P. van der Bruggen

Sequence and Expression Pattern of the Human Mage2 Gene

We reported previously identification of the human MAGE1 gene, which encodes an antigen recognized on human melanoma MZ2-MEL by autologous cytolytic T lymphocytes. In addition to MAGE1, melanoma MZ2-MEL expresses several closely related genes, one of which has been named MAGE2. The complete MAGE2 sequence was obtained and it comprises 3 exons homologous to those of MAGE1 and an additional exon homologous to a region of the first MAGE1 intron. Like the open reading frame of MAGE1, that of MAGE2 is entirely encoded by the last exon. The MAGE1 and MAGE2 sequences of this exon show 82% identity and the putative proteins show 67% identity. The MAGE2 gene is expressed in a higher proportion of melanoma tumors than MAGE1. It is also expressed in many small-cell lung carcinomas and other lung tumors, laryngeal tumors, and sarcomas. No MAGE1 and MAGE2 gene expression was found in a large panel of healthy adult tissues, with the exception of testis.

Electroporation of immature and mature dendritic cells: implications for dendritic cell-based vaccines

Until now, studies utilizing mRNA electroporation as a tool for the delivery of tumor antigens to human monocyte-derived dendritic cells (DC) have focused on DC electroporated in an immature state. Immature DC are considered to be specialized in antigen capture and processing, whereas mature DC present antigen and have an increased T-cell stimulatory capacity. Therefore, the consensus has been to electroporate DC before maturation. We show that the transfection efficiency of DC electroporated either before or after maturation was similarly high. Both immature and mature electroporated DC, matured in the presence of an inflammatory cytokine cocktail, expressed mature DC surface markers and preserved their capacity to secrete cytokines and chemokines upon CD40 ligation. In addition, both immature and mature DC can be efficiently cryopreserved before or after electroporation without deleterious effects on viability, phenotype or T-cell stimulatory capacity including in vitro antigen-specific T-cell activation. However, DC electroporated after maturation are more efficient in in vitro migration assays and at least as effective in antigen presentation as DC electroporated before maturation. These results are important for vaccination strategies where an optimal antigen presentation by DC after migration to the lymphoid organs is crucial.

IFN-gamma gene transfer restores HLA-class I expression and MAGE-3 antigen presentation to CTL in HLA-deficient small cell lung cancer.

In this study, we have analyzed the possibility of inducing T cell responses against small cell lung cancer (SCLC), a still incurable tumor, by cytokine gene transfer approaches. By RT-PCR analysis most SCLC expressed the CTL-defined tumor antigens MAGE-3 (10/11), MAGE-1 (7/11) and less frequently BAGE (4/11) and GAGE1,2 (4/11). Although the surface expression of HLA class I molecules was low on most SCLC, thus preventing CTL recognition, treatment of the cells with IFN-γ enhanced HLA-class I levels in all cases. Two MAGE3+ SCLC cell lines displaying the A2 HLA-class I allele, involved in MAGE-3 antigen presentation to CTL, were stably transfected with the IFN-γ gene (alone or co-transfected with IL-2). IFN-γ- transfected cells displayed a clearcut increase in expression of HLA-class I and β2 microglobulin at both protein and mRNA level, and of TAP-1 and TAP-2 mRNA. Perhaps more importantly, IFN-γ transfected cells were recognized by the MAGE-3-specific, A2-restricted antimelanoma CTL clone 297/22, while unmodified cells or cells transfected with the IL-2 gene alone were not. These data indicate that IFN-γ gene transfection into HLA-deficient SCLC cells is able to restore their ability to present endogenous tumor antigens to CTL and that IFN-γ gene transfer approaches may be attempted to induce specific CTL responses in SCLC.

Recognition by human V gamma 9/V delta 2 T cells of melanoma cells upon fusion with Daudi cells

Abstract Daudi Burkitt’s lymphoma cells, unlike other tumor cell lines, stimulate human T cells coexpressing the variable (V) region genes TCRG-V9 and V TCRD-V2 to proliferate and secrete lymphokines. Hybrids, derived by the fusion of Daudi cells with the human melanoma cell line MZ2-MEL 2.2, retain the morphology of melanoma cells. Unlike the parental melanoma cell line, these Daudi × MZ2-MEL 2.2 hybrids stimulate secretion of tumor necrosis factor (TNF) and granulocyte/macrophage colony stimulating factor (GM-CSF) by CD4-positive Vγ9/Vδ2 T-cell clones. Whereas the stimulator phenotype of Daudi cells behaves as a dominant trait in Daudi × melanoma hybrids, the expression of B-cell differentiation markers is suppressed. Thus, the γ/δ T-cell ligand expressed by Daudi cells behaves as a dominant tumor antigen in Daudi × melanoma hybrids and is unrelated to the differentiated B-cell phenotype. Dominant expression of the Daudi ligand for human Vγ9/Vδ2 T cells in these hybrids may provide a basis for defining the stimulatory principle at the molecular level.

605 Tumor antigens recognized by cytolytic T lymphocytes

In human tumors, several antigens recognized by autologous CTL have been identified. A first class results from the activation of genes such as MAGE-1, MAGE-3, BAGE and GAGE, which are not expressed in normal tissues with the exception of testis. MAGE-derived peptides binding to HLA-A1, Cwl6 and A2 have been identified. The MAGE family comprises genes that are expressed in tumors of several histological types. A second type of antigens identified in melanoma consists of differentiation antigens derived from proteins such as tyrosinase and Melan-A that are specific for melanocytes and melanomas. Recently, we have identified a melanoma antigen which results from a point mutation in an intron. The antigenic peptide is encoded by the end of an exon and the initial part of intron. Another antigen recognized on a large fraction of HLA-A2 melanomas involves an antigenic peptide encoded by an intron. The identification of new antigens will extend the range of patients eligible for specific immunotherapy, allowing also to immunize against several antigens borne by the same tumor. This may be a critical condition for therapeutic success.

995 Genes coding for tumor rejection antigens

We have isolated a number of genes that code for antigens recognized on human melanomas by autologous cytolytic T cells (CTL). A gene named MAGE-1 codes for two different antigenic peptides that are recognized by CTL on MHC molecules HLA-A1 and HLA-Cw16 respectively. This gene belongs to a family of 12 closely related genes. No expression of these genes was found on a large panel of normal tissues except for testis. The genes of the MAGE family are all located on the q terminal region of the X chromosome. The putative proteins produced by these genes present almost identical hydrophobicity patterns, suggesting that they exert the same function, but this function remains unknown. Gene MAGE-4 carries at least eight alternative first exons preceded by different promoters. The MAGE gene family may therefore ensure that the same function is placed under the control of nineteen different promoters, allowing for very specific spatial and temporal regulation. Gene MAGE-3 codes for a second antigen presented by HLA-A1. The relevant antigenic peptide is encoded by the MAGE-3 sequence that is homologous to the MAGE-1 sequence that also codes for an antigen presented by HLA-A1. Recently, another peptide that is encoded by MAGE-3 and binds to HLA-A2 has been found to be recognized by CTL. Two additional genes that code for tumor antigens and are expressed only in tumors and in testis have been isolated. These genes, named BAGE and GAGE, are unrelated to each other and to the MAGE family. MAGE, BAGE and GAGE are expressed in a significant proportion of tumors of different histological types, such as melanomas head and neck carcinomas, non small cell lung carcinomas and bladder tumors. They are not expressed in certain types of tumors such as leukemias. Genes coding for differentiation products, such as tyrosinase and Melan A in melanomas, also code for antigens recognized by autologous CTL.