Study On The Treatment Of Ovarian Cancer Based On Recombinant Vaccinia Oncolytic Virus Skeleton Pexa-Vec

Objective:Ovarian cancer is the most fatal gynecologic cancer.After surgery and platinum-based chemotherapy,the recurrence rate remains as high as 75 % and eventually drug resistance occurs.Studies have shown that the overall survival rate of patients is positively correlated with the amount of tumor infiltrating T lymphocytes in ovarian cancer tissues,indicating that anti-tumor immune activation is an important means to improve the survival rate of patients.In recent years,both in vitro and animal experiments have proved that oncolytic virus has good tumor suppression effect,but in high mutation load tumors have not achieved the desired effect.Ovarian cancer is a solid tumor with low mutation tumor mutation burden,so using oncolytic virus therapy alone may not have a good effect.Although the therapeutic effect of oncolytic virus alone is not as good as expected,it can be expected that oncolytic virus is an ideal therapeutic platform as it has many advantages,such as targeting to the tumor,proliferation and replication in tumor cells,lysis of tumor cells,release of tumor antigen,recruitment of a large number of immune cells and breaking the tumor inhibitory immune microenvironment.Based on the vaccinia oncolytic virus skeleton PexaVec,this study intends to explore the path and effect of synergistic anti-cancer with neoantigens,immune checkpoint inhibitors,interleukins or tumor suppressor genes.Method:The full exon sequencing was performed on tumor cells from 10 patients with ovarian cancer.According to the analysis and comparison of sequencing data,the neoantigen peptide sequence of patients was predicted.One patient ’ s neoantigen peptide and its corresponding wild peptide were further synthesized for methodological study.By FCM and ELISpot in vitro screening,a neoantigen peptide with strong activation immunity was identified and tandemly grown.Recombinant vaccinia tumor-solubilizing virus JX-YERVOY / IL2 carrying YERVOY and IL-2 sequences and recombinant vaccinia virus JX-IL33 carrying IL-33 sequences were constructed and verified by q RT-PCR and ELISA.To further optimize the method of virus amplification and titer determination in vitro,and to observe the characterization of different ovarian cancer cell lines infected with JX-YERVOY / IL2 and JX-IL33.The killing effects of recombinant vaccinia on different ovarian cancer cell lines in vitro were detected by alamar Blue and RTCA.Recombinant vaccinia oncolytic virus JX-OPCML with tumor suppressor gene OPCML was constructed,and construction was verified by q RT-PCR and Western Blotting.The characterization of different ovarian cancer cell lines infected with JX-OPCML was detected.The killing effect of recombinant vaccinia oncolytic virus on different ovarian cancer cell lines in vitro was detected by alamar Blue and RTCA.A model of animals with subcutaneous transplant tumors in naked mice and C57 BL/6 mice were established to study the effects and mechanisms of tumor suppression in the body of recombinant vaccinia oncolytic virus.Result:A total of 362 neoantigens were predicted from the mutation sites of 10 patients with ovarian cancer.Through methodological research,a set of feasible schemes for sampling and sequencing of ovarian cancer samples,prediction of neoantigens,screening and identification of neoantigens in vitro,and antigen connection were established.JX-YERVOY / IL2 and JX-IL33 vaccinia oncolytic viruses were successfully constructed.The killing effect of JX-YERVOY / IL2 and JX-IL33 vaccinia on a variety of ovarian cancer cell lines in vitro was obvious,and compared with the control virus,it also showed obvious oncolytic enhancement in some cell lines.JX-OPCML vaccinia oncolytic virus was successfully constructed,which could completely kill a variety of ovarian cancer cell lines in vitro.In some cell lines,it showed stronger killing ability than JX0 and JX-NC.In nude mice SKOV3 subcutaneous xenograft tumor model,intratumoral injection of JX0,JX-NC,JX-YERVOY / IL2,JX-IL33 and JXOPCML viruses can significantly inhibit tumor development and reduce tumor volume.JX-YERVOY / IL2 has prominent immune activation performance.Conclusions : Based on the results of in vitro and in vivo experiments,recombinant vaccinia virus expressing Ipilimumab and IL-2,IL-33 or OPCML showed different specific effects superior to vaccinia Pexa-Vec skeleton virus.These results indicated that the optimized recombination based on vaccinia oncolytic virus skeleton Pexa-Vec combined with specific,non-specific immune activation effects or tumor suppressor gene effects may bring new breakthroughs treatment of ovarian cancer.