using peripheral blood mononuclear cells and DC from an HLA-A*0201-positive patient with plasma cell leukemia to determine the recognition of primary autologous malignant cells. These CTLs lysed malignant plasma cells while sparing nonmalignant B- and T-lymphocytes, monocytes, and DCs. CONCLUSION: Our results demonstrate that c-Met oncogene is a novel tumor rejection antigen recognized by CTL and expressed on a broad variety of epithelial and hematopoietic malignant cells.