Study On Anti-lung Cancer Activity Of Oncolytic Virus Targeting Stromal Cells

Lung cancer is a serious threat to human health.In developed countries and large cities in China,the incidence of lung cancer ranks first in male tumors,and mortality is the first in malignant tumors.At present,the traditional way of treating lung cancer is surgery,chemotherapy and radiotherapy.When patients find lung cancer,they often get to the advanced stage,and the operation time is lost.Even if they are able to operate,the prognosis is poor.With the development of immunology,virology,pharmacology and other disciplines,in recent years there has been a new therapy,namely oncolytic virus therapy.The therapy has made gratifying results in the treatment of brain tumors and breast cancer,but the efficacy of its therapy in other tumors is still in the exploratory stage.Oncolytic virus can infect and kill cancer cells but had no effect on normal cell.The present study oncolytic viruses are divided into two categories,one is natural,such as Newcastle disease virus,reovirus,they have no human host,have weak ability to infect human cells,but have poor controllability,limited ability to kill tumor and easy to activate human immune system and be eliminated.The other is artificially modified viruses,such as herpes simplex,adenovirus and measles,which have specific killer effects on tumor cells and have been shown to be effective in clinical trials.This study is through homologous recombination methods to construct thymidine kinase（TK）and vaccinia growth factor（VGF）gene double deleting recombinant oncolytic vaccinia virus armed with bi-specific antibody of anti-FAP（fibroblast activation protein）and anti-mouse CD3,and to detect the inhibitory effect of FAP protein on lung cancer cell line（LLC）in mice in vitro.The main contents and results are as follows:1.Construction of recombinant oncolytic vaccinia virus rVVDD-BiTE.FAP.The BiTE-FAP and DsRed genes were amplified by PCR,and they were inserted into the pSEL2N1 plasmid by Infusion cloning,and the recombinant plasmid pSEL2N1-BiTE-FAP expressing red fluorescence was constructed.The BiTE-FAP gene regulated by the F17R promoter(P_F17R)and the DsRed gene regulated by the SE/L promoter(P_SEL)were homologously recombinated into the TK locus of the vSC20 parental virus,screening and purification by using soft agar plaque to construct double deleting recombinant oncolytic vaccinia virus（rVVDD-BiTE.FAP）armed with bi-specific antibody of anti-FAP（fibroblast activation protein）and anti-mouse CD3.2.Verify the recombinant oncolytic vaccinia virus rVVDD-BiTE.FAP.The titer of recombinant virus was determined by the method of crystal violet speckle.The genomic DNA of the virus was purified and verified by PCR,which would be sequenced in line with the size of the genome.Detection of the expression of BiTE.FAP protein in the supernatant of virus infected LLC cells by Western Blot,and determine the expression of anti-FAP-anti-CD3 BiTE recombinant oncolytic vaccinia virus.The lung cancer cell lines LLC and LL/2 were infected by virus v SC20 and rVVDD-BiTE.FAP.The replication ability of the two viruses was calculated and compared by plaque assay.It was found that the replication ability of the recombinant virus was not affected by adding exogenous genes.Using MTS method to detect the recombinant oncolytic virus on mouse lung cancer cell line（LLC）oncolytic ability,results show that with the increase of MOI value,the survival rate of LLC cells decreased.The results show that the recombinant oncolytic vaccinia virus construct correctly,and copy and oncolytic ability is not due to the insertion of exogenous gene affected.3.The killing effects of recombinant oncolytic vaccinia virus on LLC cells（LLC-FAP）in mice with over-expressed FAP.The virus filtrate was co cultured with LLC-GFP and LLC-FAP cells in the presence or absence of activated T cells.Then the survival rate of target cells was determined by MTS,and the toxicity of target cells was measured by LDH.It was found that rVVDD-BiTE.FAP had strong antitumor activity in vitro.FACS detects the killing effect.After ELISA detection of the supernatant of co culture system,it was found that the expression of cytokine IL2and IFN-gamma in T cells increased significantly after killing target cells（P<0.05）.Preliminary validation of recombinant oncolytic vaccinia virus rVVDD-BiTE.FAP in FAP overexpressing LLC cells has obvious killing effect,can be applied in the treatment of cancer.