抗α干扰素双特异性抗体的制备与初步评价

Development and Preliminary Evaluation of an Anti-IFN-α Bispecific Antibody

目的:研制对α干扰素(IFN-α)中和活性有突破性提升的双特异性抗体。方法:以靶向IFN-α不同表位的中和单抗Amilimumab和Sifalimumab为基础,采用FIT-Ig(Fabs-in-tandem immunoglobulin)双特异性抗体平台技术,通过分子克隆方法构建针对IFN-α的双特异性抗体FIT2的表达载体,并在Free Style 293-F系统中进行瞬时转染表达;采用protein A柱亲和纯化目标双特异性抗体,SDS-PAGE初步考察目标抗体的相对分子质量、组分及纯度;采用forte BIO系统鉴定其整体结合活性,并分别确认双特异性抗体的2个抗原结合表位的活性;通过ELISA方法评价其特异性,37℃放置实验鉴定其稳定性,Daudi细胞增殖实验定量评价双特异性抗体的中和活性。结果:构建了双特异性抗体FIT2表达载体,在Free Style 293-F系统中实现了瞬转高效表达,表达量为127 mg/L,亲和纯化获得目标双特异性抗体FIT2,纯度可达98.5%;FIT2结构稳定,具有良好的特异性,未发现与其他类型的干扰素及细胞因子存在交叉反应;FIT2对IFN-α1b-His的结合活性显著优于2个亲本抗体,特别是解离过程的改善尤为明显;同时,FIT2有效保留了2个抗原结合表位的活性;细胞增殖实验结果显示,FIT2对IFN-α1b具有优越的中和活性,其中和效能显著高于任一亲本抗体,明显优于2个亲本抗体联用。结论:获得了对IFN-α1b中和活性有突破性提升的双特异性抗体FIT2,特异性良好且结构稳定,有望成为治疗IFN-α相关疾病的良好的成药候选分子。

Objective:To develop a bispecific antibody with a remarkable improved neutralizing activity to interferon-α(IFN-α).Methods:The anti-IFN-αbispecific antibody expression vectors were constructed based on two different monoclonal antibodies Amilimumab and Sifalimumab and FIT-Ig(Fabs-in-tandem immunoglobulin)bispecific antibody technique platform.The bispecific antibody FIT2was expressed by the FreeStyle293-F transient transfection system,followed by the affinity purification using protein A column.SDS-PAGE was used to detect the molecular weight,composition and purity of the target antibody.The overall binding activity was identified us-ing the forteBIO system and the activity of the two antigen-binding sites was confirmed.ELISA method was applied to evaluate the specificity of the bispecific antibody.SDS-PAGE and forteBIO system were used to determine its stability at37℃.Cell proliferation experiment was performed to investigate the neutralizing activity of the bispecific antibody.Results:The expression vectors of bispecific antibody FIT2were confirmed.FIT2was efficiently expressed in FreeStyle293-F transient system,with the expression level of127mg/L.The purity of FIT2reached98.5%after an affinity purification step.The structure stability and specificity of FIT2were indicated.The binding activity of FIT2to IFN-α1b-His was significantly better than that of two parental antibodies,especially in the dissociation process.In addition,the two antigen-binding sites of FIT2retained their original binding activity.Cell proliferation experiment demonstrated that neutralizing activity of FIT2was stronger than that of single or combined parental antibody.Conclusion:We obtained a structurally stable bispecific antibody FIT2with remarkably enhanced neutralizing efficacy against IFN-α1b,and it was expected to be a good candidate drug for the treatment of IFN-αrelated diseases.