Description:
Abstract High-risk Ewing sarcoma is still fatal in many cases. Cellular immunotherapy is a promising approach for preventing relapse by eliminating residual disease after conventional treatment. A critical prerequisite is the availability of an adequate tumor target antigen. STEAP-1 is a surface protein aberrantly expressed in various cancers. Potential obstacles to the use of STEAP-1 as a tumor target are the limited immunogenicity of STEAP-1 epitopes, clonal deletion of STEAP-1-reactive T cells, and poor presentation of STEAP-1 peptides by MHC class I on tumor cells. Here we investigated whether functional and Ewing-tumor reactive STEAP-1-specific cytotoxic T cells (CTLs) can be expanded from the repertoire of normal donors by peptide stimulation. STEAP-1 was confirmed to be expressed in 10 of 10 Ewing sarcoma cell lines by PCR. To expand STEAP-1-specific CTLs, CD8+ T cells from HLA-A2-positive healthy donors were stimulated with autologous dendritic cells pulsed with a pool of 4 STEAP-1 peptides (5 µM each), in the presence of rhIL-7, rhIL-12 and rhIL-15. Weekly restimulations were performed with peptide-pulsed K562 cells gene-modified to express human HLA-A2, CD80, CD40L and OX40L (K562aAPCs). CTLs with a predominant CD8+ effector memory CTL phenotype (CD45RA-, CCR7-) were successfully generated from 6 healthy donors. Their epitope specificity was confirmed by ELISPOT analysis after 2 to 6 rounds of restimulation. Pooled STEAP-1 peptides directly added to the CTLs induced specific secretion of IFN-γ (70 to 487 spot forming cells (SFC)/105 cells, mean 297.5±138.7 SFC/105 cells), in the absence of relevant background responses to a control peptide (0 to 50 and mean of 16.2±19.9 SFCs/105 cells). Restimulation of CTLs with individual STEAP-1 peptides demonstrated donor-dependent reactivity with one to four of the peptides, while neither one emerged as immunodominant. The CTLs specifically lysed STEAP-1 peptide pulsed target cells (52.9±4.3% at an effector-to-target ratio of 20:1), but not cells pulsed with control peptides (0.0±3.8%). In contrast, STEAP-1 specific CTLs failed to functionally interact with the HLA-A2+/STEAP-1+ Ewing sarcoma cell lines as measured by cytolysis and cytokine secretion, even after upregulation of MHC class I molecules by pretreatment with IFN-γ. Thus, while the induction of STEAP-1 peptide-specific CTLs is feasible, these CTLs do not efficiently recognize endogenously expressed antigen on Ewing sarcoma cells. Higher-avidity CTLs are likely needed for exploiting STEAP-1 as a target for adoptive immunotherapy in this disease. To define the critical requirements for recognition and lysis of STEAP-1 expressing target cells in an autologous setting, we are currently exploring activated autologous γδ T-APCs expressing full-length STEAP-1 protein by retroviral gene transfer as targets and stimulator cells for STEAP-1 specific CTLs. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1927.