Strategies to optimize T cell-based cancer immunotherapy

Abstract: Remarkable progress has been made in the field of tumor immunology in the past decade but active immunotherapy is not yet an established treatment modality. Tumor associated antigens (TAAs) presented in the form of peptides in association with MHC class 1 can be recognized by T cells and represent major targets of immunotherapy strategies to treat cancer patients. However, ineffective antigen presentation, tumor evasion and T cell tolerance limit anti-tumor immune responses. We have investigated several approaches aimed at improving induction of T cell responses and sensitization of tumor cells to T cell effector mechanisms, which are crucial steps for successful T cell-based immunotherapy. HER-2/neu (HER-2) is a self tumor antigen over-expressed in a variety of tumors and expressed at low levels by normal epithelial surfaces. To enhance the low immunogenicity of HER-2 we developed a series of peptide analogs of the HER-2.369 epitope to improve binding to HLA-A2 We found that one of the analogs (HER-2.369 V2V9) induced cytotoxic T lymphocytes (CTLs) both in vitro and in vivo in HLA-A2/Kb transgenic (tg) mice at 100-fold lower concentration than the wild-type epitope. Importantly, CTLs generated by the analog peptide were also able to recognize the wild-type epitope and HER-2+ tumors in a MHC-restricted manner. The analysis of thermodynamic parameters demonstrated that the high biological activity of the V2V9 peptide variant was associated with a slower dissociation kinetic profile, resulting in an epitope with greater HLA-A2 stability. Next we studied immune responses directed against HER-2 in HLA-A2/Db tg mice upon DNA vaccination. Unexpectedly we found that a DNA vaccine encoding for full length HER-2 protected mice from HER-2+ tumor challenge by a CTL independent mechanism. A strategy aimed at enhancing MHC class 1 processing by a multi-epitope DNA vaccine encoding for 3 HER-2 derived HLA-A2 peptides linked in a "string of epitopes" resulted in high numbers of peptide specific T cells but failed to induce tumor protection. We found that HER-2+ but not HER-2 tumor cells down-regulated MHC class 1 and components of the antigen processing pathway which impaired the capacity to produce and display MHC class 1 peptide-ligands to specific CTLs. These findings are important for the optimization of vaccines targeting HER-2, and suggest that, to be effective, vaccines should aim at inducing an integrated immune response where also CD4+ T cells and antibodies are important components. In searching for new means to improve MHC class I-restricted antigen presentation in malignant cells, we identified retinoic acid (RA) as a modulator of antigen presentation and sensitivity to T cell effector mechanisms in tumor cells. Treatment of neuroblastoma cells with RA resulted in increased expression of MHC class 1 and of proteolytic and regulatory subunits of the immunoproteasome, increased half-life of MHC class 1 complexes and enhanced sensitivity of neuroblastoma cells to both MHC class I-restricted peptide-specific and MHC non-restricted lysis by CTLs. In the next study we extended our investigation of the effects of RA to the uveal melanoma (UM) tumor model. The current therapy of UM metastases is inefficient and the development of new treatment modalities is needed. We found that RA suppresses proliferation and causes morphological changes compatible with differentiation in a panel of UM cells. RA treatment of UM resulted in G I/GO cell cycle arrest which correlated with an increase of p21, p27 and p53 protein levels and with significant down-modulation of the HER-2/neu proto-oncogene surface expression. Unlike neuroblastomas, UM cells failed to up-regulate components of the MHC class 1 antigen processing pathway upon RA-treatment. Nevertheless, RA-treated UM cells exhibited increased sensitivity to both MHC class I-restricted killing by cytotoxic T lymphocytes and NK cell-mediated lysis. These observations could be explained (at least in part) by more efficient conjugate formation between UM cells and killer lymphocytes. Taken together, our findings suggest that the application of retinoids in combination with T cell-based immunotherapy may be an effective therapy for the treatment of neuroblastoma and uveal melanoma.