Identification Of Broad-spectrum CTL Epitopes In Hyper-expressed Antigens COX-2 In Esophageal Carcinoma

Esophageal carcinoma (ECC) is one of the top frequently occurred malignant cancers, particularly common in northern Henan province, China. Squamous cell carcinoma has been the more common cell-type of ECC, accounting for almost 90%. Now the therapy including surgery, chemotherapy, radiation, or a combination for ECC is only to palliate the symptoms. However, a 5-year survival rate in patients with esophageal carcinoma is less than 10%. Novel treatment options of ECC are urgently needed. Indeed, tumor immunotherapy has evolved specifically to offer several attractive potential advantages. It is strictly autologous with very few side effects. Additionally, once the immune response has started, its efficacy is independent on the localization, type, and proliferate state of tumor cells.The immune system has long been implicated in controlling tumours. The evidence collected in the last decade indicates that CTLs which recognize peptide fragments derived from tumor-associated antigens (TAA). Such CTLs can directly lyse tumour cells and also secrete cytokines such as interleukin (IL)-2, tumour necrosis factor (TNF), granulocyte macrophage-colony stimulating factor (GM-CSF) and interferon (IFN)-γ, which have indirect anti-tumor effects. Adoptive transfer experiments in humans have also demonstrated the efficacy of the CTLs with anti-tumour activity. In addition, human clinical trials have demonstrated that epitope-specific CTLs can be induced in cancer patients and their induction correlated, in multiple instances, with partial or complete tumour responses. Identification of TAA peptides expressed by different human tumors provided the basis for antigen-specific active immunotherapy or vaccination and facilitated the design of new vaccination clinical trials. Although the discovery of human TAAs has revitalized efforts to develop antigen-specific vaccines and adoptive T-cell immunotherapy, a major limitation has been the lack of universal TAAs applicable tothe majority of patients with common ECC. COX-2 is overexpressed in the ECs but rarely in normal cells. Furthermore, its expression has been directly linked to tumor development such that inhibition of COX-2 in COX-2⁺ human tumors leads to growth arrest. In addition, targeting a single HLA restriction element carries the risk of inducing immune escape by tumor down-regulation of the particular HLA allele.In this study, we identify a new HLA-A2/A3-binding peptide epitope derived from COX-2 that triggers MHC-restricted CTL lysis of COX-2⁺ ECCs. The characterization of multiple COX-2 epitopes with different HLA restriction elements broadens the clinical potential of this target antigen in two important ways: (a) Targeting combination of HLA-A2 and HLA-A3 molecules increases the number of patients potentially eligible for COX-2-directed immunotherapy. The HLA-A2 supertype is expressed by 45-55% of the patient population, and HLA-A3 supertype is expressed by 50-55% of patients. Co-expression is found in nearly 20% of patients. Thus, >80% of COX-2⁺ patients could already be considered for therapies, (b) The multiple HLA restriction CTL epitopes help address the issue of immune escape that has limited previous antigen-specific T-cell therapies. Tumor loss of a single HLA allele is a significant component of MHC alterations described in cancer cells. As emphasized in these studies, staining of cells with a mAb recognizing a monomorphic determinant of HLA class I antigens does not detect selective loss of HLA class I allospecificities. Thus, in strategies targeting both of the HLA-A2 and HLA-A3 restricted epitope, tumor loss of only HLA-A2 or HLA-A3 antigens would not be sufficient for immune escape in patients who express both alleles. Targeting polyallele restricted peptides derived from COX-2 may also minimize immune escape because of loss or mutation of antigen expression.The integration of molecular and immunological techniques has led to the identification of a large number of human tumour associated antigens recognized by CTLs. With the development of bioinformatics, a series of new technologies in this field have been used for epitope mapping. Identification of CTL epitopes by establishing computer algorithms can achieve high-throughput epitopThe integration of molecular and immunological techniques has led to the identification of a large number of human tumour associated antigens recognized by CTLs. In the present study, we used epitope prediction programs, available at SYFPEITHI base on Matrix—based T2 cell epitope selection algorithms combination with MHCPred base on 3D-QSAR (three-dimensional quantitative structure-activity relationship) and NetChoP66.0 base on proteasomal cleavage motifs, we designed COX-2-derived peptides that bind to HLA-A2 and -A3 molecules.Now, cytokine release assays and tetramer analysis provide powerful new ways to assess the specificity and magnitude of CTLs. Thus, with current technologies, it is now possible to characterize distinct aspects of the T cell response to antigens. HLA/peptide tetramers are able to detect the presence of specific T cell receptors on the cell surface, the cytokine release assays reflect the capacity of T cells to be activated in response to antigen, and the cytotoxicity tests indicate that the specifically activated T cell has the machinery to kill target cells. All the selected COX-2 peptides fulfilled the requirements for HLA binding and in vitro priming. Using an in vitro CTL sensitization protocol that allows stimulation of human CTLs with peptide-pulsed DCs in the presence of IL-2 and IL-7, we generated HLA-A2/A3-restricted CTLs against P479 (COX-2_479-487 ALYGDIDAV) based on Cytotoxicity assays and IFN-γrelease ELISPOT assay, which is a favoured method widely employed for quantification of T lymphocytes responsive against TAA-derived epitopes.In the present study, the ECC cell lines were proved to express COX-2 in mRNA level by RT-PCR. More importantly, the peptide-specific CTLs induced were able to recognize the HLA-A2⁺ COX-2⁺EC-9706 and HLA-A3⁺ COX-2⁺ EC-1 cells in IFN-γELISPOT assays, which is supported by Chromium release assay, while the generated CTLs could not recognize the HLA-A3⁺ COX-2^- EC-109 cells. These data confirm the endogenous origin of COX -2_479-487 in EC-1 and EC-9706 cells and its presentation to the cell surface in the context of HLA-A2/A3 molecules.