A Van Pel

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.

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.

Tumor Rejection Antigens and Immune Surveillance

The existence of specific tumor rejection antigens was first demonstrated with chemically induced mouse sarcomas: each tumor was found to express a different antigen [1]. Similar findings were made with ultraviolet-induced tumors [2]. Later, the generality of the existence of tumor rejection antigens was questioned when spontaneous mouse tumors were found to be completely incapable of eliciting an immune rejection response [3]. However, further experiments demonstrated that even these tumors express weak transplantation antigens that are potential targets for immune rejection by the syngeneic host [4].

T-lymphocytotoxicity in malignant melanoma: Induction with autologous mutagenized tumor cell clones

Monoclonal antibody (mAb) R-24, recognizing ganglioside GD3 on neuroectodermal tumors, shows several functional propert ies in v i t ro : i nh ib i t i on of cel l growth, complement f i xa t i on and mediation of antibody dependent ce l l u la r cytot o x i c i t y (ADCC). The tissue spec i f i c i t y of th is mAb R-24 was studied on 190 cryopreserved, unfixed human t issue sections by ind i rec t immunoperoxidase sta in ing. The spec i f i c i t y for neuroectodermal tumors was s t r i k ing . Ganglioside GD3 was detected in a l l 21 t issue sections of 21 patients with primary melanoma and in a l l 37 probes of 24 patients with metastatic malignant melanoma. The major i ty of tumor ce l ls in the samples of primary malignant melanoma expressed GD3: however, GD3 expression was more heterogeneous in samples of metastatic lesions even in d i f fe ren t metastases of the same pat ient . Of 18 nevi, 16 reacted with monoclonal antibody R-24, while melanocytes in the basal layer of normal skin stained only weakly and i r regu la r l y . None of the 32 normal and 12 feta l human t issue types were R-24 pos i t i ve , but a strong cytoplasmic staining was observed with single ce l ls in the dermis and in the i n t e r s t i t i a l t issue of the gastro intest ina l t rac t , in the in ter lobu lar septa of the thymus, and in other d i s t i nc t locat ions. Only two malignant carcinoid tumors of 38 nonmelanomatous tumors tested reacted with monoclonal antibody R-24. The presented f indings are pa r t i cu la r l y relevant in view of the c l i n i ca l appl icat ion of th is mAb in v ivo. Local inflammatory response at the tumor s i te and tumor remissions have been observed a f ter appl icat ion of pur i f ied mAb R-24 in a percentage of pat ients. Side effects (skin rash) have been minimal with th is speci f ic antibody. New monoclonal ganglioside antibodies have been generated to cover the complete antigenic phenotype of neuroectodermal tumors. I .Med.Kl in ik , Joh.Gutenberg Univers i t~t , 6500 Mainz W.Germ. S 147