Biological Functional Characterization Of Human Anti-bp180 Single-chain Fv Antibody

Bullous pemphigoid is a common autoimmune blistering disorder seen in the elderly,which is characterized clinically by tense subepidermal blister and bullae. Up to recently, systemic glucocorticosteroids have been the mainstay of therapy, frequently in combination with other immunosuppressants. Although the condition can be temporarily alleviated, the frequent recurrence of the disease and the severe side effects of glucocorticosteroids remain to be the most serious problems[1].The major histologic feature of BP is subepidermal blisters with different inflammatory cells.The autoantibodies against the basement membrane zone (BMZ)in patients can be detected,and also show linear deposition of C3, IgG, and/or IgE along BMZ by direct immunofluorescence detection. Recent studies confirmed that the pathogenesis of BP contains an intact complement cascade,degranulation of resident mast cells and activation of infiltrating neutrophils which are required in the dermal-epidermal separation induced by BP autoantibodies[2].BP180 is a member of the collagen protein family and is also referred to as type XVII collagen or BPAg2, which located at the hemidesmosome.It plays a important role in the pathomechanism of BP. The non-collagenous 16A domain (NC16A), located at the membrane-proximal region of BP180, is considered to be the major pathogenic epitope for BP autoantibodies[3].The pathogenic autoantibodies of BP bind to the hemidesmosomal antigens such as BP180, which results in an intact complement cascade activation,degranulation of resident mast cells, activation of infiltrating inflammatory cells and proteinases , loss of cell-matrix adhesion structure and finally blister formation. Nishie[4]et al humanized the mouse BP autoantigen and developed BP-like skin lesions and a human disease phenotype. These findings clearly show that human autoantibodies against BP180 play a crucial role in blister formation in vivo. It was confirmed that the anti-BP180 autoantibodies trigger subepidermal vesiculation via the classical pathway of the complement system activation. It therefore makes sense to block the pathological action of autoantibodies by preventing them from binding to the corresponding pathogenic autoantigen using targeted genetically engineering scFv antibody.In our previous studies, the scFv antibody fragments were isolated and expressed, which specifically bind to BP180-NC16A, and purified by Ni affinity chromatography method[5, 6]. The purpose of the present study is to characterize the biological function of anti-BP180-NC16A scFv.Objective: To characterize the function of human anti-BP180 single-chain Fv (scFv) antibody.Methods: Anti-BP180 IgG autoantibodies were purified by Ni affinity chromatography from the sera of BP patients. The inhibitive effects of anti-BP180 scFv on the binding of BP-IgG autoantibodies to the recombinant NC16A fragment of human BP180 antigen or intact human tissue were observed by inhibition ELISA and inhibition immunofluorescence,respectively.BP frozen cryosections model was constructed to confirm the inhibitive effects of anti-BP180 scFv with variable concentration on the binding of BP-IgG autoantibodies with BP180.Results: It was confirmed that the scFv had a dose-dependent inhibitive effect on the binding of BP-IgG autoantibodies against BP180 with the corresponding antigen at the given concentrations between 0 and 60μg/ml. The inhibitive rate was up to 69.50% (P<0.01). When the scFv concentration was higher than 40μg/ml, we found no linear fluorescence deposition along the dermal-epidermal junction (DEJ). We are successful to construct the BP frozen cryosections model,and dermal–epidermal separation can be seen using haematoxylin and eosin staining.In this model,we can observe that the scFv shows marked ability to inhibit the binding of BP autoantibodies and subsequent dermal-epidermal separation.Conclusions: The anti-BP180 single-chain Fv antibody can markedly inhibit the binding of BP-IgG autoantibodies with BP180 and then affect subsequent complement activation in vitro.Our results provide experimental proof that the blocking of pathogenic epitopes using engineered scFv appears to be efficient as a method for BP therapy.