Design Of New Type Interferon Analogs And Study Of Their Bioactivity

Type III interferons were found in the recent years which are produced by infected cells or certain immune cells such as plasmacytoid dendritic cells(p DCs). They function similarly to type I interferons. Currently four members have been found, which are named IFN-λ1(IL-29), IFN-λ2(IL-28A), IFN-λ3(IL-28B) and IFN-λ4. They all bind to the same receptor complex consisting of IL-28R1 and IL-10R2 subunits to display antiviral bioactivities through activation of JAK-STAT signaling pathway.The genes encoding the type Ⅲ interferons are clustered on human chromosome 19(19q13.13 region). IFN-λ1 and IFN-λ2 genes are located in the positive strand while IFN-λ3 and IFN-λ4 genes are located in the negative strand. IFN-λ1 is composed of 5 exons; IFN-λ2 and IFN- 3 are composed of 6 exons. The promoter region of type Ⅲ interferons contains the binding site of interferon regulatory factor 3(IRF3) and IRF7, and at least one functional NFκB binding sites. There is a 25-amino acids signal peptide in the N-terminal of IFN-λ2 and IFN-λ3 protein sequences respectively. A 19-amino acids signal peptide is also found in the N-ternianl of IFN-λ1 protein sequence. Mauture IFN-λ2 and IFN-λ3 are composed of 175 amino acids while matured IFN-λ1 is composed of 181 amino acids. IFN-λ4 is transcribed from the IFNL4-delta G allel. The IFNL4-TT allele is considered to be a pseudogene. Although the homology between IFN-λ4 and the other 3 type III interferon members is very low, the structure of IFN-λ4 is similar to that of the other 3 type III interferon members. IFN-λ4 also binds to IL-28R1/IL-10R2 receptor complex to excert its bioactivity through JAK-STAT signaling pathway.Recent studies have found that the single nucleotide polymorphisms(SNP) in the unstream region of the IFN-λ3 genes are associated with the response to PEG-IFN-α plus ribavirine treatment of hepatitis C. The SNP is also associated with spontaneous clearance of hepatitis C virus. In addition, the SNP of IFN-λ4 gene is also associated with treatment response to PEG-IFN-α in combination with ribavirine and the spontaneous clearance of hepatitis C virus.Human type Ⅲ interferon receptor subunit gene il28 ra is located in chromosome 1p36.11, containing 7 exons. There are 3 alternatively spliced forms after transcription, resulting in 3 different m RNA molecules. IL-28R1 is composed of 520 amino acids. There is a 20-amino acid signal peptide at the N-ternianl of IL-28R1 protein sequence. IL-28R1 is divided into a 208-amino acid extracellular domain, a 23-amino acid transmembrane domain and a 271 amino acid intracellular domain. The extracellular domain has 4 N-glycosylation sites and 1 O-glycosylation site. The intracellular domain has 3 tyrosine residues. Tyr343 and Tyr517 are required for the activation of STAT2 and STAT5. The expression of IL-28R1 is cell specific; it is only expressed in mucosal epithelial cells.The other human type Ⅲ interferon receptor subunit gene il10r2 is located in 21q22.11, which contains 7 exons and encodes a polypeptide of 325 amino acids containing a 19-amino acid signal peptide at the N-termianl. The extracellular domain of IL-10R2 has 4 N-glycosylation sites and 4 O-glycosylation sites. The intracellular domain of IL-10R2 is composed of 79 amino acids.IL-28R1 is a specific receptor subunit of type III interferon, which is expressed only in mucosal epithelial cells and hepatocytes while IL-10R2 is shared by IL-10, IL-22, IL-26 and type III interferons. Meanwhile, the expression of IL-10R2 is not cell type specific. Primarily, type III interferons bind to the IL-28R1 subunit, which then binds with IL-10R2, forming a ligand-receptor complex and activating the JAK-STAT cell signal pathway.In addition to direct antiviral activity, type III interferons can also regulate immune response by up-regulating MHC I molecules. Type III interferons play a pivotal role in the prevention of viral infection in mucosal epithelial cells. Different from type I interferons, which are associated with severe and widespread side effects, type III interferons elicit a limited side effects because of the specificity of its receptor expression. Therefore, type III interferon is very promising in the prevention and treatment of respiratory and gastrointestinal viral infection.Type III interferons can inhibit the growth of a variety of tumor cells. Type III interferons can induce the expression of TRAIL and Fas death receptor(CD95) to activate caspases 3/7 that finally induce apoptosis. Type III interferon can also arrest the cell cycle at the G1 phase, by upregulating the expression of the cell cycle regulator p21 and promoting phosphorylation of Rb,eventually resulting in inhibition of cell proliferation.Type III interferon plays a pivotal role in antiviral defense and innate immune response in specific target cells. Different from type I interferon, the activity of type III interferon is cell specific. Although type III interferon plays an important role in tumor therapy, viral infection and autoimmune diseases, only clinical trials for HCV therapy have been carried out. The Phase IIb study has shown an efficacy of IFN-λ1 in the treatment of HCV that is similar to IFN-α. IFN-λ1 treatment induced more-rapid viral suppression compared with IFN-α during the first 12 weeks. However, at week 24, rates of viral suppression are almost identical for IFN-λl and IFN-α. As previously predicted, IFN-λl induces limited side effects. Neutropenia and thrombocytopenia are rare in patients receiving PEG-IFN-λl. Therefore, IFN-λl should be used to replace IFN-α.Type III interferons have very specific antiviralactivities, with very low side effects. However compared with type I interferons, their bioactivity are 10-fold lower. Therefore, it is important to improve the biological activity of type III interferons.It was found that the antiviral activity of IFN-λ3 was higher than that of IFN-λ1, but it was difficult to express it in prokaryotic expression system. Meanwhile IFN-λ1 is easy to be expressed and purified in the prokaryotic system. Therefore, to develop optimal IFN-λ molecules with improved drug attributes, we designed 9 IFN-λ analogs by replacing critical amino acids of IFN-λ1 with the IFN-λ3 counterparts and expressed them in prokaryotic system. 4 of the 9 designed analogs could be expressed in E.coli.. Then the bioactivities of the 4 expressed IFN-λ analogs were verified by a series of experimental studies.It is the first time to study the expression and purification of IFN-λ 1/3 analogs, and the modification of PEG. Then we studied the activity and stability of IFN-λ1/3 analogs. Analog-6 displayed an unexpected high potency that is higher than that of type I IFN(IFN-α2a) in activating the IFN-stimulated response element(ISRE)-luciferase reporter. Importantly, both PEG-analog-6 and PEG-analog-7 effectively inhibited the replication of hepatitis C and influenza A virus effectively. The activity of PEG-analog-6 is higher than that of PEG-IFN-α2a. In addition, analogy-6 also inhibited the proliferation of lung cancer cell HCC827 and increased the expression of APOBEC3 G in the HCC827 cell line.Here we provided a method of exchanging the sequences between family members for protein engineering and proved that it could improve the drug attributes of protein drugs. All designed analogs bear no unnatural amino acid sequence and should display minimal immunogenicity. Therefore this method could be used to engineer similar proteins, improving drug properties. Finally we draw the following clusions:1. Designed 9 IFN-λ analogys by exchanging critical amino sequences of IFN-λ1 and IFN-λ3 and tried to express, purify and modify them with PEG. We found 4 of the 9 designed analogs with IFN-λ1 at N-teminal could be expressed in Ecoli. The analogs with a IFN-λ3 N-teminal could not be expressed in Ecoli.. A novel technology of designing, expressing, purifying and modifying IFN-λ analogs was developed..2. The expression of interferon-stimulated genes Mx A and OAS was detected in Hep G2 cells treated with analogs by Real-time PCR; the results confirmed the bioactivity of the 4 successfully expressed analogs. The bioactivities of these 4 analogs were dose- and time-dependent.3. Phosphorylation of STAT1 was detected in response to analog treatment, indicating that the analogs signal through the JAK-STAT pathway.4. We quantitatively studied the bioactivity of the analogs by ISRE-luciferase reporter gene system. The bioactivity of analog-6 and analog-7 was higher than that of IFN-α2a.5. Immunostaining and RT-PCR results demonstrated that the analogs could inhibit the replication hepatitis C virus and their anti-HCV activity were equivalent to IFN-α2a.6. Immunology stainging and RT-PCR results demonstrated the analogs could inhibit the replication hepatitis C virus and their activity were equivalent to PEG-IFN-α2a.7. The stability of PEG-analog-6 was tested by placing 3 lots of the protein preparations at 40°C for a month and found that the concentration and purity of PEG-analog-6 remained quite stable, indicating that high stability.8. Cell proliferation result suggested that PEG-analog-6 could inhibit the proliferation of HCC827, and induce HCC827 apotosis. Therefore, PEG-analog-6 could inhbit cell proliferation and its activity is higher than that of PEG-IFN-λ1.In summary, the results of this study show that analog-6 can be expressed in E. coli and easily purified with good stability. Analog-6 can induce the expression of ISGs, with high antiviral and antitumor activities, and hence is a promising candidate for the development of new protein drug.