Affinity Maturation Of Anti-TSLP Recombinant Human Monoclonal Antibody In Vitro

Objective:Thymic stromal lymphopoietin（TSLP）was identified as a secreted factor of a mouse thymic stromal cell line 20 years ago;Soon after,human orthologs were discovered.At present,the signal transduction pathway mediated by TSLP has been widely studied,including up-regulate cytokines of Th2-related diseases,such as asthma,atopic dermatitis,hypersensitivity reactions and even some cancers.Therefore,TSLP is considered as a potential target for treatment-related diseases^[1].Since the discovery of hybridoma technology in 1975,monoclonal antibodies can be prepared with good specificity.However,due to the murine origin of them,there is a strong human anti-mouse antibody reaction（HAMA）^[2,3]in the treatment of human diseases,which limited the application of clinical.Currently,Murine antibody was humanized by using genetic engineering techniques,which is the hot topic in the field of antibody drugs.The development of monoclonal antibody drugs has experienced four stages:murine antibody,chimeric antibody,humanized antibody and full human antibodies.In the previous work,the anti-TSLP-scFv（anti-Thymic stromal lymphopoietin full-human single chain antibody）was screened by constructing a large library of full-human antibody library and phage display technology,which solved the problem of immunogenicity,but the affinity of the antibody is low^[8,9].so we adopted computer-aided techniques for homology modeling of antibodies and antigens and predicted molecular anomalies of amino acid residues by molecular dynamics simulation for site-directed mutagenesis to promote in vitro affinity maturation of anti-TSLP monoclonal antibodies.Methods:In the previous study,specific anti-TSLP-scFv（84）was screened from full human antibody library by phage display technique and obtained the DNA sequence of this antibody by sequencing.1.In this study,homologous 3D models of TSLP and anti-TSLP-scFv were constructed by using computer-aided techniques.In addition,we obtained a reasonable and stable docking model by molecular docking technology,which simulated molecular dynamics and force field.By alanine scanning and single point mutation,we can predicte amino acid sites,of which affinity can be increased after mutation.2.Specific primers were designed according to the mutation sites and a single point mutation was performed for the target fragment by overlap PCR to obtain the mutated DNA sequence of anti-TSLP-scFv.The mutant sequence was linked to the prokaryotic expression vector pLZ16,then it was transformed into E.coli DH5αF,.After expression,the affinity-enhanced anti-TSLP-scFv bacterial strain was screened by ELISA.3.The affinity enhanced antibody gene was inserted into the eukaryotic expression vector PCDNA3.1-sp-Fc and PMH₃^en,then it was transfected into HEK-293T cells to express the anti TSLP-scFv-Fc fusion protein.The anti TSLP-scFv-Fc fusion protein was identified by SDS-PAGE and Western blot.The affinity of recombinant antibodies was detected by the biological macromolecule interaction technique.Results:1.The Discovery studio4.5 system was used to establish a three dimensional model of TSLP antigen and anti TSLP-scFv.5J11A and 4KFZ C（VH）/4HS6 A（VL）,the highest similarity sequences of TSLP and anti TSLP-scFv were searched by BLAST（NCBI Server）as the initial model.The protein docking was calculated by ZDOCK.Based on the CHARMm energy,the RDOCK was used to optimize the configuration of the butt complex found by ZDOCK.The final docking stable conformation Pose50 was obtained by molecular dynamics simulation.The amino acid analysis was performed on the docking surface.Alanine scan and saturation mutation were carried out to determine the amino acid combination after the virtual mutation.The amino acid combination was TRP（109）-ARG,TRP（109）-LYS,TRP（109）-TYR,TRP（109）-LEU,TRP（109）-GLU.2.By pairing the specific primers of the mutation sites,the size of the front fragment was about 300bp,and the size of the latter fragment was about 450bp.The size of the anti TSLP-scFv gene fragment by the overlapping extended PCR was about 750bp.3.The gene sequence of mutanted anti TSLP-scFv was inserted into the expression vector pLZ16,and the recombinant plasmid was transformed into E.coli DH5αF,by successful sequencing.Anti-TSLP-scFv was expressed by the prokaryotic system.We found that the affinity of mutated M4 increased by ElISA detection.4.The gene sequence of permutated 84 and mutated M4 were successfully recombined into eukaryotic expression plasmid PCDNA3.1-sp-Fc and PMH₃^en.Then the recombinant plasmid was transfected into HEK-293T cells.The expression of anti TSLP-scFv-Fc fusion protein was identified by SDS-PAGE and Western blot,and the size was about 67KD.5.The affinity of mutated M4 was about 10 times higher than premutated.Conclusion:The affinity maturation of anti TSLP recombinant monoclonal antibody in vitro has been successfully promoted by computer aided technique.