E De Plaen

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.

Structure of the Gene of Tum- Transplantation Antigen-p35b - Presence of a Point Mutation in the Antigenic Allele

Mutagen treatment of P815 tumour cells produces tum‐ variants that are rejected by syngeneic mice because they express new transplantation antigens. These ‘tum‐’ antigens elicit a cytolytic T lymphocyte (CTL) response but no detectable antibody response. The DNA of tum‐ variant P35 was transfected into P815 cell line P1.HTR. Transfectants expressing tum‐ antigen P35B were identified on the basis of their ability to stimulate anti‐P35B CTL. This was repeated with a cosmid library and a cosmid carrying the sequence encoding antigen P35B was recovered from a transfectant expressing the antigen. Gene P35B is 6 kb long and contains 11 exons. The sequence shows no homology with the previously identified tum‐ gene P91A nor with any gene presently recorded in the data banks. The antigenic allele of gene P35B differs from the normal allele by a point mutation located in exon 5. This mutation, which replaces a Ser by an Asn residue, was shown by site‐directed mutagenesis to be responsible for the expression of the antigen. A synthetic decapeptide covering the sequence surrounding the tum‐ mutation rendered P815 cells sensitive to lysis by anti‐P35B CTL. Surprisingly, the homologous peptide corresponding to the normal sequence of the gene had the same effect, indicating that this tum‐ mutation does not exert its effect by generating the aggretope or the epitope of the antigenic peptide. As observed previously with gene P91A, we found that fragments of gene P35B containing only exons 4 and 5, which were cloned in non‐expression vectors, transferred efficiently the expression of the antigen.

Noxp20 and Noxp70, two new markers of early neuronal differentiation, detected in teratocarcinoma‐derived neuroectodermic precursor cells

The murine 1C11 cell line, derived from F9 pluripotent teratocarcinoma cells, exhibits features of a bipotential neuronal precursor as it converts into serotonergic or catecholaminergic neurons under appropriate induction. In order to point out molecular markers expressed in this early neuroectodermic commitment, we used a cDNA subtractive hybridization method. The 105 different isolated cDNAs represented 75 known genes, expressed sequence tags (EST) or genomic fragments. A majority of known proteins encoded by these sequences are involved in cellular mobility or migration. We characterized two sequences showing identities with ESTs and we called them Noxp20 and Noxp70. The Noxp20 transcript encodes a putative protein with a predicted caspase recruitment domain and the Noxp70 transcript encodes a putative protein displaying a Zn‐finger domain. Consistent with their roles in neuronal cell development, in situ hybridization showed that Noxp20 and Noxp70 are over‐expressed in brain. At embryonic days 12 and 15, Noxp20 is strongly expressed in the ventricular and intermediate zones of the brain and of the spinal cord. At embryonic day 15, Noxp70 was found to be strongly expressed in the ventricular zone around the telencephalic ventricle, and to a lower extent in the thalamus and hypothalamus. At post‐natal day 10, Noxp20 mRNA was detected in the dentate gyrus, the hippocampus, the cerebellum and the olfactory bulb.

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.