Construction Of A Native-like Bispecific Antibody Simultaneously Neutralizing Tumor Necrosis Factor-alpha And Interleukin-17A As The Treatment Of Rheumatoid Arthritis

Recently,pathogenesis of the autoimmune disorders has not been revealed completely due to the complex influences of the genetic and environmental factors.As the most common autoimmune disorders in human,rheumatoid arthritis(RA)has a high morbidity of 0.5%-1%.Over the past decades,the monoclonal antibodies(m Abs)have benefited millions of patients with chronic autoimmune diseases,including RA,psoriasis and the Crohn`s disease.The anti-tumor necrosis factor(TNF-?)m Abs have become sucessful for the therapy of the inflammatory diseases.Unfortunately,about 30% of RA patients are refractory to the anti-TNF-? mabs,which may be caused by the compensatory effects of other inflammatory signals.Generally,the main obstacles of a bispecific antibody(Bs Abs)as a therapeutic drug are how to design the rational structure for the satisfactory therapeutic effect,and how to generate the Bs Ab with high-quality.With the development of the genetic engineering antibody technology,as well as the illumination of pathogenesis of many diseases,numbers of Bs Abs have been proposed,improved and optimized.Bispecific antibodies are genetically engineered antibodies,which combine the specificity of two types of antibodies and can bind two different antigens or epitopes.Due to the diversity and complexity of autoimmune diseases,the therapeutic response of the m Abs that target a single antigen is limited.Nevertheless,Bs Abs can interact with different receptors and ligands,which will supplement therapeutic limitations of the m Abs.Therefore,with combination of another target,i.e.IL-17 A,a new Bs Ab will be generated to enhance therapeutic response of the anti-TNF-? mab.Several principles have been proposed,including the production of the antibody,complexity of the purification,affinity with the antigens,and solubility and stability of the antibody.Based on theoretical and experimental work by other researchers,we have designed and construct a Bs Ab targeting TNF-? and IL-17 A.The physico-chemical properties,biological activity,and the therapeutic effects on RA were studied systematically,including:1.Two kinds of human m Abs expressed by the eukaryotic expression vector,targeting TNF-? and IL-17 A,respectively,were constructed.2.The eukaryotic expression vector for Ig G-like Bs Ab,which targeted TNF-?and IL-17 A simultaneously,was generated with a combination of electrostatic Fc pairing and exchange of the light/heavy chain variable region,i.e.the Crossmab method,using two m Abs as parents.3.The eukaryotic expression system of HEK293 F mammalian cells was used to express,purify,and identify the constructed eukaryotic expression vector.4.The physico-chemical properties of the purified antibodies,for instance,the purity,stability,affinity and the specificity,were evaluated.The results showed that the stability of the Bs Ab was comparable to the native Ig G molecule,and the corresponding affinity to the targets was inherited from its parents excellently.Moreover,the Bs Ab could neutralize the cytotoxicity of TNF-?-mediated fibroblasts and block the chemokines secreted by the induced HT-29,and the function of the Bs Ab was comparable to that of the anti-TNF-? mab.5.The effects of Bs Ab on the secretion of cytokines in RA-FLS of synovial fibroblasts in a RA patient,were examined by real-time PCR and ELISA.The Bs Ab could inhibit the inflammatory factors,chemokines and matrix metalloproteinases expressed by the induced RA-FLS.Moreover,the corresponding expression was much lower than that of anti-TNF-? mab or anti-IL-17 A mab alone.6.The Bs Ab has shown an obvious effect on inhibiting the proliferation of RA-FLS and blocking the cell migration in the cell scratches and cell migration experiments.Bs Ab was superior to the anti-TNF-? mab and anti-IL-17 A mab alone in blocking the cell migration.As a conclusion,we have demonstrated that the Ig G-like Bs Ab,targeting TNF-? and IL-17 A simultaneously,maintained the physico-chemical properties of the native antibodies,and overcame the limited therapeutic response of anti-TNF-? in RA.Furthermore,the combined blockade of TNF-? and IL-17 A has been proven to be more powerful than suppressing either factor alone.Therefore,the anti-TNF-?/IL-17 A Bs Ab is promising to be an effective therapeutic drug for the treatment of RA and some other autoimmune diseases.