MAGE-3 Peptide Research Articles

Variable expression on lung cancer cell lines of HLA-A2-binding MAGE-3 peptide recognized by cytotoxic T lymphocytes.

Cytotoxic T lymphocytes (CTL) specific for HLA-A2-binding MAGE-3 peptide (FLWGPRALV) were generated by repetitive stimulation of PBMC with the peptide in the presence of EBV-transformed B blasts and IL-2. Using these CTL, we investigated the expression of the HLA-A2-binding MAGE-3 peptide on lung cancer cell lines. Of 14 cell lines investigated, 1-87, PC-9, OU-LC-KI, 11-18 and LK87 were derived from HLA-A2 positive patients. But cytofluorometry analysis showed that 1-87, PC-9 and OU-LC-KI, but not 11-18 or LK87 expressed the HLA-A2 antigen. All five cell lines expressed MAGE-3 gene mRNA. Twelve of thirteen CTL lines from two HLA-A2 positive donors showed no cytotoxicity against any of the 14 lung cancer cell lines. CTL line TI-1 showed cytotoxicity against 1-87 but not against any of the other cell lines. Treatment of 1-87 with IFN-gamma greatly augmented the cytotoxicity of TI-1 and induced it in the other 12 CTL lines, confirming the expression of the peptide on 1-87. No cytotoxicity was induced by IFN-gamma treatment of PC-9 or OU-LC-KI. However, PC-9 and OU-LC-KI pulsed with the peptide were killed efficiently by all of the CTL lines, suggesting no expression of the peptide on those cells. A low level of cytotoxicity was induced on 11-18 but not LK87 by IFN-gamma treatment, although expression of the HLA-A2 antigen was not observed by cytofluorometry. These findings showed that expression of the HLA-A2-binding MAGE-3 peptide recognized by CTL was variable on lung cancer cell lines.

Autologous human dendriphages pulsed with synthetic or natural tumor peptides elicit tumor-specific CTLs in vitro.

The recent identification of tumor-associated antigens and tumor-associated antigen-derived peptide epitopes recognized by cytolytic T lymphocytes (CTLs) in the context of major histocompatibility complex (MHC) class I molecules has prompted the development of peptide-based vaccines for the treatment of human cancers, particularly melanoma. The design of such clinical protocols requires an understanding of the inherent immunogenicity of the peptide(s) and a choice of a facilitating adjuvant promoting cellular immunity against these peptides. We have evaluated the abilities of a series of defined synthetic peptide epitopes derived from MART-1/Melan-A, gp100, tyrosinase, and MAGE-3 or unfractionated peptides naturally presented by melanoma MHC molecules to elicit HLA-A2-restricted and melanoma-reactive CTLs from the peripheral blood of normal donors or patients with metastatic melanoma. Autologous peripheral blood dendritic cells (DCs), which were easily generated from all donors when cultured in the presence of recombinant human interleukin-4 and recombinant human granulocyte-macrophage colony-stimulating factor were pulsed with melanoma peptides and used to "prime" and/or "boost" CTL cultures in vitro. Our results suggest that antimelanoma CTLs may be reproducibly generated in short-term in vitro cultures in this manner using either a subset of the defined synthetic peptides (MART-1/Melan-A27-35, MART-1/Melan-A32-40, gp100(280-288), tyrosinase368-376, and MAGE-3(271-279)) or unfractionated peptides (containing both idiotypic and shared melanoma epitopes) derived from freshly isolated autologous melanoma lesions. These in vitro data support the use of autologous DCs prepulsed with such peptides as an appropriate antigen adjuvant delivery system in melanoma peptide-based vaccines.

Vaccine Trials for the Clinician: Prospects for Tumor Antigens

Recent insights in antigen presentation, the identification of human tumor antigens, and the demonstration of MHC class-I-restricted cytotoxic T lymphocyte (CTL) recognition of peptides encoded by tumor antigen have renewed the interest and enthusiasm for the development of cancer vaccines. Melanoma serves as a paradigm of an immunogenic human tumor, and several tumor antigens, including MAGE, MART-1/Melan-A and gp100, recognized by CTLs, have now been isolated. Candidate antigens for novel vaccine trials may include HLA class-I-binding tumor peptides that serve as CTL epitopes, whole tumor protein, or DNA-based vaccines. Requirements for the use of peptides are that the patient's tumor presents the relevant CTL epitopes as used in the vaccine and expresses the appropriate MHC class-I-restricting molecule. Immunological monitoring may be facilitated when using peptide-based vaccines. Because optimal presentation of tumor antigens may depend on provision of appropriate costimulatory signals, it may be more advantageous to administer professional antigen-presenting cells (APCs), such as dendritic cells (DCs) pulsed with tumor peptide or protein, to cancer patients. Developments in molecular genetics have led to a new approach in vaccines consisting of cancer cells genetically engineered to express immunomodulatory molecules. This may result in increased antitumor responses to both gene-modified as well as unmodified tumor cells. The therapeutic approach is extended to vaccination trials with recombinant viruses containing the genes encoding tumor antigens, minigenes containing multiple CTL epitopes, or double recombinant vectors engineered to express both the tumor antigen and immunostimulatory molecules. Clinical peptide, protein and DNA-based vaccine trials have recently been initiated. Thus far, exciting clinical remissions were obtained in melanoma patients following vaccination with HLA-A1-binding MAGE-3 peptide and in B-cell lymphoma patients immunized with autologous DCs pulsed with anti-idiotype protein, i.e., the individual patient's unique tumor antigen. Also, following injection of foreign HLA-B7 DNA into cutaneous melanoma metastases, T-cell migration into treated lesions and enhanced cellular immunity at the site of the tumor were shown in some patients. These encouraging results suggest that effective new vaccines in cancer will be identified.

Efficient induction of antitumor cytotoxic T lymphocytes from a healthy donor using HLA-A2-restricted MAGE-3 peptide in vitro.

The antigenic peptides encoded by tumor-rejection antigen genes, MAGE-1 and -3, have been identified, and various methods have been utilized for the in vitro induction of MAGE-specific, cytotoxic T lymphocytes (CTL) from peripheral blood mononuclear cells (PBMC) using synthetic peptides. However, all of these methods are technically demanding and thus have a relatively limited usefulness. We herein report a simple and efficient method for the vitro induction of specific CTL by using the HLA-A2-restricted MAGE-3 peptide from the PBMC of a healthy donor. CTL responses could thus be efficiently induced from unseparated PBMC by stimulation with freshly isolated, peptide-pulsed PBMC as antigen-presenting cells and by using interleukin-7 and keyhole limpet hemocyanin for the primary culture. The induced CTL could thus recognize and lyse not only HLA-A2 target cells pulsed with the peptide but also HLA-A2 tumor cells expressing MAGE-3, in an HLA-class-I-restricted manner. This simple method may, therefore, become a useful tool for investigating the potential peptides for tumor antigens as well as for developing various immunotherapeutic approaches for human malignant tumors.

A peptide encoded by the human MAGE3 gene and presented by HLA-B44 induces cytolytic T lymphocytes that recognize tumor cells expressing MAGE3.

The human MAGE3 gene is expressed in a significant proportion of tumors of various histological types, but is silent in normal adult tissues other than testis and placenta. Antigens encoded by MAGE3 may therefore be useful targets for specific antitumor immunization. Two antigenic peptides encoded by the MAGE3 gene have been reported previously. One is presented to cytolytic T lymphocytes (CTL) by HLA-A1, the other by HLA-A2 molecules. Here we show that MAGE3 also codes for a peptide that is presented to CTL by HLA-B44. MAGE3 peptides containing the HLA-B44 peptide binding motif were synthesized. Peptide MEVDPIGHLY, which showed the strongest binding to HLA-B44, was used to stimulate blood T lymphocytes from normal HLA-B44 donors. CTL clones were obtained that recognized not only HLA-B44 cells sensitized with the peptide, but also HLA-B44 tumor cell lines expressing MAGE3. The proportion of metastatic melanomas expressing the MAGE3/HLA-B44 antigen should amount to approximately 17% in the Caucasian population, since 24% of individuals carry the HLA-B44 allele and 76% of these tumors express MAGE3.

HLA Photoaffinity Labeling Reveals Overlapping Binding of Homologous Melanoma-associated Gene Peptides by HLA-A1, HLA-A29, and HLA-B44

Melanoma-associated genes (MAGEs) encode tumor-specific antigens that can be recognized by CD8+ cytotoxic T lymphocytes. To investigate the interaction of the HLA-A1-restricted MAGE-1 peptide 161-169 (EADPT-GHSY) with HLA class I molecules, photoreactive derivatives were prepared by single amino acid substitution with N beta-[iodo-4-azidosalicyloyl]-L-2,3-diaminopropionic acid. These derivatives were tested for their ability to bind to, and to photoaffinity-label, HLA-A1 on C1R.A1 cells. Only the derivatives containing the photoreactive amino acid in position 1 or 7 fulfilled both criteria. Testing the former derivative on 14 lymphoid cell lines expressing over 44 different HLA class I molecules indicated that it efficiently photoaffinity-labeled not only HLA-A1, but possibility also HLA-A29 and HLA-B44. MAGE peptide binding by HLA-A29 and HLA-B44 was confirmed by photoaffinity labeling with photoreactive MAGE-3 peptide derivatives on C1R.A29 and C1R.B44 cells, respectively. The different photoaffinity labeling systems were used to access the ability of the homologous peptides derived from MAGE-1, -2, -3, -4a, -4b, -6, and -12 to bind to HLA-A1, HLA-A29, and HLA-B44. All but the MAGE-2 and MAGE-12 nonapeptides efficiently inhibited photoaffinity labeling of HLA-A1, which is in agreement with the known HLA-A1 peptide-binding motif (acidic residue in P3 and C-terminal tyrosine). In contrast, photoaffinity labeling of HLA-A29 was efficiently inhibited by these as well as by the MAGE-3 and MAGE-6 nonapeptides. Finally, the HLA-B44 photoaffinity labeling, unlike the HLA-A1 and HLA-A29 labeling, was inhibited more efficiently by the corresponding MAGE decapeptides, which is consistent with the reported HLA-B44 peptide-binding motif (glutamic acid in P2, and C-terminal tyrosine or phenylalanine). The overlapping binding of homologous MAGE peptides by HLA-A1, A29, and B44 is based on different binding principles and may have implications for immunotherapy of MAGE-positive tumors.

A peptide encoded by the human MAGE3 gene and presented by HLA-B44 induces cytolytic T lymphocytes that recognize tumor cells expressing MAGE3

The humanMAGE3 gene is expressed in a significant proportion of tumors of various histological types, but is silent in normal adult tissues other than testis and placenta. Antigens encoded byMAGE3 may therefore be useful targets for specific antitumor immunization. Two antigenic peptides encoded by theMAGE3 gene have been reported previously. One is presented to cytolytic T lymphocytes (CTL) by HLA-A1, the other by HLA-A2 molecules. Here we show that MAGE3 also codes for a peptide that is presented to CTL by HLA-1344.MAGE3 peptides containing the HLA-1344 peptide binding motif were synthesized. Peptide MEVDPIGHLY, which showed the strongest binding to HLA-1344, was used to stimulate blood T lymphocytes from normal HLA-1344 donors. CTL clones were obtained that recognized not only HLA-B44 cells sensitized with the peptide, but also HLA-B44 tumor cell lines expressingMAGE3. The proportion of metastatic melanomas expressing theMAGE3/HLA-1344 antigen should amount to approximately 17% in the Caucasian population, since 24% of individuals carry theHLA-B44 allele and 76% of these tumors express MAGE3.

Synthesis of a Radioiodinated Photoreactive MAGE-1 Peptide Derivative and Photoaffinity Labeling of Cell-Associated Human Leukocyte Antigen-A1 Molecules

The synthesis of a photoreactive derivative of the human leukocyte antigen-A1 (HLA-A1)-restricted MAGE-1 peptide 161-169 (EADPTGHSY) is described. Using conventional automated solid-phase peptide synthesis, a photoreactive derivative of this peptide was synthesized by replacing histidine-167 with photoreactive N-β-4-azidosalicyloyl-L-2,3-diaminopropionic acid. The C-terminal tyrosine was incorporated as phosphotyrosine. This peptide derivative was radioiodinated in the presence of chloramine T. This iodination took place selectively at the photoreactive group, because the phosphate ester prevented tyrosine iodination. Following dephosphorylation with alkaline phosphatase and chromatographic purification, the radiolabeled peptide derivative was incubated with cells expressing HLA-A1 or other HLA molecules. Photoactivation resulted in efficient photo affinity labeling of HLA-A1. Other HLA molecules or other cellular components were not detectably labeled. This labeling was inhibited by HLA-A1 but not by HLA-A2-binding peptides. This synthesis is generally applicable and can also be adapted to the synthesis of well-defined radiolabeled nonphotoreactive peptide derivatives.

Human melanoma antigen MAGE-1 peptide; tumor-associated antigen gene cloning and expression for use in antitumor recombinant vaccine production : Cytel World 9504 542; 16 February 1995

Human melanoma antigen MAGE-1 peptide; tumor-associated antigen gene cloning and expression for use in antitumor recombinant vaccine production : Cytel World 9504 542; 16 February 1995

A peptide encoded by human gene MAGE-3 and presented by HLA-A2 induces cytolytic T lymphocytes that recognize tumor cells expressing MAGE-3.

The human MAGE-3 gene is expressed in many tumors of several histological types but it is silent in normal tissues, with the exception of testis. Antigens encoded by MAGE-3 may, therefore, be useful targets for specific anti-tumor immunization of cancer patients. We reported previously that MAGE-3 codes for an antigenic peptide recognized on a melanoma cell line by autologous cytolytic T lymphocytes (CTL) restricted by HLA-A1. Here we report that the MAGE-3 gene also codes for another antigenic peptide that is recognized by CTL restricted by HLA-A2. MAGE-3 peptides bearing consensus anchor residues for HLA-A2 were synthesized and tested for binding. T lymphocytes from normal individuals were stimulated with autologous irradiated lymphoblasts pulsed with each of three peptides that showed strong binding to HLA-A2. Peptide FLWGPRALV was able to induce CTL. We obtained CTL clones that recognized not only HLA-A2 cells pulsed with this peptide but also HLA-A2 tumor cell lines expressing the MAGE-3 gene. The proportion of melanoma tumors expressing this antigen should be approximately 32% in Caucasian populations, since 49% of individuals carry the HLA-A2 allele and 65% of melanomas express MAGE-3.

Human gene MAGE-3 codes for an antigen recognized on a melanoma by autologous cytolytic T lymphocytes.

Human melanoma cell line MZ2-MEL expresses several antigens recognized by autologous cytolytic T lymphocyte (CTL) clones. We reported previously the identification of a gene, named MAGE-1, that codes for one of these antigens named MZ2-E. We show here that antigen MZ2-D, which is present on the same tumor, is encoded by another member of the MAGE gene family named MAGE-3. Like MAGE-1, MAGE-3 is composed of three exons and the large open reading frame is entirely located in the third exon. Its sequence shows 73% identity with MAGE-1. Like MZ2-E, antigen MZ2-D is presented by HLA-A1. The antigenic peptide of MZ2-D is a nonapeptide that is encoded by the sequence of MAGE-3 that is homologous to the MAGE-1 sequence coding for the MZ2-E peptide. Competition experiments using single Ala-substituted peptides indicated that amino acid residues Asp in position 3 and Tyr in position 9 were essential for binding of the MAGE-1 peptide to HLA-A1. Gene MAGE-3 is expressed in many tumors of several types, such as melanoma, head and neck squamous cell carcinoma, lung carcinoma and breast carcinoma, but not in normal tissues except for testes. It is expressed in a larger proportion of melanoma samples than MAGE-1. MAGE-3 encoded antigens may therefore have a wide applicability for specific immunotherapy of melanoma patients.

Potential role of dendritic cell vaccination with MAGE peptides in gastrointestinal carcinomas

Dendritic cells (DCs) loaded with tumor antigens have been emerging as a new strategy in cancer treatment. The MAGE genes are selectively expressed in a variety of cancer tissues such as melanoma or gastrointestinal carcinomas. However, no expression is observed in normal tissues except testis. There are several reports on clinical trials with these immunogenic peptides including MAGE gene-derived, which were shown to be effective for some patients with carcinomas. We previously reported a clinical trial treating gastrointestinal carcinoma patients with immature DC and MAGE peptides via intravenous injection. Autologous DCs were generated ex vivo and were pulsed with MAGE-3 peptide, depending on the patient's HLA haplotype (HLA-A02 or A24). In this study, patients were immunized with mature DCs pulsed with the MAGE-3 peptide four times every 2 weeks via s.c. injection close to the axilla and inguinal lymph nodes. Twenty-eight patients with advanced gastrointestinal carcinoma were treated and no toxic side effects were observed. Peptide-specific CTL responses, improvement in performance status, tumor marker decrease and minor tumor regressions after vaccination were observed in some patients. These results suggested that DC vaccination with the MAGE-3 peptide would be safe and can exhibit antitumor effects even in the patients with advanced gastrointestinal carcinoma who were previously treated with chemotherapy or radiation therapy.