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.     

Induction and characterization of cytotoxic T-lymphocytes recognizing a mutated p21ras peptide presented by HLA-A*0201

The ras oncogene is frequently found to be activated in human cancer through point mutations at codons 12, 13 or 61. We explored whether these altered p21 ras protein sequences contain peptide sequences that can activate naive CD8⁺ cytotoxic T lymphocytes (CTL). Several wild-type and mutated p21 ras peptides were identified that carry a binding motif for human leukocyte antigen (HLA)-A*0201. Two peptides were found to bind strongly to this allele. CD8* CTL bulk cultures specifically reacting with one of these peptides could be induced, using processing-defective T2 cells loaded with peptide CLLDILDTAGL as stimulators. The peptide is derived from p21ras, position 51–61, and carries a 61 Gln → Leu mutation. In contrast, a 9-mer peptide CLLDILDTA corresponding to amino acid sequence 51–59 of wild-type p21ras did not yield reactive CTL cultures. T-cell clones with low affinity for the 11-mer peptide were isolated from CLLDILDTAGL-reactive bulk cultures. These T cells did not lyse melanoma cells transfected with 61-Leu N-ras, although lysis was found when these transfectants were pulsed with the 11-mer peptide. Possibly, T cells of higher affinity may be required to demonstrate processed peptide on the cell surface. The combined experiments suggest that a peptide derived from mutated p21ras can be recognized by HLA class 1-restricted CTL, whereas an analogous wild-type p21ras peptide may not be immunogenic. © 1995 Wiley-Liss, Inc.     

Identification of new HLA-A*0201-restricted cytotoxic T lymphocyte epitopes from neuritin

Identification of cytotoxic T lymphocyte (CTL) epitopes from additional tumor antigens is essential for the development of specific immunotherapy of malignant tumors. Neuritin, a recently discovered antigen overexpressed in astrocytoma, is considered to be a promising target for biological therapy. In the present study, we predicted and identified HLA-A2-restricted CTL epitopes from neuritin by using the following four-step procedure: (1) computer-based epitope prediction from the amino acid sequence of neuritin; (2) peptide-binding assay to determine the affinity of the predicted peptide with HLA-A2.1 molecule; (3) stimulation of primary T cell response against the predicted peptides in vitro; and (4) testing of the induced CTLs toward target cells expressing neuritin and HLA-A2.1. The results demonstrated that effectors induced by peptides of neuritin containing residues 13–21, 121–129 and 4–12 could specifically-secrete interferon-γ and lyse target cells. Our results indicate that these peptides are new HLA-A2.1-restricted CTL epitopes, and may serve as valuable tools for astrocytoma immunotherapy.     

Identification of a new HLA-A*0201-restricted cryptic epitope from CYP1B1

Cytochrome P450 1B1 (CYP1B1) was recently shown to be a candidate tumor antigen broadly expressed in solid and hematologic malignancies. Nevertheless, use of such self-antigens as targets for immune intervention can be limited because of loss of high-avidity T cells during negative selection in the thymus. Recent data suggest that targeting of cryptic epitopes may represent a way to circumvent such self-tolerance and induce efficient antitumor CTL responses. Here, we present the identification and characterization of a novel, cryptic HLA-A*0201-binding peptide from CYP1B1. The nanomer CYP246 was identified by epitope deduction using algorithms to predict HLA-A*0201-binding peptides. CYP246 is characterized by strong initial HLA-A*0201 binding but a short MHC/peptide binding half-life. Expansion of high-avidity CTL was readily possible using autologous CD40-activated B cells from normal donors and cancer patients as antigen-presenting cells, suggesting that an intact T-cell repertoire can be expanded for this epitope. Lysis of CYP1B1-expressing, HLA-A*0201+ tumor cell lines and primary tumor cells confirmed that sufficient levels of CYP246 are presented by tumor cells for effector CTL killing. These findings indicate that CYP246 is a candidate cryptic epitope for immune interventions in which tumor CYP1B1 is targeted.     

A MAGE-A3 peptide presented by HLA-DP4 is recognized on tumor cells by CD4+ cytolytic T lymphocytes

Antigens encoded by MAGE-A3 and recognized by T cells are interesting targets for tumor immunotherapy because they are strictly tumor specific and shared by many tumors of various histological types. A number of MAGE-A3 antigenic peptides presented by HLA class I molecules have been used in clinical trials, and regressions of melanoma metastasis have been observed. We report here the identification of a MAGE-A3 epitope, TQHFVQENYLEY, presented to CD4+ T lymphocytes by HLA-DP4 molecules, which are expressed in approximately 76% of Caucasians. This new epitope may be useful both for therapeutic vaccination and for the evaluation of the immune response in cancer patients. Interest ingly, the CD4+ T cells lysed HLA-DP4 tumor cells expressing MAGE-A3, indicating that this epitope, in contrast to other class-II MAGE-A3 epitopes, is presented at the surface of tumor cells. The study of this disparity in the presentation of two epitopes from the same protein may lead to a better understanding of the endogenous class II presentation pathway.     

Identification of a naturally processed HLA-A*0201 HPV18 E7 T cell epitope by tumor cell mediated in vitro vaccination

Immunotherapy of HPV-associated disease such as cervical cancer is moving from preclinical investigation to clinical trials. The viral oncoproteins E6 and E7 are ideal target antigens because their expression is mandatory in HPV-transformed tumor cells. T cells are the most important effector cells for therapeutic vaccination strategies. Therefore, the identification and characterization of HPV E6 and E7 T cell epitopes is necessary. Methods to date rely on screening for immunogenicity of peptides predicted by algorithms. Presentation of the identified peptides on tumor cells, however, needs to be confirmed. In our study, we have improved the method to identify peptide epitopes of HPV18 E7 that are actually presented by tumor cells. We induced allogeneic T-cell lines by stimulation with HPV18-positive, CD80 and HLA-A*0201 transfected cervical cancer cells. Sensitized T cells were probed against an array of a HPV18 E7 20mer peptide-library. We found specific reactivity to one of the 20mer peptides. This sequence was then screened via algorithms for putative epitopes. One putative HLA-A2 restricted epitope was confirmed to bind to HLA-A2, to be immunogenic and to induce IFN gamma-release in ELISpot assays. Epitope-specific T cells were cytolytic toward autologous peptide pulsed targets and HPV18 transformed tumor cells. The identification of epitope-specific T cells in tumor infiltrating lymphocytes of a HPV18-positive HLA-matched cervical cancer patient suggests an in vivo relevance of the identified epitope. We suggest that our approach is advantageous over conventional methods, because it yields candidate peptides that are relevant CTL epitopes that are expressed, processed and presented by tumor cells.     

Establishment of an HLA-A*0201 human papillomavirus type 16 tumor model to determine the efficacy of vaccination strategies in HLA-A*0201 transgenic mice

With the increasing generation of new cancer vaccine strategies, there is also an increasing demand for preclinical models that can carefully predict the efficacy of these vaccines in humans. However, the only tumor models available to study vaccines against human papillomavirus (HPV) 16 have been developed in C57BL/6 mice. To test the HLA-restricted capabilities of vaccination strategies, it is important to establish a tumor model in HLA-A*0201 transgenic mice. By transfecting heart lung fibroblasts from HLA-A*0201 mice with HPV16 E6 and E7 oncogenes and H-Ras V12, we have generated a transgenic cell line that is tumorigenic in HLA-A*0201 mice. The dominant H-2D(b) HPV16 E7 epitope was removed from the E7 construct to ensure that all antitumor responses were mediated through the HLA-A*0201-restricted epitopes. We used this tumor model to test the efficacy of two genetic vaccines: a plasmid DNA multi-epitope vaccine encoding human epitopes of HPV16, and a Venezuelan equine encephalitis (VEE) virus-based vector to deliver HPV16 E6 and E7 RNA. We show that both our multi-epitope DNA- and VEE-based vaccines protect 100% of HLA-A*0201 transgenic mice from tumor challenge and elicit a specific T-cell response against multiple HLA-A*0201-restricted HPV16 epitopes. Furthermore, both vaccines significantly decreased tumor burden when tested therapeutically. In conclusion, this is the first tumor model that allows for the assessment of the potential of a vaccine to induce HPV-directed, HLA-A*0201-restricted, antitumor responses in mice. These results pave the way for the clinical evaluation of these vaccines.     

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.     

Identification of HLA-A*0201-restricted CTL epitopes encoded by the tumor-specific MAGE-2 gene product

MAGE-2 is expressed in many tumors, including melanoma, laryngeal tumors, lung tumors and sarcomas, but not in healthy tissue, with the exception of testis. Thus, MAGE-2-derived peptides that bind to HLA class I molecules and elicit cytotoxic T lymphocyte (CTL) responses could be of significant therapeutic importance. In this study, we show that several MAGE-2-derived peptides bind with high affinity to HLA-A*0201. Three of them form complexes with HLA-A*0201 that are stable at 37°C and are immunogenic in HLA-A*0201K^b transgenic mice. Moreover, CTLs against 2 of them (M2 112-120, and M2 157-166) specifically recognize cells that express both the MAGE-2 protein and HLA-A*0201K^b. These 2 peptides are processed and presented in the context of HLA-A*0201. Therefore, these peptides are candidate components in peptide-based vaccines for the treatment and prevention of several types of MAGE-2-expressing cancers. Int. J. Cancer 73:125–130, 1997. © 1997 Wiley-Liss, Inc.     

A novel DNA methyltransferase I-derived peptide eluted from soluble HLA-A*0201 induces peptide-specific, tumor-directed cytotoxic T cells

MHC peptides derived from tumor-associated antigens (TAAs) can serve as the basis for the development of immunotherapeutics to treat human malignancies. Previously, we identified novel HLA-A*0201 (HLA-A2)-restricted peptides recovered from soluble HLA molecules secreted by human tumor cell lines, transfected with truncated genes of HLA-A2 and HLA-B7. Here, 4 candidate peptides eluted from soluble HLA-A2 were selected on the basis of their precursor proteins being TAAs. Peptide p1028 (GLIEKNIEL), derived from DNA methyltransferase I (DNMT-1), which is overexpressed in various human tumors, showed the highest affinity to HLA-A2 and was relatively abundant in the sMHC/peptide complexes of all transfected breast, ovarian and prostate cancer cell lines. Peptide p1028-specific CTLs were generated in vitro and shown to efficiently lyse not only target cells pulsed with the peptide but also HLA-A2-positive breast cancer cell lines MDA-231 and MCF-7. The peptide induced IFN-gamma production in CTLs, which were selectively stained by a p1028 tetramer. Since DNMT-1 is a widely expressed tumor-associated enzyme, the novel DNMT-1-derived, HLA-A2-restricted peptide GLIEKNIEL identified here may provide a suitable candidate for a therapeutic cancer vaccine.     

Two new tumor-specific antigenic peptides encoded by gene MAGE-C2 and presented to cytolytic T lymphocytes by HLA-A2

We have identified 2 antigens recognized by several melanoma-specific cytolytic T lymphocyte clones isolated from a melanoma patient with a clinical history of tumor regression after immunotherapy. Both antigens are presented by HLA-A2 and encoded by gene MAGE-C2, a cancer-germline gene shown previously to be silent in normal somatic tissues and expressed in 40% of melanomas and in other tumor types. One antigen corresponds to peptide ALKDVEERV(336-344), whereas the other corresponds to peptide LLFGLALIEV(191-200). The CTL clones recognizing these 2 peptides also recognized allogeneic tumor cell lines expressing MAGE-C2 and HLA-A2. These 2 new peptides are the first known MAGE-C antigens and represent promising targets for cancer immunotherapy.     

Identification of HLA-A*0201-restricted cytotoxic T lymphocyte epitope from proliferating cell nuclear antigen

Peptide-based immunotherapy strategies appear promising as an approach to successfully induce an antitumor immune response and prolong survival in patients with various cancers. Protein antigens and their specific epitopes are formulation targets for anti-tumor vaccines. Bioinformatical approaches to predict major histocompatibility complex binding peptides can facilitate the resource-consuming effort of T cell epitope identification. Proliferating cell nuclear antigen including Ki-67 and PCNA, associated with the proliferation process of the cell, seems to be an attractive new target for tumor-specific immunotherapy. In this study, we predicted seven HLA-A*0201-restricted CTL candidate epitope of Ki-67 and eight epitope of PCNA by computer algorithm SYFPEITHI, BIMAS, and IEDB_ANN. Subsequently, biological functions of these peptides were tested by experiments in vitro. We found Ki-67_(280–288) (LQGETQLLV) had the strongest binding-affinity with HLA-A*0201. Further study revealed that Ki-67_(280–288) increased the frequency of IFN-γ-producing T cells compared to a negative peptide. Because Ki-67 was broadly expressed in most advanced malignant tumors, indicating a potential anti-tumor application in the future.     

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.     

Characterization of antigenic peptides presented by HLA-B44 molecules on tumor cells expressing the gene MAGE-3

The amino acid sequence of the protein encoded by the gene MAGE-3was screened for peptides containing the binding motif for HLA-B44. Nine peptides were synthesized, and their binding affinity for HLA-B*4402 and -B*4403 was analyzed in an HLA class I α-chain refolding assay. Four peptides with binding affinity for HLA-B*4403 were chosen for in vitro cytotoxic T-lymphocyte induction assays using as antigen-presenting cells peptide-pulsed, autologous activated B lymphoblasts from a healthy, B*4403⁺ donor. Peptide-specific effectors could be raised only against one peptide, M3-167. Cytotoxic T lymphocytes specific for this peptide were also able to recognize melanoma cell lines expressing HLA-B44 and the gene MAGE-3, strongly suggesting that M3-167is a naturally processed MAGE-3-encoded epitope presented by HLA-B44.M3-167 is a 1 amino acid N-terminal extension of M3-168, a naturally processed epitope MAGE-3-encoded epitope presented by HLA-A1 that has been previously described. TAP binding studies of these 2 peptides revealed that the TAP affinity of M3-167 is about 9-fold higher than that of M3-168. M3-167 or a longer precursor could be transported into the endoplasmatic reticulum, where it could be trimmed for presentation by HLA-A1 or -B44 molecules. Taken together, our data suggest that M3-167 could be an immunodominant peptide encoded by the gene MAGE-3. © 1996 Wiley-Liss, Inc.     

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.     

Identification of an HLA-A*0201 cytotoxic T lymphocyte epitope specific to the endothelial antigen Tie-2

Tie-2 stabilises pericyte-endothelial interactions during angiogenesis and is highly expressed on endothelium during several diseases, including arthritis, age-related macular degeneration and cancer. A vaccine that targets endothelium overexpressing Tie-2 may result in vessel damage and stimulate an inflammatory cascade resulting in disease regression. We have identified a region unique to Tie-2 (amino acids 1-196) that is homologous in humans and mice. Using computer algorithms, several HLA-A*0201 epitopes that are identical in mice and humans were predicted within this region; however, binding assays showed that the majority of these epitopes were of low affinity. Modification of the anchor residues of 4 epitopes enhanced HLA binding. These epitopes were incorporated by site-directed mutagenesis into a Tie-2 DNA construct. Immunisation of HLA*0201 transgenic mice with one of the modified Tie-2 constructs stimulated CTLs that recognised both wild-type and modified peptide-pulsed target cells. In contrast, no CTLs were generated in mice immunised with wild-type Tie-2 construct, demonstrating that the modified epitope was necessary in the generation of CTLs. Moreover, CTLs from mice immunised with the modified construct killed HLA-A*0201 endothelial cells overexpressing Tie-2. Our study demonstrates that it is possible to break tolerance to the endothelial antigen Tie-2, suggesting that it may be feasible to design a vaccine to activate CTLs to kill endothelial cells overexpressing Tie-2.     

Small cell lung carcinomas express shared and private tumor antigens presented by HLA-A1 or HLA-A2.

Tumor-derived peptides presented by MHC class I molecules are targets for tumor rejection by CD8+ CTLs. MHC-restricted CD8+ CTLs are required also for the identification and characterization of tumor antigens that will be useful for immune therapy. For many human solid tumors, however, tumor antigens remain undefined because of the difficulty of generating MHC-restricted, tumor-specific CTLs required for their analysis. CD8+ CTL responses are modulated by CD4+ helper T cells and by antigen-presenting cells. In this study, highly purified CD8+ T cells were mixed with tumor cells in primary cultures in the absence of any other cells to reduce the complexity of CTL generation. Tumor cells were transfected with HLA-A1 or HLA-A2 and used to stimulate partly matched HLA-A1- or HLA-A2-positive CD8+ T cells. Partial MHC class I matching of tumor and CD8+ T cells and omission of other cells in primary culture was highly effective in generating MHC class I-restricted CTL to poorly immunogenic small cell lung carcinomas (SCLCs). Cytotoxicity was further enhanced by cotransfection of tumor cells with B7.1 (CD80). ICAM-1 (CD54) was not as effective as costimulation. SCLC cells presented tumor-specific peptides with HLA-A1 and HLA-A2 and were lysed by A1- or A2-restricted CD8+ CTLs. A1- and A2-restricted CD8+ CTLs detected shared tumor antigens on unrelated SCLC tumor lines in addition to private antigens. The use of direct antigen presentation by MHC class I-transfected tumors to MHC class I-matched CD8+ T cells is an effective way to generate MHC class I-restricted CTLs toward poorly immunogenic tumors in vitro, permitting the molecular identification of their tumor antigens.