A Recombinant Oncolytic Adenovirus Expressing A Mutant Soluble Interleukin-6 Receptor In Lung Cancer Treatment

Cytokine IL-6 has many beneficial functions in hematopoiesis and regeneration.The IL-6/JAK/STAT3 signaling pathway is activated abnormally in many types of tumors.The cytokine IL-6 in the signaling pathway leads to activation of transcription factor STAT3,which further increases the expression of IL-6.This positive feedback pathway provides a tumor microenvironment that promotes tumor growth.IL-6 overreaction plays a central role in the occurrence and development of cancer,usually associated with poor clinical prognosis.In tumor microenvironment,IL-6/JAK/STAT3 signaling pathway inhibits the anti-tumor immune response and promotes the proliferation,survival,invasion and metastasis of tumor cells.Therefore,by inhibiting the IL-6/JAK/STAT3 signal path,the anti-tumor immunity of the body can be activated to further inhibit the progress of the tumor.Oncolytic adenovirus is a selfreplicative virus that selectively infectes and lyzes tumor cells.Oncolytic adenovirus kills tumor cells and releases tumor antigens,recruits immune cells,a process so-called changing tumor "from cold to hot".However,the infiltration and activation of immune cells within tumor also induce a large number of pro-inflammatory cytokines,including IL-6.IL-6 activates STAT3,a key transcription factor in precancerous cells,to control a serial of tumorigencsis processes,including survival,proliferation,growth,angiogenesis and invasion.Therefore,we hypothesized that blockingIL-6/JAK/STAT3 signaling pathway might achieve an optimal therapeutic effect in oncolytic adenoviral therapyIn this study,we designed a mutant soluble IL-6 receptor(mutant-soluble IL-6 receptor,Mutant-sIL-6R),which competitively binds to IL-6,however,unlike the wild type soluble IL-6 receptor(wild type-soluble IL-6 receptor,Wild Type-sIL-6R),IL-6/Mutant-SIL-6R complex can not bind to gp130,thus blocks the STAT3 activation and the IL-6 trans signaling pathway(IL-6 trans-signaling).We found that Mutant-sIL-6R can effectively block the IL-6/JAK/STAT3 signaling pathway of A549,H1299,and llc cells and inhibit the activation of downstream STAT3.The results were compared with the significant enhancement of the phosphorylation of STAT3 in the tumor cells of Wild Type-sIL-6R,and further confirmed that the Mutant-SIL-6R constructed by us can effectively block the IL-6/JAK/STAT3 signaling pathway.And in the llc subcutaneous tumor animal experiment,the growth of the mouse llc in the Mutant-sIL-6R treatment group was inhibited,and there was a significant difference compared with the control group.On the basis of confirming the function of the mutant-soluble IL-6 receptor,we designed a recombinant adenovirus(Mut-sIL-6R-AD5-E1A)expressing Mutant-sIL-6R,aiming to link the blocking of the IL-6/JAK/STAT3 signaling pathway with the treatment of the oncolytic adenovirus.While the tumor is dissolved and the anti-tumor immunity is activated,inhibiting the IL-6/JAK/STAT3 signal path and inhibiting the blocking effect of the IL-6/JAK/STAT3 signal path on the anti-tumor immunity.Our study found that Mut-sIL-6R-AD5-E1A can express Mutant-sIL-6R and release to the outside of the cell,and this activity does not affect its own dissolved tumor and replication functions.The Mutant-sIL-6R expressed by Mut-sIL-6R-AD5-E1A can also effectively block the IL-6/JAK/STAT3 signaling pathway of A549,llc and H1299 cells and inhibit the phosphorylation of downstream STAT3.It was found that the replication ability of the adenovirus in the rat-derived cells was significantly lower than that of the human cells.In order to effectively mimic the treatment status of the human-derived tumor in the immunized mice,the llc-CAR was constructed to stably express the coxsackie-adenovirus receptor.The replication ability of the adenovirus in llc-CAR was significantly enhanced.In vivo,there was no significant difference in tumor size and survival between the Mut-sIL-6R-AD5-E1A treatment group and the Untreated group when virus was implanted into the subcutaneous tumor of mice for treatment.After further literature analysis,we found that the expression of CAR is related to the carcinogenesis and development of lung cancer.High expression of CAR can enhance the invasion and metastasis of lung cancer cells,so it is not possible to stably express CAR through 11c to enhance the invasion efficiency of oncolytic adenovirus.In order to avoid the failure to express mutant IL-6 receptor due to the low infection rate of mouse-derived adenovirus.In the following improved experiment,we can use humanized mice.Human tumor cells were subcutaneously injected and adenoviral therapy was performed.We can also search for highly invasive mouse-derived cells of adenovirus,establish mouse tumor model,and verify the therapeutic effect of Mut-sIL-6R-AD5-E1A.The innovation of our research is to combine IL-6 blocking with the treatment of tumor adenovirus to construct recombinant adenovirus Mut-sIL-6R-AD5-E1A,expressing mutated soluble IL-6 receptor.It is proved that it has a good blocking effect on IL-6/JAK/STAT3 signaling pathway.This breakthrough has brought a new idea for IL-6 signal blocking and adenoviral therapy,and laid a solid foundation for the combination of the two therapy in the future.