Research On Specific Killing Activity And Translocation Mechanism Of Novel Immuno-caspase6 In HER2 Overexpressing Tumor Cells

Targeted therapy of cancer research has been a hotspot in recent years. To realize the specifically targeting to tumor cells of cytotoxic elements and to achieve efficient killing activity, while reducing the damage of normal tissue, are the challenges for the development of antitumor targeted drug. As the member of targeted drug, immnutoxins specifically bind to the receptors expressing or overexpressing on tumor cell surface, and the molecules are internalized in endocytic compartments. After receptor-mediated endocytosis, the cytotoxic fragments are translocated in cytoplasm and induce the death of the tumor cells. Although immunotoxin applied to hematological malignancies and solid tumors in clinical trials obtained remarkable achievements, but the patients developed the neutralizing antibodies and dose-dependent vascular leak syndrome in the course of clinical trials. Because of its toxic fragments are foreign proteins, the immunogenicity of immunotoxin has become an important factor restricting their development.In our previous work, we replaced exogenous toxin protein in immunotoxin by human-derived pro-apoptotic molecular and constructed a series of immuno- proapoptotic molecules, which were named immunoapoptotin. Study confirmed immunoapoptotin can specifically target HER2-positive tumor cells and induce apoptosis to achieve efficient killing activity in vitro and in vivo. The domainⅡof Pseudomonas exotoxin A which is responsible for translocation contains more than 100 amino acids, and it may induce the immune responses. To further reduce immunogenicity of immunoapoptotin while ensuring the efficient translocation after endocytosis, we focus on the replacement of PEA domainⅡwith novel translocation sequence.In this study, we replaced the full long PEA translocation domain with optimized Furin recognizing sequence of Pseudomonas exotoxin A, Diphtheria toxin and nano-arginine peptide, and constructed three novel immunoapoptotin (e23sFv-FDT/FPE/R9-casp6). Compared with non-Furin recognizing sites (e23sFv-Null-casp6, negative control) and with full long PEA domainⅡof Immuno-caspase6 (e23sFv-PEⅡ-casp6, positive control), we investigated the specific killing activity of novel Immuno-caspase6 on HER2-positive tumor cells.The results show that the substrate of active Caspase-6, Lamin A, was cleaved 72 h after transfected with the novel Immuno-caspase6 in HER2-positive SGC-7901 cells. The apoptotic phenomenons such as incomplete nuclear membrane of tumor cells, nuclear condensation were emerged. The growth curve of transfected cells demonstrate that the growth of HER2-positive cells was significantly inhibited, while no significant effect was observed on the growth of HER2-negative HeLa cells. Immuno-caspase6 containing FDT/R9 has the equivalent killing activity compared with positive control, while Immuno-caspase6 containing FPE is slightly lower than the former three groups. We have established an Immuno-caspase6 stable-expressing CHO cell lines, and obtained the secreted proteins by ultrafiltration. We investigated the intracellular translocation pathway by observing the co-localization between the Immuno-caspase6 and cellular organelle. By confocal fluorescencemicroscopy, we observed that the translocation has occurred 6h after treatment with Immuno-caspase6 containing Furin recognizing sequence. In the null negative control group, Immuno-caspase6 gathered in endosomes or lysosomes, and could not enter the cytoplasm. Our results demonstrated that the novel immunoapoptotin could translocate from endosome into cytoplasm directly after cleaved by Furin in endosome, rather than through the Golgi apparatus and ER of retrograde transport route. No difference was observed in various recognizing sequences group. Through the use of proton pump inhibitor Bafilomycin A1 and Furin protease inhibitor Dec-RVKR-CMK confirmed that the internalization of immunoapoptotin and translocation under endosome - lysosome pathway play key role in inducing apoptosis.The in vivo antitumor activity was evaluated in HER2 positive SGC-7901 gastric tumor-bearing nude mice with intramuscular injection of LipofectAMINE-encapsulated plasmid adjacent to the tumor. The results confirmed that compared with positive group, Immuno-caspase6 containing FDT, R9 gained the equivalent inhibition on tumor growth and prolonged survival of tumor-bearing nude mice. No significant difference was observed between different groups. The specific distribution of Immuno-caspase6 was observed in the tumor tissue and induced a large number of tumor cell apoptosis without any obvious toxicity on key tissues.We confirmed that the novel Immuno-caspase6 exhibit more efficient growth inhibition than the first–line chemotherapeutic drug Herceptin to HER2 positive tumor cells. To further test whether the novel Immuno-caspase6 have cytotoxicity on the Herceptin-resistant cell, we established several Herceptin-resistant gastric and breast cancer cell lines. Results of Flow cytometry showed HER2 expression of Herceptin-resistant cancer cell lines was significantly reduced, while the Immuno-FDT-casp6 demonstrated dramatic cytotoxicity on Herceptin-resistant tumor cells in vitro.Compared with Immuno-caspase6 containing full long translocation domain of PEA, Immuno-caspase6 containing DT / PEA Furin recognizing sequence or nano-arginine peptides demonstrate similar cytotoxic activity. However, the translocation sequences containing more than 100 amino acid residues of the former reduced to 9-10 amino acid residues, which have lower immunogenicity. And it would facilitate the long-term repeated use in clinic. More importantly, our study demonstrated that the novel Immuno-caspase6 exhibit efficient cytotoxicity on Herceptin-resistant tumor cells, thus we believe that the novel Immuno-caspase6 have a broad clinical applification.In summary, the novel Immuno-caspase6 can specifically bind to and be internalized into HER2-positive tumor cells, then cleaved by Furin in endosome and translocated into the cytoplasm directly. They exhibit efficient killing activity on HER2-positive tumor cells, even on Herceptin-resistant tumor cells. Our research provides a new strategy for the treatment of HER2-positive tumors, but also provides alternative of other tumor targeted treatment.