靶向IL-12 p40和TNF-α的双功能抗体可抑制小鼠银屑病发生

Inhibitory effect of bispecific antibody targeting IL-12 p40 and TNF-α simultaneously on psoriasis in mice

目的构建具有能同时阻断白细胞介素12(IL-12)/IL-23 p40与肿瘤坏死因子α(TNF-α)的双功能抗体,并对其生物学功能进行鉴定,观察其对小鼠银屑病的阻断效果。方法根据已有的阿达木单抗(adalimumab)和优特克单抗(ustekinumab)蛋白序列,设计、构建并制备了全新的双功能抗体Bi AU003、Bi AU022和Bi AU023。采用ELISA检测抗原抗体结合能力;用TNF-α和双功能抗体处理人脐静脉内皮细胞(HUVEC),加入FITC标记的内皮白细胞黏附分子1(ELAM-1)抗体,经流式细胞术检测ELAM-1的表达量;用IL-12和双功能抗体处理经IL-2活化的人外周血单个核细胞(PBMC),并用ELISA检测上清液中γ干扰素(IFN-γ)的量;体外实验采用双功能抗体治疗IL-12和TNF-α诱导的银屑病小鼠,HE染色检测病变皮肤厚度。结果3种双功能抗体对其相应的抗原都有较强的亲和力,能明显抑制ELAM-1蛋白的表达和IFN-γ的分泌,并且明显抑制小鼠的银屑病形成,其效果与现有的抗体药阿达木单抗和优特克单抗相当或更优。结论新构建的3种双功能抗体Bi AU003、Bi AU022和Bi AU023,是TNF-α和IL-12/IL-23 p40的阻断剂,能有效阻断小鼠银屑病形成。

Objective To construct bispecific antibodies,which can block interleukin 12（ IL-12）/IL-23 p40 subunit and tumor necrosis factor α（ TNF-α） simultaneously,and identify their biological function and inhibitory effect on psoriasis formation in mice. Methods Based on the sequences of adalimumab and ustekinumab,three kinds of bispecific antibodies were designed,named Bi AU003,Bi AU022 and Bi AU023. The specificity and binding capacity of bispecific antibodies were determined by ELISA. After co-treated with bispecific antibodies and TNF-α,the level of endothelial leukocyte adhesion molecule-1（ ELMA-1）labeled with fluorescein isothiocyanate（ FITC） in human umbilical vein endothelial cells（ HUVECs） were examined by flow cytometry. Human peripheral blood mononuclear cells（ PBMCs） were purified and cultured in the medium containing IL-2and IL-12 in the absence or presence of bispecific antibodies. Commercial ELISA kit was used to detect interferon γ（ IFN-γ）concentration in the supernatant. BALB/c mice were used for psoriasis model construction through injection of IL-12 and TNF-α subcutaneously. Then they were treated with the bispecific antibodies. Psoriasic skin was measured in thickness and scale under microscopy after HE staining. Results The three kinds of bispecific antibodies could specifically recognize IL-12/23 p40 and TNF-α protein,and inhibit IFN-γ secretion and the expression of ELAM-1 protein. Data also indicated that bispecific antibodies inhibited the formation of psoriasic skin,and showed an equal or superior effect to control antibody drug.Conclusion The novel bispecific antibodies,Bi AU003,Bi AU022 and Bi AU023,can serve as an antagonist of TNF-α and IL-12/23p40,and have a blocking effect on mouse psoriasis formation.