Experimental Studies On Highly Specific HER2/neu Chiral Peptide Vaccine

Cell immunogy is the major to eliminate the tumor cells in the body,and cototo-xic T cells(CTL) is the most immportant.CTL is activated by the compound of MHC-Ⅰmolecuar and antigen peptide which is presented by antigen presentation cells(APC).The length of the antigen peptide is most 9～11 amino acids.It is has been proved that synthesized peptide can combine with MHC-Ⅰmolecuar on the surface of APC directly without the processing of APC,have the same effect as the natual endogenous peptide.The identification of the tumor antigen peptide can apply the peptide as the tumor vaccine.But peptide molecular is small ,the immunogenicity of peptide is low,half-life time of peptide is short and the peptide is hydrolized by enzyme easily.All these can influence the existing time and valence of CTL.Two methods were used to improve these situation : ①Change regional optical rotation by turn some L-peptide to D-peptide without altering the combination of peptide and MHC-Ⅰmolecuar. The synthesized D-peptide can be stable by resisting the hydrolization of the protein enzyme and increase the half-life time thus increade immunogenicity of peptide. ②Immunity ajuvant GM-CSF is used to activate the macrophage and other immunity cells and get the more powerful cell immunity.1.The selection,designation and synthesis of the HER2/neu peptide:(1) Design of the amino acid sequence of peptide: SYFPETTHI sequence analysis system was used to predict MHC-Ⅰmolecular restricted CTL epi-position of HER2/neu protein ,and select two region P42-50 (HLYQGCQVV) and P782-790 ( VSRLLGICL).Because the change of combination region of the peptide with the different allel gene of MHC-Ⅰmolecular is locolized the N-terminal amino acid of the peptide, so we change the C-terminal amino acid without changing the combination of the peptide and the MHC-Ⅰmolecular.We substitute D-Leu for L-Leu for the P42-50, substitute D-Ser for L-Ser for P782-790,and get two chiral peptide vaccine D-P42-50,D-P782-790.(2) Peptide synthesis: Peptides L-P42-50, D-P42-50,L-P782-790,D-P782-790 were synthesized with solid phase peptide synthesis method and Fmoc-strategy. Each amino acid was fully protected during the peptide synthesis procedure.(3) The purification and identification of peptide:The peptide was purifed by middle pressure reverse liquid chromatography.And the purity of L-P42-50, D-P42-50 is 99.2%,98.9%, the purity of L-P782-790, D-P782-790 is 99.1%,99.0%. The fraction of interested was confirmed by measuring the molecular weight of each fraction with EMS. Then the crude peptide was purified on preparative RP-HPLC. The purified peptide was received through the collection of interested fraction and the disposal of vacuum rotation evaporation and ice freeze. The product of pure peptide L-P42-50, D-P42-50 is 63% and 45% ,the product of pure peptide L-P782-790,D-P782-790is 58% and 46% .2. Study on induction of specific CTL in vivo by peptide vaccine immunization. The construct of mouse breast tumor model and vaccinated with peptide vaccine:BALB/c mice were inoculated with mouse breast tumor cell line D2F2(which was transfected with full cDNA of human HER2/neu gene), then vaccinated with peptide vaccine and divided for ten group:Nacl group;GM-CSF group; L-P42-50 group;L-P42-50+ GM-CSF group;D-P42-50 group; D-P42-50+ GM-CSF group;L-P782-790 group; L-P782-790 + GM-CSF group; D-P782-790 group; D-P782-790 + GM-CSF group.(1) Compared with the control group; L-P42-50+ GM-CSF group; D-P42-50+ GM-CSF group; L-P782-790+ GM-CSF group and D-P782-790 + GM-CSF group have significant difference on the increase of the weight of spleen(P﹤0.05, P﹤0.05, P﹤0.01, P﹤0.01);promoting the proliferation of T lymphocytes(P﹤0.05, P﹤0.05, P﹤0.01, P﹤0.01);the secreting γ-IFN of T cells(P﹤0.05, P﹤0.05, P﹤0.01, P﹤0.01); and HER2/neu specific CTL(P﹤0.05, P﹤0.05, P﹤0.01, P﹤0.01). The results indicated all these four peptides can form compound with MHC-Ⅰmolecular,bind to the T cell receptor,activated special CTL.(2) Compared with L...