Molecular Design,Production And Bioactivity Of The Genetically Engineered Antibody Against LOX-1

Atherosclerosis(AS)continues to be a major cause of morbidity and mortality in the world.Lectin-like oxidized low-density lipoprotein receptor-1(LOX-1)plays an important role in the genesis and develop of AS.LOX-1 basal expression is relatively low,and it is substantially up-regulated in disease states.LOX-1 has been proved to be a sensitive diagnostic biomarker of atherosclerosis.In addition,recent evidence indicates that targeting LOX-1 is a reliable strategy for the therapy of vascular disease and that LOX-1-mediated pro-atherogenic effects can be inhibited by anti-LOX-1 monoclonal antibodies(MAbs).Moreover,LOX-1 can be used for plaque imaging using MAbs and selective delivery of anti-atherosclerotic agents.However,the application of the intact MAb molecules is limited by potential side effects and high production costs.Their large size also limits their ability to penetrate the diseased areas.In addition,binding of the fragment crystallizable(Fc)domain of the MAbs to Fc receptors on the cell surface limits the circulation and mobility of the whole molecules.As an alternative to MAbs,small molecular antibody fragments such as single-chain antibody fragments(scFvs)are increasingly being used in diagnostic and therapeutic applications,due to their specific binding affinity to antigens,superior bio-distribution and blood clearance,low-immunogenicity,easy preparation,low cost,and high modifiability.However,despite the great potential of scFv molecules,few antibody fragments possess ideal biophysical properties for medical applications,as their downsizing molecular size results in too-rapid clearance from the blood and involves a decrease in valence and failure to induce secondary immune functions because of the removal of the Fc region.Thanks to the advantages of high modifiability and easy preparation,a series of efficient scFv engineering strategies has been developed.As the anti-LOX-1 MAbs have been proved with important use in the diagnosis and therapy of AS,we think that the anti-LOX-1 scFv with good quantity has rich potential as a LOX-1 targeting vector for immunodiagnostics,drug delivery and potential immunotherapy.This study concentrates on the efficient production technology and engineering of the anti-LOX-1 scFv with the aim to increase its application efficacy in LOX-1-based therapy,and the application of the scFv in AS targeted drug delivery.Three aspects of studies about the anti-LOX-1 scFv were developed in this paper,as shown below.1.Efficient production strategy of the anti-LOX-1 scFv.The expression of the anti-LOX-1 scFv was attempted using E.coli and Brevibacillus choshinensis expression systems.A systematic optimization study of the expression of anti-LOX-1 scFv in Brevibacillus choshinensis was performed using Plackett-Burman screening design.The scFvs expressed from the two expression systems can be purified efficiently using a two-step column chromatography purification progress.Properties including antigen-binding activity,structural characterization and stability of the purified anti-LOX-1 scFv proteins obtained from two expression systems were analyzed.Results showed that the anti-LOX-1 scFv expressed in E.coli mostly formed inclusion bodies,while Brevibacillus choshinensis can be an ideal host for soluble expression of the scFv,and the anti-LOX-1 scFv expressed from Brevibacillus choshinensis hosts showed similar antigen affinity(1.017E-07 M)with that of the scFv expressed from mammalian cells.This study lays a foundation for the next studies and for the scale-up production of the anti-LOX-1 scFv.2.Engineering study of the anti-LOX-1 scFv.Three different protein engineering strategies were applied to optimize the anti-LOX-1 scFv to obtain anti-LOX-1 scFv mutants or derivatives with high antigen binding activity:(1)Multimerization of the anti-LOX-1 scFv to improve its antigen binding avidity.The scFv multimers were constructed by shortening the length of the linker between the two variable domains of the scFv and by fusing the scFv with self-merization domains.After optimization,these scFv multimers were successfully expressed and purified.Results showed that compared with the anti-LOX-1 scFv monomer,the scFv multimers displayed increased antigen-binding avidity and neutralization activity,especially the anti-LOX-1 scFv tetramer,which improved the antigen-binding avidity by 170-fold higher,moreover,it had a higher thermal stability and longer half-life in serum.(2)Fusion of the anti-LOX-1 scFv and LOX-1-binding peptides guided by molecular simulation study.Molecular simulation showed that the LOX-1 binding peptides LTPATAI,FQTPPQL and LSIPPKA with the anti-LOX-1 scFv had different interfaces docking with the LOX-1 protein,thus there is a possibility to increase the antigen binding activity of this scFv by fusing it with these LOX-1 binding peptides.Each of the three LOX-1 binding peptides was designed to fused at the N-terminus,C-terminus or the linker region,respectively.Nine LOX-1 binding peptide and anti-LOX-1 scFv fusions were constructed and produced.Results showed that the sequence of the LOX-1-binding peptides and their fusing mode with the scFv importantly affected the affinity and stability of the fusion proteins.The anti-LOX-1 scFv with N-terminal fusing peptide fusion LSI-scFv showed a higher antigen affinity,about 7-fold over that of the wild type scFv,without thermal stability and serum stability decreased.(3)Computer-aided engineering of the anti-LOX-1 scFv.Analysis of the anti-LOX-1 scFv and LOX-1 docking complex showed that 30 mutations of the scFv displayed a higher antigen-antibody binding energy,thus 30 mutants were constructed and produced.Results showed that compared with the wild type anti-LOX-1 scFv,the mutants L102 R,L103H,L103 W,G104R and V190 H displayed obvious higher LOX-1 binding activity,especially the mutant L103 H,which showed a 30-fold higher antigen-binding activity over the parental scFv.3.Preliminary application study of the anti-LOX-1 scFv in AS targeted drug delivery system.As serious side effects because of the low specificity,the application of many AS therapeutic drugs are limited.In this study,the anti-LOX-1 scFv or its variant was set as the targeted vector to fuse with AS therapeutic peptides,including antioxidant peptides,hirudin peptides,thrombin inhibitory peptides and angiotensin converting enzyme inhibitory peptides.A series of fusion proteins were produced and their properties including antigen-binding activity,structural characterization and drug activities were studied.Results indicated that these antibody-drug complexes displayed both LOX-1 targeting activity and anti-AS pharmaceutical activity.The effects of the scFv and the therapeutic peptides connecting together played on each other were also analyzed.Overall,in this study an efficient producing technology of the anti-LOX-1 scFv was provided and the engineering of the anti-LOX-1 scFv was studied from different eyesight and using different methods.Results showed that the scFv multimers,scFv and peptide fusion proteins,scFv mutants with optimized quality were obtained.Finally bifunctional antibodies with LOX-1 binding activity and anti-AS activity were generated,which showed potential use in AS targeted therapy.This study will not only provide material bases for the AS diagnosis and targeted therapy but also provide valuable reference for production and further engineering of the antibody fragments and the application of the antibody fragments in targeted drug delivery system.