Protein SSX2 (45-59)

Proteins encoded by genes of the SSX family are specifically expressed in tumors and are therefore relevant targets for cancer immunotherapy. One of the first identified family members, SSX-1, is expressed in a large fraction of synovial sarcomas as a fusion protein together with the product of the SYT gene. In addition, the full-length SSX-1 antigen is frequently expressed in tumors of several other histological types such as sarcoma, melanoma, hepatocellular carcinoma, ovarian cancer and myeloma. To date, however, SSX-1 specific T cell responses have not been investigated and no SSX-1 derived T cell epitopes have been described. Here, we have assessed the presence of CD4(+) T cells directed against the SSX-1 antigen in circulating lymphocytes of cancer-free individuals. After a single in vitro stimulation with a pool of peptides spanning the entire SSX-1 protein we could detect and isolate SSX-1-specific CD4(+) T cells from 5/5 donors analyzed. SSX-1-specific polyclonal populations isolated from these cultures recognized peptides located in three distinct regions of the protein containing clusters of sequences with significant predicted binding to frequently expressed MHC class II alleles. Characterization of specific clonal CD4(+) T cell populations derived from one donor allowed the identification of several naturally processed epitopes recognized in association with HLA-DR. These data document the existence of a significant repertoire of CD4(+) T cells specific for SSX-1 derived sequences in circulating lymphocytes of any individual that can be exploited for the development of both passive and active immunotherapeutic approaches to control disease evolution in cancer patients.

Ectopic gene expression in tumors versus normal somatic tissues provides opportunities for the specific immunotargeting of cancer cells. SSX gene products are expressed in tumors of different histological types and can be recognized by tumor-reactive CTLs from cancer patients. Here, we report the identification of an SSX-2-derived immunodominant T cell epitope recognized by CD4(+) T cells from melanoma patients in association with HLA-DR. The epitope maps to the 37-58 region of the protein, encompassing the sequence of the previously defined HLA-A2-restricted immunodominant epitope SSX-2(41-49). SSX-2(37-58)-specific CD4(+) T cells were detected among circulating lymphocytes from the majority of melanoma patients analyzed and among tumor-infiltrating lymphocytes, but not in healthy donors. Together, our data suggest a dominant role of the 37-58 sequence in the induction of cellular CD4(+) T cell responses against SSX antigens and will be instrumental for both the onset and the monitoring of upcoming cancer-vaccine trials using SSX-derived immunogens.

Purpose: Synovial sarcoma X (SSX) breakpoint genes are expressed in a variety of cancers but not in normal tissues, except for testis, and are potential targets for immunotherapy. The aims of this study were to determine the expression and immunogenicity of these antigens in patients with epithelial ovarian cancer (EOC). Experimental design: SSX-1-, SSX-2-, and SSX-4-specific reverse transcription-PCR were done on a panel of EOC specimens. Sera from a subgroup of the patients were tested for SSX-2 and SSX-4 antibody by ELISA and recombinant antigen expression on yeast surface (RAYS). In vitro stimulation of peripheral blood mononuclear cells from a patient bearing SSX-4-expressing tumor with a pool of long peptides spanning the protein sequence was used for assessment of SSX-4-specific CD4(+) T cells recognizing distinct antigenic sequences restricted by HLA class II alleles. Results: Our results indicate expression of SSX-1, SSX-2, and SSX-4 in 2.5%, 10%, and 16% of 120 EOC specimens, respectively. When all three SSX antigens are considered, aberrant expression was found in 26% of ovarian tumors. Antibodies to SSX-2 and SSX-4 were detectable by ELISA and RAYS in two patients. SSX-4-specific CD4(+) T cells recognizing two previously undescribed SSX-4-derived T-cell epitopes in association with HLA-DR (SSX-4: 51-70 and SSX-4: 61-180) were identified. Conclusions: Our study shows aberrant expression of SSX antigens in a proportion of patients with EOC. The evidence of humoral immunity to SSX-2 and SSX-4, and SSX-4-specific CD4(+) T cells among circulating lymphocytes in patients with antigen expressing EOC suggest that these antigens are attractive targets for specific immunotherapy in EOC.