A Study On The Effect Of The Number Of BAFF Antagonistic Peptides On Its Inhibitory Activity As A Peptide-Fc Fusion Protein

Studies have shown that BAFF is associated with a variety of autoimmune diseases,including systemic lupus erythematosus(SLE),rheumatoid arthritis(RA)and primary Sj(?)gren’s syndrome(p SS).Therefore,several BAFF antagonistic products have developed for the treatment of autoimmune diseases,such as monoclonal antibodies(Belimumab,Tabalumab),receptor-Fc fusion proteins(Atacicept,Briobacept)and peptide-Fc fusion protein(Blisibimod).Among them,Blisibimod is a peptide-Fc fusion protein,which has the advantages of long half-life and high affinity,and has shown good efficacy and safety in clinical trials for the treatment of patients with SLE.In 2015,the lab designed a new fusion protein(GK12-Fc)with good biochemical activity according to the design concept of Blisibimod,and found that the fusion protein3GK12-Fc with three antagonistic peptides GK12 had the greater binding capacity to BAFF than GK12-Fc.So this thesis mainly studied the functional difference between fusion proteins with different numbers of antagonistic peptides.In this study,we designed and constructed the 2GK12 and 3GK12 genes based on the GK12 gene sequence,and purified the fusion proteins GK12-Fc,2GK12-Fc and3GK12-Fc.Furthermore,we detected the ability of fusion proteins binding to BAFF,the ability of peptides BCMA1,BCMA2,BR31 and BR32 inhibiting the fusion proteins binding to BAFF through ELISA.The results showed that the fusion proteins GK12-Fc,2GK12-Fc and 3GK12-Fc all bind to BAFF in a dose-dependent manner.The binding ability to BAFF raises with the increase of fusion proteins concentration,reaching saturation at 150μg/m L.Moreover,the binding ability to BAFF also enhances with the increase in the numbers of GK12 in the fusion protein,GK12-Fc,2GK12-Fc and 3GK12-Fc showed 77.1%,83.1% and 95.2% binding activity compared to BCMAFc-myc,respectively.Peptides BCMA1,BCMA2,BR31 and BR32 could significantly inhibit the binding of fusion proteins to BAFF.Among them,peptide BCMA1 had the strongest inhibitory effect on GK12-Fc,when BCMA1 concentration was 100μg/m L,BCMA1 significantly inhibited the binding effect of GK12-Fc by 46.8%;while peptide BCMA2 had the strongest inhibitory effect on 3GK12-Fc,when BCMA2 concentration was 100μg/m L,BCMA2 significantly inhibited the binding effect of 3GK12-Fc by52.0%.So we speculated that the binding site of GK12-Fc and BAFF is similar to the binding site of BCMA1 and BAFF.Peptides BR31 and BR32 had the strongest inhibitory effect on 2GK12-Fc,when the concentration of BR31 and BR32 were100μg/m L,BR31 and BR32 significantly inhibited the binding effect of 2GK12-Fc by46.2% and 35.8%,respectively.So we speculated that the binding sites of 2GK12-Fc and BAFF is similar to the binding sites of BR3 and BAFF.Since BCMA1 and BR31 contain the conserved Dx L motif that plays an important role in the binding of BAFF and its ligands,while BCMA2 and BR32 are outside the conserved Dx L motif,therefore,we speculated that increasing the number of antagonistic peptides GK12 may cause the binding sites of the fusion proteins and BAFF to change.In summary,our study provides a meaningful idea for the design of peptide-Fc fusion proteins targeting BAFF with different numbers of antagonistic peptides.