Anti-melanoma Activity Of A Novel Tumor Vaccine Based On TLR7

Objective: Melanoma is a common skin cancer with high malignancy.It is characterized by high invasiveness,high mortality and high immunogenicity.In a phase II clinical trial,50% of ipilimumab-treated melanoma patients had adverse side effects.Some of them were forced to terminate the treatment because of their severe pancreatitis.Even Nivolumab may help pateients have a longer non recurrent survival period only when they also receive a curative resection.Therefore,it is urgent to find a more effective treatment for melanoma patients.5-azacytidine(Aza)has been approved by FDA for the treatment of myelodysplastic syndromes.Low doses of Aza can upregulate the expression of silent endogenous retrovirus(ERVs)in tumor cells through demethylation,and thus activating the immune response via a viral defense pathway.Toll-like receptors(TLRs)are bridges linking innate and acquired immunity.TLR7 agonists can effectively activate the immune system and exert an anti-tumor action.The purposes of this study were:(i)to synthesize a novel effective TLR7 agonist;(ii)to prepare a novel whole cell tumor vaccine by coupling our synthesized agonist to Aza treated melanonma cells;and(iii)to study the in vivo antitumor effect of the vaccine on mice.Methods:(i)A chemical synthesis method was used to produce a novel pure TLR7 agonist,SZU-106,and its structure was identified by the mass spectrometry and Nuclear Magnetic Resonance(NMR);(ii)the method of ELISA was employed to detect the in vitro immune-activating effect of SZU-106 on immune cells;(iii)CCK8 kits were purchased to determine the optimal concentration of Aza on melanoma B16-F10 cells;(iv)ELISA was applied to determine the optimal concentration of Aza on B16-F10 melanoma cells;(v)0.5 μM Aza-treated B16-F10 cells were coupled with SZU-106 and then killed by the UV light so as to prepare the whole cell tumor vaccine named Aza-cell-106;(vi)the in vitro immune activating effect of the vaccine was investigated by ELISA;(vii)The flow cytometry was used to detect the effect of the vaccine on dendritic cells(DCs);(viii)the melanoma model of mice was established by injection of B16-F10 cells for investigating the anti-tumor activity of the vaccine;(ix)after the mouse was sacrificed,the tumor tissues were collected for the analysis of T-cell infiltration;and(x)the in vivo immune-activating effect of vaccine was studied by CBA kit.Results:(i)A novel TLR7 agonist SZU-106 was successfully synthesized.It could effectively activate the immune cells to secrete cytokines in vitro.Due to the unique carboxyl structure of SZU-106,it could be chemically coupled to tumor cells.Low doses of Aza could increase the expression of silent ERVs through demethylation and activate the virus defense pathway,which resulted in the high expression of TLR3 in the B16-F10 cells.(ii)The whole cell tumor vaccine,Aza-cell-106 was successfully prepared.The vaccine could stimulate mouse splenic lymphocytes and bone marrowderived dendritic cells(BMDCs)to secrete a large amount of cytokines.The vaccine could also promote the the maturation of dentrite cells(DCs)and so enhance its capability of antigen presentation.(iii)In the melanoma tumor model of mice,the vaccine could induce a transient increase of cytokines,thereby stimulating the immunity,inhibiting the tumor growth,shrinking the tumor volume and prolonging the survival period of mice.No nausea was observed in the experiment.The combination therapy of the vaccine and Repertaxin drastically increased the infiltration of CD4+T and CD8+ T cells,and the therapeutic effect of Repertaxin was enhanced significantly.Conclusion: The novel synthesized SZU-106 is an effective TLR7 agonist.The whole cell tumor vaccine,Aza-cell-106 exhibits an efficient immune activating effect in both in vitro and in vivo experiments.The vaccine can effectively inhibit the growth of melanoma in mice.This study sheds light on the immunotherapy of melanoma.