Comparative Study On Anti-melanoma Activity Of Recombinant Human IFN-α1b And IFN-α2b In Vitro

Background:Melanoma is among the most aggressive forms of skin cancers.The prognosis of melanoma is extremely poor in middle and advanced stages.It is challenging for the treatment of melanoma.High-dose IFN-α2b is an adjuvant treatment for high-risk melanoma after surgery.However,IFN-α2b has large side effects,and 80%of the patients are unable to tolerate high doses and are forced to accept reduced or discontinuing treatment.Although immune checkpoint blockers(ICBs)and gene targeted drugs have improved the survival rate of melanoma patients universally,quite a number of melanoma patients are still unresponsive to these newly developed therapies.Recombinant human IFN-α1b is the first genetic engineering class I new drug with independent intellectual property rights developed in China.It is currently widely used in viral diseases, hematological tumors and so on.A previous phase I clinical trial reported that within 18patients with metastatic malignancies who had accepted recombinant IFN-α1b therapy for over 8 weeks,6 patients(5 with renal cell carcinoma and 1 with malignant hemangioendothelioma)showed progression-free survival for over 1 year,which indicated a potential antitumor effect of IFN-α1b.During the last ten years,IFN-α1b has been used to treat melanoma by this research group.We found that the side effects caused by IFN-α1b are significantly lower than IFN-α2b,while its efficacy is significantly better than IFN-α2b.IFN-α1b can not only be used as an adjuvant treatment for stage II and III melanoma,but also has a significant effect on stage IV melanoma.Encouragingly,our latest retrospective analysis showed that a recombinant product of IFN-α1b led to a median survival of 14.1 months in the patients with stage IV melanoma,and only 7.8%of the patients suffered severe adverse events,mainly fever,when they first took the drug (unpublished data).During the past two years,as a result of the improvements of medication,nearly 500 cases did not experience severe adverse events of high fever above40°C.These data demonstrate that IFN-α1b is a promising drug for the treatment of Chinese melanoma patients.Although we have used IFN-α1b to treat more than 800 cases of melanoma,however,there is no relevant research on the specific mechanism of IFN-α1b in treating melanoma.Aims:1.Compare the effects of IFN-α1b and IFN-α2b on the proliferation,apoptosis,migration and invasion of melanoma cells.2.Compare the effects of IFN-α1b and IFN-α2b on the antitumor immunity of melanoma.Methods:1.A CCK-8 assay was performed to measure the impacts on the proliferation of melanoma cells in vitro.2.Flow cytometry was performed to measure the impacts on the apoptosis of melanoma cells in vitro.3.Wound scratch assay was performed to measure the impacts on the migration of melanoma cells in vitro.4.Transwell assay was performed to measure the impacts on the invasion of melanoma cells in vitro.5.Peripheral blood was collected from 45 melanoma patients before treatment.PBMC were stimulated with culture medium alone,or medium supplemented with IFN-α1b or IFN-α2b.6.Flow cytometry was used to evaluate apoptotic effects.7.Lactate dehydrogenase(LDH)release assays were used to evaluate cytotoxic effects.8.Flow cytometry was used to analyze immunoregulatory effects on NK cells,NKT cells, CD3~+CD8~+T cells,and melanoma cells.9.Flow cytometry was performed to measure the impacts on the HLA-ABC of melanoma cells in vitro.10.Enzyme-linked immunospot(ELISPOT)assays were used to analyze the level of Granzyme B,TNF-αand IFN-γafter IFN-α1b or IFN-α2b stimulation.11.Flow cytometry was performed to measure the impacts on the PD-1/PD-L1 on CD3~+ CD8~+T cells and the PD-L1 of melanoma cells in vitro.Results:1.IFN-α1b showed a much stronger inhibition on the proliferation and apoptosis of melanoma cells than IFN-α2b.2.IFN-α1b had the same ability to inhibit the migration and invasion of melanoma cells as IFN-α2b.3.IFN-α1b enhanced the activity of NK cells,NKT cells and CD3~+CD8~+T cells from melanoma patients,while they were not different statistically.4.IFN-α1b had the same ability to enhance the expression of HLA-ABC in melanoma cells as IFN-α2b.5.IFN-α1b or IFN-α2b indentically enhanced the level of Granzyme B in PBMC of the patients with melanoma.6.IFN-α1b or IFN-α2b have no significant effects on TNF-αlevels in PBMC of the patients with melanoma.7.Compared with IFN-α2b,IFN-α1b induced a relatively lower level of IFN-γin PBMC.8.Compared with IFN-α2b,IFN-α1b without affecting the expression of programmed cell death-ligand 1(PD-L1)in CD3~+CD8~+T cells and induced a relatively lower level of PD-L1 in melanoma cells.Conclusion:1.IFN-α1b and IFN-α2b have the similar effects on immune cell activation.Therefore, the mechanism that the effects of IFN-α1b in the treatment of melanoma is better than IFN-α2b may not be based on the differences between the activation of immune cells.2.IFN-α1b has a stronger inhibitory and pro-apoptotic effect on melanoma cells than IFN-α2b,which may be an important mechanism for the clinical treatment the effects of IFN-α1b to be significantly better than IFN-α2b.In addition,IFN-α1b has little toxic and side effects,and patients can tolerate doses 2 to 5 times higher than IFN-α2b during clinical treatment,thereby endowing IFN-α1b a superior effect.3.The induction of PD-L1 by IFN-α1b is significantly lower than that of IFN-α2b, which may be one of the better clinical effects of IFN-α1b.