The Recombinant Trichosanthin-Antigen Peptide Vaccine For Cancer Immunotherapy

ObjectiveThe research is to develop an"all-in-one"tumor vaccine delivery system(cell-penetrating peptide-trichosanthin-antigen peptide)for transcutaneous immunotherapy of tumors.A recombination technology was used to fuse a tumor antigen peptide and cell-penetrating peptides at both ends of the trichosanthin gene sequence.The fusion protein vaccines were prepared using a prokaryotic expression system.The system was characterized to evaluate the efficacy of the tumor vaccine in vitro and in vivo.MethodsFirst,the antigen peptide(legumain peptide,amino acid sequence EDVTPENFLAVLR,orTrp2 peptide,amino acid sequence SVYDFFVWL)was genetically fused to the C terminus of trichosanthin(TCS)gene and cell-penetrating peptide(LMWP,amino acid sequence RRRRRRGGRRRR)was fused to the N-terminal for constructing the cell-penetrating peptide-TCS antigen peptide recombinant protein.The gene was cloned into the prokaryotic expression vector p MXB10 to express the recombinant fusion protein rTCS-leg-intein-CBD,rTCS-Trp2-intein-CBD,r LTL-intein-CBD,and r LTT-intein-CBD,respectively.The products were purified with a chitin affinity column,and after cleavage by cysteine,and the recombinant proteins were obtained.Secondly,we explored the abilities of these vaccines to activate the dendritic cells and promote T cell proliferation.The uptake efficiency of the vaccines in dendritic cells was investigated by flow cytometry and inverted fluorescence microscope.The cytotoxicity of the vaccines on dendritic cells was investigated by MTT assay.The activation of dendritic cells by these vaccines was investigated by flow cytometry and the proliferation of T cells by ELISA.Thirdly,we investigated the efficacy and safety of the tumor vaccines in the animal models bearing subcutaneously transplanted tumors.The proliferation of CD4~+T cells and CD8~+T cells after the treatment with the recombinant proteins were evaluated.The anti-tumor mechanisms were investigated.ResultsThe recombinant proteins of rTCS,rTCS-leg,r LTL,rTCS-Trp2,and r LTT were prepared.The results showed that the fusion proteins with cell-penetrating peptide could obviously enhance the cellular uptake efficiency and effectively activate dendritic cells and promote the proliferation of T cells.The in vivo results showed that the fusion proteins r LTL and r LTT could effectively promote the proliferation of CD4~+T cells and CD8~+T cells as well as the secretion of Th1-type cytokines IFN-γ,TNF-α,and IL-2,as well as the inhibition of the secretion of proinflammatory cytokines such as TGF-βand IL-10.The r LTL and r LTT vaccines significantly enhanced the antitumor effect of T cells,thereby effectively inhibiting the growth of tumors and improving the survival rate of the mice.ConclusionTrichosanthin has a good immunostimulation effect,and the fused cell-penetrating peptide can significantly improve the immunotherapy effect of recombinant trichosanthin-antigen.It provides a new idea for constructing a potent trichosanthin-assisted cancer vaccine and a trichosanthin-based strategy for immunotherapy.