An Altered Peptide Ligand For Native Cytotoxic T Lymphocyte Epitope TRP-2_180-188 Enhanced The Immunogenicity～1

Recently, many researchers have focused attention on epitope-based vaccine design (EBVD). So far, more than 170 antigenic peptides (epitopes) derived from 60 different human tumor antigens have been identified. Some of epitope-based vaccines have been applied to clinical trials of different tumor types. The majority of clinical trials indicated that: 1) Peptide vaccination is safe in human subjects without toxicities or side effects; 2) Wild type epitope vaccination does not induce a strong enough anti-tumor immune response in vivo because of its low immunogenicity. Furthermore, there is no significant clinical outcome observed in the peptide-based vaccination clinical trials. Therefore, how to enhance an epitope's immunogenecity is critical to peptide-based vaccine for cancer patients.The most common approach for improving the immunogenicity of peptide vaccines is to alter the recognition by T cell receptors (TCR) by replacing the residue in MHC-bound peptides. TCR could recognize a specific peptide in an MHC restricted manner. T lymphocytes, however, have been shown to have no single ligand specificity. They recognize a large array of peptides with similar characteristics, including some modified non-natural peptides, which are called altered peptide ligands (APLs). These APLs can generate a number of different responses in T cells, ranging from enhancing immunological function (agonist) to completely turning off their functional capacity (antagonist), which are not detected with the cognate ligand. Therefore, this is a ractical approach to enhancing peptide immunogenicity by selective amino acid replacement. In previous studies. Three APLs, i.e., MART1/Melan A_27-35 (AAGIGILTV) 1L, gp100_209-217 (ITDQVPFSV) 2Mand CEA CAP-1 (YLSGANLNL) 6D have been shown to be TCR agonists in vitro and in vivo. Significant anti-tumor immune responses were also observed in clinical trials for all three agonists.Tyrosinase-related protein (TRP-2) is a non-mutated melanocyte differentiation antigen (MDA), such as gplOO and MARTl/Melan A recognized by melanoma-reactive CTL in humans and mice. TRP-2 is widely expressed in melanoma and glioblastoma multiforme (GBM). Melanoma-reactive CTLs recognize TRP-2(i8O-i88) (SVYDFFVWL) in both the mice and humans. Therefore, TRP-2(i8o-i88) peptide could be a promising peptide-based cancer vaccine candidate for melanoma and glioma patients. Unfortunately, as with the wild type epitope of the three agonists mentioned above, although there are pre-existing T-cell precursors specific to the wild type TRP-2(i8o-i88) (SVYDFFVWL) in the peripheral blood of melanoma patients, most CTLs have lost the ability to reject tumor cells because of their tolerance state. The purpose of this study is to optimize a TRP-2 (iso-iss) epitope APL by selective amino acid replacement to improve peptide immunogenicity and to break the self tolerance, in order to generate a stronger anti-tumor immune response.In this study, primary and auxiliary anchor residues in the sequence of TRP-2(i8o-i88) (SVYDFFVWL) epitope were replaced by dominant amino acid residues. Briefly, PI (Y) and P2 (L or M) was used either alone or in combination to substitute for the corresponding amino acid of the natural epitope. Four APLs were selected for further synthesis, purification and functional identification based on their high MHC affinity after screening by three software programs and molecular dynamics simulation.The four APLs sequences are as follows: TRP-2 2L (SLYDFFVWL), TRP-2 2M (SMYDFFVWL), TRP-2 1Y2L (YLYDFFVWL), TRP-2 1Y2M (YMYDFFVWL). The four APLs were then evaluated using classic tests, including binding affinity to HLA-A*0201 -positive T2 cells and the stability of the peptide/MHC complex . The results demonstrated that: l)The four APLs did have stronger binding affinities than the wild type peptide. Their bingding affinities (FI) from high to low was as follows: TRP-2 2L (2.49), TRP-2 2M (2.15), TRP-2 1Y2M (1.82), TRP-2 1Y2L(1.45), TRP-2 WT (1.32). 2) After six hours of treatment with brefeldin A, the remaining percentage of peptide/MHC complex from high to low was as follows: TRP-2 2M (80%), TRP-2 1Y2M (71%), TRP-2 1Y2L(69%), TRP-2 WT (56%), TRP-2 2L (53%). Compared to wild type TRP-2(i8o-i88) epitope and the other three APLs, TRP-2 2M had amuch higher binding affinity to HLA-A*0201 molecule and was more stable. These findings indicated that TRP-2 2M probably is a promising agonist for the TPR-2(i8o-i88)epitope.CTL function is usually evaluated using two different approaches. One method is to measure the level of cytokine production, such as IFN-y. The other method is to test the cytolytic activity, i.e., the release of granzyme B (GrB) by CTLs upon effector-target interaction, which is a specific indicator of CTL and NK cytotoxic activity. In this study, high sensitive ELISPOT assay was used to evaluate the APL-specific CTL function based on cytokine and GrB release. APLs were used to stimulate healthy donor PBMC in vitro. Functional analysis of CTLs indicated that: l)TRP-2 2M specific CTL recognized wild type peptide that loaded to T2 cells, and released high levels of IFN -y and GrB. 2)There was no significant difference in recognition of the LB373-MEL cell line (HLA- A*0201 \ TRP-2+) among TRP-2 2M, the other APLs and the wild type peptide.HLA-A*0201/Kb transgenic mice were immunized with the TRP-2 wild type or analogue peptides coemulsified in IFA with the PADRE Th epitope. Eleven days after immunization, splenocytes were then cultured with APLs for 5 days in vitro, and ELISPOT assay was used to measure IFN-y production by peptide-specific CTLs. The results demonstrated that those specific CTLs induced by TRP-2 2M peptide could efficiently recognize wild type TRP-2 peptide and release higher levels of IFN-y, compared to immunization with wild type peptide and the other three APLs. The TRP-2 2M analogue possessed a significantly higher immunogenicity and induced stronger immune response after vaccination in the HLA-A*0201/Kb transgenic mice.Taken together, the results demonstrated that the higher immunogenicity in vivo of the 2M peptide is paralleled by a markedly increased immunogenicity in vitro of this analogue, and is consistent with its greater petide/MHCcomplex stability in vitro when compared to the wild type peptide. It is supposed that the stronger immune response induced by TRP-2 2M is probably related to its greater stable peptide/MHC complex. We concluded that TRP-2 2M is the agonist of the TRP-2 wild type peptide. Further study will be focused on evaluating the cytolytic activity of CTLs induced by TRP-2 2M to other tumor cell types, and will apply to a clinical trial to treat melanoma and glioma patients.