An Altered Peptide Ligand Based On D-amino Acids For Native Cytotoxic T Lymphocyte Epitope TRAG-3 Enhanced The Immunogenicity

Objective: using non-protein amino acids to modify the structure of the CTL epitope of TRAG-3(58-66) (ILLRDAGLV) peptide,to screen the high-affinity CTL mimotope which can overcome peptide hydrolysis and induce a strong CTL response in vivo, To put basis for further exploring the efficient vaccine of non-protein amino acid CTL mimotop.Methods: Firstly, using non-protein amino acids to substitute every Natural amino acid site of the epitope of TRAG-3(58-66) (ILLRDAGLV) peptide, which is substituted from L-type amino acid D- type amino acid, to get the Altered peptide ligands; then, with the help of SCI O2 Workstations and parallel computing system in laboratory, to simulate and investigate APLs and HLA-A*0201 molecules with three-dimensional structure for high-throughput screening the CTL mimotope,and screen two APLs with high-affinity for Synthesis, purification and identification; lastly, through the classic T2 Binding assays in vitro and the stability of peptides in human plasma experiment, to analysis the capabilities of the peptides resistant to enzymatic degradation and the elimination half in vivo.Results: taking advantage of non-protein amino acids to substitute every Natural amino acid site of the epitope of TRAG-3(58-66) ( ILLRDAGLV) peptide, we get eight CTL Altered peptide ligands(APLs)——P1 TRAG-3(58-66) I(D)LLRDAGLV、P2 TRAG-3(58-66) IL(D)LRDAGLV、P3 TRAG-3(58-66 ILL(D)RDAGLV、P4 TRAG-3(58-66) ILLR(D)DAGLV、P5 TRAG-3(58-66)ILLRD(D)AGLV、P6 TRAG-3(58-66) ILLRDA(D)GLV、P8 TRAG-3(58-66)ILLRDAGL(D)V、P9 TRAG-3(58-66)ILLRDAGLV(D) ; Making use of Computer molecular modeling, we screen two CTL Altered peptide ligands(APLs)——P5 TRAG-3(58-66) ILL RD(D) AG LV、P6 TRAG-3(58-66) ILLRDA(D)GLV,the numbers of hydrogen bonds are 12 and 8 when they bind to HLA-A*0201 molecules respectively, the numbers of hydrogen bonds of APLs of P5 and P6 are more than the numbers of hydrogen bonds of the natural epitope peptide(the numbers of hydrogen bonds are 11), and the distance of anchor residues (P2-P9) of the two APLs are 18.38 ? and 16.29 ? respectively, the results meet the requirements of distance with 15-20 ? between the HLA-A * 0201 restricted CTL epitope anchor residues. The solvent accessible surface area of sites of P2 and P9 are 0 and 0.193?2,in the APLs of P5 respectively,and the solvent accessible surface area of sites of P2 and P9 are 0.386 ?2 and 0.902?2 in the APLs of P6 respectively. Accordingly,we get two CTL mimotopes which are P5 TRAG-3(58-66) ILL RD(D) AG LV、P6 TRAG-3(58-66) ILLRDA(D)GLV. Basing on the assays of they Bind to HLA-A*0201 molecules, they have higher Affinity than the Natural epitope peptide; According to the Classic T2 peptide binding assay in vitro,we demonstrated that the results consistent to the results of Computer molecular modeling,the values of FI is 1.50,2.09 respectively.Conclusion: Replace the use of non-protein amino acid sites corresponding to the structural transformation of the natural amino acids have 8 APLs, through computer simulation of molecular affinity screened two better than the natural epitope mimotope peptide, then analyzed by in vitro affinity experiments , and molecular analysis of experimental results in good agreement, verify the accuracy of the computer molecular screening, follow-up study in further validation P5: ILLRD (D) AGLV, P6: ILLRDA (D) GLV in vitro anti-plasma enzymatic capacity and calculated half-life, as well as their TRAG-3 + tumor cell killing effect and applies to non-small cell lung cancer patients in clinical trials, in order to further explore the non-protein amino acid-based simulation CTL epitope peptide vaccine design basis.