In Vitro Affinity Maturation Of A Fully Human Anti-multiple IFNα Subtypes Monoclonal Antibody With Novel Antigen Epitope

AIA22, a fully human anti-multiple IFNa subtypes monoclonal antibody selected from the fully synthetic phage display library, shows a novel antigen epitope distinct from the antibodies in clinical trails, e.g., sifalimumab and AGS-009. AIA22 also displays a different neutralizing profile of IFN-a subtypes from sifalimumab. Despite the lower affinity, the neutralization activity of AIA22 to IFN-a2b was remarkably better than that of sifalimumab, indicating AIA22 has obvious epitope advantages against the control antibody in our early research stage. However, the neutralization activities of AIA22 to most IFNa subtypes, except IFNa-2b and 4b, was not so good as those of sifalimumab, indicating further engineering of the candidate antibody AIA22 is needed. Structure-dependent affinity maturation technology was used for design and reconstruction of AIA22. The binding activity, neutralization activity, stability and metabolic characteristics of the designed moleculars were then evaluated in this study. Finally, we got several candidate antibodies with significantly improved affinity and neutralization activity to multi IFNa subtypes, these antibodies also showed similar or better overall neutralizing activity as the control antibody.Firstly, alanine scanning of AIA22 CDR region were performedand the change of binding activities to IFNa-1b and IFNa-2b were determined by using ELISA assay, of which the result revealed the essential information of the interaction between antigen and antibody, providing the experimental data to support the construction of structure model of antigen antibody complex by homology modeling and molecular docking methods. According to the analysis of the complex structure model, the low affinity of AIA22 and IFNa-1b may be due to the obvious structural incompatibility on the interaction surface between AIA22 and IFNa-lb. Combined with the results of alanine scanning, we designed a series of mutant antibodies to change the local conformation of the incompatible sites, the Y99 of AIA22 heavy chain and the LoopAB region of IFNa-lb. ELISA was used to identify the change of the binding activity of these mutants, following selectively combination of the mutation sites with binding activity significantly improved. We got several mutant antibodies with the improved binding activities to IFNa-lb while those to IFNa-2b was not decreased after the identification of affinity and kinetic properties of combined mutants to IFNa-lb and IFNa-2b by ELISA and forteBio system. Further, the neutralizing activity of the mutant antibodies in vitro was identified, and finally we got L-S34A+H-32G96W (AIAmut), the mutant antibody with the significantly improved neutralizing activity to multi IFN-a subtypes. Compared with the parent antibody AIA22, the neutralizing activity of AIAmut was in a different degree of improvement to IFNa-la,2a,2b,4b,5,10,16,17 and 21, and basically remained unchanged to IFN-a6 and 8, and was significantly deteriorated to IFN-al4. Overall, the neutralizing activity of AIAmut to IFNa was significantly improved. Compared to the control antibody Sifalimumab, the neutralizing activity of AIAmut was similar to IFNa-4b,5, 10,16,17 and 21, and was almost close to IFNa-la, and still remained a big gap between the Sifalimumab to IFNa-6,8,14 in the premise of the dominance of the neutralizing activity of AIAmut to IFN-α2b.At the same time, we have successfully crystallized the AIA22-Fab/IFNa-2b complex and obtained the structural information which helped the designation of a series of mutant antibodies based on AIAmut. W2S1A7, W2A7, W2A10, W2A11, S1A7, S1A10, S1A11 and W3, these 8 antibody mutants were finally selected as the candidate mutant antibodies by comparing the change of affinity and binding characteristics of different mutant antibodies to IFNα-1b and IFNa-2b.Further, we estimated the 8 candidate antibodies according to their expression level, stability and neutralizing activity. IFNa-1b-His, IFNa-2b-His and IFNa-4a-His were used as the target antigens respectively in the stability evaluation, and the standard samples were at stored at 4℃. The active concentrations of the mutant antibodies in serum and pH6.5 PBS buffer stored at room temperature and at 37℃ separately were measured. As a result, the binding activity of W3 and S1 A11 was lower than other mutants and AIAmut, and W3 was given up directly considering the lower expression levels than those of other mutants. Meanwhile, the W2S1A7, W2A7 and W2A10 were evaluated according to the neutralizing activity of the mutant antibodies to IFNa-lb and IFNa-2b. Compared with AIAmut, the neutralizing activity of the three mutant antibodies to IFNa-lb and IFNa-2b was improved, particularly to IFNa-lb, which was even better than Sifalimumab. The neutralizing activity of three candidate antibodies against different IFNa subtypes was comprehensively determined, and the results showed that the neutralizing activity of W2S1A7 and W2A7 was significantly improved to all IFNa subtypes except for IFNa-14. Specifically, the neutralizing activity of W2S1A7 and W2A7 was better than Sifalimumab to IFNa-2b,4a,21, and was similar to IFNa-lb,5,8,10,16 and 17, and was improved to IFNa-6 though lower than Sifalimumab, and still remained zero to IFNa-7, and was observed decreased to IFNa-14. Likewise, the neutralizing activity of AIAmut to IFNa-14 was not so good as AIA22.The pharmacokinetic characteristics of W2S1A7 and W2A7 in Babl/C mice showed that the half-life of both candidate antibodies was over 120 hours. In contrast, the highest blood concentrations and half lives of W2S1A7 were a little better than W2A7, so we chose W2S1A7 as a candidate antibody for futher development.