Studies On Production Of Recombinant HER2/neu ICD And Its Anti-tumor Effects

HER2/neu(Human Epidermis Growth Factor Receptor 2)is a member of the HER receptor family.The HER2/neu genes locate in the long arm of chromosome 17(17q21)and encode a transmembraneous glycoprotein with intrinsic tyrosime kinase activity.The molecular weight of HER2 is 185KD. Commonly,HER2/neu is expressed at a low level in breast epithelial cells and other normal cells,but it is over-expressed in 20-30%of breast cancer cells and other tumor tissues like ovary cancer,gastric carcinoma and non-small cell lung cancer.What's more,drug tolerance and infaust prognosis are easily to be found in breast cancer patients with high expression of HER2/neu.Since humoral and cellular immune reaponses against HER2/neu can be detected in many tumor patients,HER2/neu has the potential to be an efficient target for oncotherapy.Heat shock proteins(HSPs)are high conserved molecular chaperones in most amimals with the ability of facilitating proteins to fold,refold and degrad. The current studies showed that HSPs played a considerable role in stimulating anti-tumor immune responses though the following pathway:1)HSPs can activate the innate immune system to produce anti-tumor effects,such as activating natural killer(NK)cells and macrophages to kill tumor cells.2)HSPs can also activate the adaptive immune system to play anti-tumor effect:HSPs help the antigenic peptides to enter the antigen-presenting cell(APC)through specific receptors.After entering the APC,HSPs assists antigens to be processed and presented in MHC classâ… pathways.Thus,antigenic peptides can be co-expressed with MHC classâ… molecules on DCs and consequently stimulate the generation of specific cytotoxic T lymphocyte(CTL).At the same time,HSPs like HSP90 and HSP70 can elicit increased expression of MHC melecules and co-stimulatory molecules,such as CD80,CD83,CD86 and CD40 on the surface of APCs.These investigations indicate that HSPs have the potential to be used as molecular adjuvants of vaccine.Only a few stress proteins have been shown to be effective in animals as adjuvants(the most frequently studied being GRP94/gp96 and HSP70).In addition,recent studies have shown that another HSP,known as HSP110,also exhibits this immune-stimulating activity when purified from a tumor tissue and applied in a preventative therapy.More over,HSP110 was more effetient in binding and stabilizing large protein substrates.In the present study,we binded recombinant HER2/neu ICD with recombinant HSP110 noncovalently forming a novel tumor vaccine.On one hand,the vaccine was based on the ability of HSP 110 to bind with large protein substrates.On the other hand,since the vaccine aimed at HER2/neu ICD,it can be applied in any patient expressing this antigen,while traditional HSP vaccine acquired from sugery is patient specific.Thus,this kind of recombinant vaccine is more convenient and effective than traditional HSP vaccine.The contents of this paper are focused on the following parts:â‘ Constructing expression vector pPICZα/HER2/neu ICD,transforming it into Pichia pastoris via electroporation and screening the engineering strains secreting the protein at high levels steadily.â‘¡Noncovalent binding between recombinant HER2/neu ICD and recombinant HSP110 in vitro,testing the ability of inducing specific CTLs of the complex,and detecting the effect on inhibiting tumors of the complex.â‘¢Establishing a zymotechnique of 80 L of HER2/neu ICD,optimizing the main parameters,and creating a new method for large-scale purification of HER2/neu ICD.1.Constructing,screening and identificating of Pichia pastoris engineering strains expressing HER2/neu ICD steadily at a high level:The HER2/neu ICD expression vector pPICZα/HER2/neu ICD was constructed via PCR from PCMV plasmid using specific primers with Xhoâ… and Xbaâ… target sites and a part ofα-mating factor signal peptide.The recombinant vector was identified by endonuclease digestation assay and sequencing,then linearized and transformed into Pichia pastoris X-33 via electroporation..10 of zeocin resistant positive clones of Pichia pastoris were screened for high level-expressing strains by PCR,SDS-PAGE of the fermental supernatants, and western blot.The results indicated that the recombinant HER2/neu ICD was identical with the native HER2/neu ICD.And the HER2/neu ICD protein was purified by centrifugation,cation-exchange chromatography and gradient elution.The purity coefficient of HER2/neu ICD could be more than 95%and the concentration of HER2/neu ICD in the broth can reached to 110 mg·L^-1.2.Studies on large-scale zymotechnique and purification of HER2/neu ICD:We further explored the zymotechnique of 80 L of HER2/neu ICD.The study was focused mainly on pH value,culture medium,dissolved oxygen, methanol feeding speed,initial biomass and etc.The results indicated that in the FM21 medium with 0.5%peptone,the best pH was 4.6,DO between 25%～30%and the supply speed of methanol is 11 mL·h^-1·L^-1multiplied by initial fermentation volume.And the supernatant fermentation was purified by SP Sepharose XL on pH3.8 and by stepwise elution using 0.4 mol·L^-1NaCl and 0.8 mol·L^-1NaCl.The purity coefficient of HER2/neu ICD could be more than 85% and the concentration of HER2/neu ICD in the broth can reached to 295 mg·L^-1.3.Studies on HSP110-HER2/neu ICD complex to induce specific immune reaction:(1)Study on induction of specific CTLs activity in vivo by HSP 110-HER2/neu ICD complex vaccine immunizing:D2F2 cells were digested with trypsine and EDTA and resuspended in PBS with the cell density of 1×10⁸·mL^-1.BALB/c mice were inoculated with 0.1 mL of D2F2 cells(1×10⁷ cells),and were divided into 5 groups randomly,14 mice per group.10μg of HSP110,HER2/neu ICD,HSP110-P_789-797and HSP110-HER2/neu ICD and 200μL of PBS were injected into each group hypodermically weekly,respectively.And the mice were injected 3 timesâ‘ The lymphocytes and splenocytes from 6 sacrificed mice of each group were isolated 7 days post the last immunization.The numbers of IFN-γ-secreting cell were detected by ELISPOT method.The results showed that the numbers of IFN-γ-secreting cell of all experimental groups are more than that of PBS control group evidently,and the numbers of IFN-γ-secreting cell of HSP110-HER2/neu ICD group are significantly higher than that of HSP110-P_789-797(P＜0.01).â‘¡The cytotoxicity of CTLs from spenocytes against D2F2 cells were detected using LDH release assay.The results indicated that the killing rate of HSP110-P_789-797and HSP110-HER2/neu ICD group were significantly higher than that of PBS and HSP110 group(P＜0.01).While,the difference between PBS and HSP110 group was non-statistically significant.(2)Study on tumor suppression activity of HSP110-HER2/neu ICD complex vaccine in vivo:â‘ All of the remaining mice were sacrificed 28 days after the last immunization and the tumor tissues were isolated.The tumor tissues were weighed,and their volumes and the tumor suppression ratio were calculated. The results suggested that the average tumor volume and weight of HSP110-HER2/neu ICD and HSP110-P_789-797group were much lower than that of PBS and HSP110 group.Relatively,the tumor suppression ratio of HSP110-HER2/neu ICD and HSP110-P_789-797group were much higher than that of PBS and HSP110 group.In addition,the average tumor volume and weight of HSP110-P_789-797group were significantly higher than that of HSP110-HER2/neu ICD group(P＜0.01).â‘¡Histopathological changes of tumor tissuesThere was no large necrotic area in the tumor tissues of PBS and HSP110 group,but large and obviously necrotic areas were found in the tumor tissues of HSP110-HER2/neu ICD and HSP110-P_789-797group.The inflammatory cells were found in the edge of necrosis areas in HSP110-HER2/neu ICD and HSP110-P_789-797group.The peripheria of necrosis tissue was still there but the peripheria of cell was disappeared.In some cells,the nucleolus was dissolved of even diappeared.â‘¢Changes of the ultramicrostructures of tumor cellsIn HSP110-P_789-797group,macrophagocytes and lymphocytes were found around the tumor cells by TEM.Cell necrosis change could be found in tumor cells,and there were also cells with broken karyon,damaged membrane, dwindled karyon,swoln organella,or many vacuoles in the cytoplasm. Agglomerated chromatin and nucleolus were close to the karyotheca.There were no visible normal tumor cells in many areas.The phenomena above were much more remarkably in the HSP110-HER2/neu ICD group.In PBS group, many natural tumor cells were seen with big karyon,plenty of nucleoli and glycogenosomes.No swoln organelle could be found in these cells.Most of the cells of HSP110 group were natural tumor cells,while immune cells were found to attack tumor cells in some areas.To sum up,in this study we constructed and screened the high level steady expression HER2/neu ICD Pichia pastoris engineering strains,and obtained a high level expression with a yield of 295 mg·L^-1in a fermentor of 80 L by optimizing the main parameters affecting zymotechnique,establishing a new method for large-scale zymotechnique and purification of HER2/neu ICD in Pichia pastoris.And we confirmed the HSP110-HER2/neu ICD complex(the noncovalent binding products between recombinant HER2/neu ICD and recombinant HSP110)could induce specific immune responses for the first time. Our study made the foundation of using HSP110-HER2/neu ICD complex as a potential vaccine to cure tumors overexpressing HER2/neu and will help the further exploration on the immunifaction mechanism of HER2/neu ICD.