Identification of a sperm protein 17 CTL epitope restricted by HLA‐A1

Abstract

Sperm protein 17 (Sp17) is a novel cancer-testis antigen. We previously reported the successful generation of Sp17-specific HLA-A1-restricted cytotoxic T-lymphocytes (CTLs) from the peripheral blood of a healthy donor using a recombinant Sp17 protein. These CTLs were able to kill not only target cells pulsed with the recombinant protein but also fresh Sp17؉ tumor cells. In the present study, we have identified a nonapeptide sequence within the Sp17 protein that is predicted to have a high binding affinity for the HLA-A1 molecules. We generated the synthetic nonapeptide and successfully propagated a peptide-specific CTL line. We confirmed that peptide Sp17 Sperm protein 17 (Sp17) is a spermatozoa-specific protein of unknown function, although it has been implicated in acrosome reactions during fertilization 1 and appears to bind heparan sulfate. 2 It is a highly immunogenic protein in vivo because a high proportion of vasectomized males spontaneously develops anti-Sp17 antibodies. We have identified Sp17 as a novel cancer-testis (CT) antigen in multiple myeloma 3 and ovarian cancer. 4 Sp17 expression is found at both the mRNA and protein levels and its normal tissue expression is limited to only testis and not any other normal tissues. The restricted normal tissue expression of Sp17 has now been confirmed independently, 5 suggesting that an Sp17-based tumor vaccine will be tumor-specific with very limited toxicities to normal tissues.The clinical relevance of our findings has been supported by further work demonstrating the ability to generate Sp17-specific cytotoxic T-lymphocytes (CTLs) that could kill Sp17-positive fresh autologous or HLA-matched tumor cells from healthy donors 6,7 and cancer-bearing patients. 4,8 Through these works, we have successfully generated an Sp17-specific HLA-A1-restricted CTL line from a healthy donor. 6 Since delineation of the CTL epitope may provide the tool for the future study and characterization of CD8 T-cell function in vivo and the generation of epitope-specific therapeutic strategies, we have in the present study used this T-cell line to determine the Sp17 CTL epitope restricted by the HLA-A1 element. MATERIAL AND METHODSPeripheral blood mononuclear cells (PBMCs) were separated from heparinized venous blood by Ficoll-Hypaque density gradient centrifugation. Dendritic cells (DCs) were generated from peripheral blood monocytes as previously described. 9 Briefly, PBMCs were seeded into 6-well culture plates containing 3 ml of RPMI 1640 and 10% FCS at 5-10 ϫ 10 6 per well. After 2 hr at 37°C, nonadherent cells were removed and the adherent cells were cultured at 37°C in RPMI 1640 supplemented with 10% FCS, 800 IU/ml GM-CSF and 1000 IU/ml IL-4. After 7 days of culture, DCs were harvested for pulsing with the synthetic peptide and used as antigen-presenting cells for the generation of Sp17 peptide-specific CTLs. Successful generation of DCs was confirmed in flow cytometry.Following culture, the DCs were washed twice and added to 50 ml polypropylene tubes. The DCs were pulsed with...

Some remarks on the somatic expression of sperm protein 17

“…Using a pair of sequence-specific primers, the presence of HSp17 mRNA has been clearly shown in 17% of patients with multiple myeloma and in the primary tumor cells from 70% of patients with ovarian carcinoma. 2-4 Chiriva-Internati et al 5 have further shown that HSp17 contains functional cytotoxic T-lymphocyte epitopes. These results indicate that HSp17 is a cancer-testis (CT) antigen in multiple myeloma and ovarian carcinoma and suggest its suitability as a target for immunotherapy of these 2 neoplastic diseases.…”

Sperm Protein 17: Clinical Relevance of a Cancer/Testis Antigen, from Contraception to Cancer Immunotherapy, and Beyond

“…Further characterization of such antigens clarified that they belonged to the RSA (rabbit sperm autoantigen) family [31], consisting of four proteins of 14,16, 17 and 18 kDa [32]. Functional analyses revealed that RSA acted as a "glue" binding spermatozoa to the zona pellucida of the egg [27].…”

“…Although the relevance of CTA as targets for immunotherapy is widely recognized and it has been extensively studied [8,10,11,13,[16][17][18], CTA functions and mechanisms of action remain poorly understood. The first evidence stems from the research on MAGE-A11 and MAGE-A4 in the pioneering studies by Nagao, Bai, and Peikert:…”

“…Through tumor cDNA expression libraries screening with autologous patients' serum, SEREX led to the identification of more than 1000 tumor-associated antigens, a large number of which belonged to the CTA family [11]. By the end of the 1990s, the well established testis and cancer cell expression pattern shared by these antigens gave rise to the cancer/testis antigen definition by Old and Chen [14,15].Although the relevance of CTA as targets for immunotherapy is widely recognized and it has been extensively studied [8,10,11,13,[16][17][18], CTA functions and mechanisms of action remain poorly understood. The first evidence stems from the research on MAGE-A11 and MAGE-A4 in the pioneering studies by Nagao, Bai, and Peikert:…”

Biological Treatment for Liver Tumor and New Potential Biomarkers

“…Peptide-based vaccines can be a better way to vaccinate because peptides are more stable and are easier to construct and, more importantly, they are free from bacterial and other forms of contamination. One of the possible limitations for peptide-based vaccines is the necessity to match the patient's human leukocyte antigen (HLA) type to the peptide of choice [53].…”