Site-specific monoPEGylated interferon alpha2a mediated by microbial transglutaminase / 生物工程学报

PEGylation is considered one of the most successful techniques to improve the characteristics of protein drugs including to increase the circulating half-life of proteins in blood and to decrease their immunogenicity and antigenicity. One known PEG modification method is to attach PEG to the free amino group, typically at lysine residues or at the N-terminal amino acid with no selectivity, resulting in a heterogeneous product mixture. This lack of selectivity can present problems when a therapeutic PEGylated protein is being developed, because predictability of activity and manufacturing reproducibility are needed for regulatory approval. Enzymatic PEGylation of proteins is one route to overcome this limitation. Transglutaminases (TGase) are enzyme candidates for site-specific PEGylation. We use human interferon alpha 2a (IFN α2a) as a test case, and predict that the potential modification residues are Gln101 by computational approach as it contains 12 potential PEGylation sites. IFN α2a was PEGylated by Y shaped PEG40k-NH2 mediated by microbial transglutaminase. Our results show that the microbial transglutaminase mediated PEGylation of IFN α2a was site-specific only at the site of Gln101 in IFN α2a, yielding the single mono-conjugate PEG-Gln101-IFN α2a with a mass of 59 374.66 Da. Circular dichroism studies showed that PEG-Gln101-IFN α2a preserved the same secondary structures as native IFN α2a. As expected, the bioactivity and pharmacokinetic profile in rats of PEG-Gln101-IFN α2a revealed a significant improvement to unmodified IFN α2a, and better than PEGASYS.

Changes in peripheral blood during the antiviral treatment with Peg-IFNα-2 a and ribavirin for patients with chronic hepatitis C and the relationship between the changes and effect / 中华实验和临床病毒学杂志

Objective@#To observe the changes of peripheral blood image of chronic hepatitis C (CHC) patients treated with pegylated interferon (Peg-IFN) and ribavirin, and explore the relationship between the changes and serum virological respose (SVR).@*Methods@#Patients with CHC treated with Peg-IFN α-2 a and ribavirin in the Second Division of Liver Disease in Beijing Ditan Hospital were monitored for peripheral blood cells routine examination and Liver function, kidney function, thyroid function at baseline and week 2, 4, 8, 12, 24, 36, 48, and week 12, 24, 48 after the end of treatment for chronic hepatitis C, and HCV RNA.@*Results@#The decrease of peripheral blood cell counts began to appear at week 2 of treatment. For CHC patients without cirrhosis, white blood cells, lymphocyte, hemoglobin and platelets at week 2, while minimum values were seen at week 36, and the neutrophils reached the minimum value to at week 24. Significant recovery of the peripheral blood changes was seen at the end of treatment (48 weeks), and reached pre-treatment levels at week 48 after the end of treatment. For CHC patients with cirrhosis, white blood cells, lymphocytes, hemoglobin and platelets significantly decreased at week 2, while neutrophils reached a minimum value at 2 weeks. And hemoglobin and platelets reached a minimum value at 24 weeks, and the white blood cells reached the minimum value at weeks 24-36 besides lymphocyte reached the minimum value at week 36. Significant recovery was seen at the end of treatment (48 weeks), and the blood cell counts reached pre-treatment levels at 48 weeks after the end of treatment. For patients with CHC, hemoglobin decreased by more than 27.47% at week 4, which means that the patient would have a predictive significance for SVR, as well as the of PLT reduction by more than 36.96% at week 8.@*Conclusions@#During the treatment with Peg-interferon and ribavirin, the variation of blood picture has some predicting effect, which predicts the result of antiviral treatment.

The influence of PEG-IFN-α 2a on the expression of ICOS on CD4+ T cell in patients with chronic hepatitis B / 中华微生物学和免疫学杂志

Objective To study the expression levels of inducible costimulator (ICOS) on CD4+ T cells in peripheral blood in patients with chronic hepatitis B and its change after treated with interferon.Methods All 56 patients were divided into two groups (interferon group has 28 cases,lamivudine group has 28 cases),and interferon group treated by PEG-IFN-α 2a,lamivudine group treated by lamivudine,respectively.There were 20 healthy individuals as control group.The levels of ICOS on CD4+ T cells of patients with chronic hepatitis B were kinetically detected by flow cytometry.The copies of HBV DNA in sera were dynamically detected by real-time PCR.Results The levels of ICOS on CD4+ T cells in patients with chronic hepatitis B was higher than that of normal controls(P＜0.001 ).However,the expressions of ICOS on CD4+ T cells in patients could be deduced by PEG-IFN-α 2a and obviously decreased after treatment.There was significant difference between interferon group and lamivudine group (P＜0.05 in 24 weeks,and P＜0.01 in 48 weeks).After treatment,the change values of ICOS in interferon group was positively correlated with the change copies of HBV DNA (r =0.972,P＜0.001 ).However,the change values mentioned above that did not find correlation in lamivudine group(r=-0.101,P=0.608).Conclusion The study shows the patients with chronic hepatitis B have disordered in cellular immunity and increased with the expression of ICOS on CD4+ T cells.PEG-IFN-α 2a could decrease the expression of ICOS on CD4+ T cells in patients,and correct the Th2 type immune polarization to some extent,and that plays a positive role in antiHBV.

The treatment for bladder tumor with ATRA combining with interferon alpha-2a / 肿瘤研究与临床

Objective To investigate the effect of all trans-retinoic acid (ATRA) combining with Interferon α-2a on bladder tumor and the possible mechanisms. Methods Fifty female Wistar rats with bladder tumor were established and separated into 4 groups randomly that are DMSO group (A), IFN-α-2a group(B), ATRA group(C) and ATRA+IFN-α-2a group(D). Then each group was treated by drugs indicated. Finally, the weight of bladder, the stage and grade of tumor, the apoptosis and proliferation index of tumor were determined to estimate the effect of ATRA+IFN-α-2a. Results The body weights in group D are the highest and the bladder weights in group D are the lowest. The stages and grades of tumor in group D were statistically decreased compared with those in the other 3 groups. Accordingly, the apoptosis of cancer cells in group D was enhanced, whereas the proliferation index in group D was decreased significandy. Conclusion The effect of ATRA combined with Interferonα-2a on the bladder tumor is better than that of monotherapy.