TNFα is a pro-inflammatory cytokine that is a therapeutic target for inflammatory autoimmune disorders. Thus, TNFα antagonists are successfully used for the treatment of these disorders. Here, new association patterns of rhTNFα and its antagonists Adalimumab and Etanercept are disclosed. Active rhTNFα was purified by IMAC from the soluble fraction of transformed Escherichia coli. Protein detection was assessed by SDS–PAGE and Western blot. The KD values for rhTNFα interactions with their antagonists were obtained by non-competitive ELISA and by microscale thermophoresis (MST). Molecular sizes of the complexes were evaluated by size-exclusion chromatography-high performance liquid chromatography (SEC-HPLC). Surprisingly, both antagonists recognized the monomeric form of rhTNFα under reducing and non-reducing conditions, indicating unexpected bindings of the antagonists to linear epitopes and to rhTNFα monomers. For the first time, the interactions of rhTNFα with Adalimumab and Etanercept were assessed by MST, which allows evaluating molecular interactions in solution with a wide range of concentrations. Biphasic binding curves with low and high KD values (<10−9 M and >10−8 M) were observed during thermophoresis experiments, suggesting the generation of complexes with different stoichiometry, which were confirmed by SEC-HPLC. Our results demonstrated the binding of TNFα-antagonists with rhTNFα monomers and linear epitopes. Also, complexes of high molecular mass were observed. This pioneer investigation constitutes valuable data for future approaches into the study of the interaction mechanism of TNFα and its antagonists.

中文翻译：

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rhTNFα与其拮抗剂阿达木单抗和依那西普相互作用机制的新见解。

TNFα是促炎性细胞因子，是促炎性自身免疫疾病的治疗靶标。因此，TNFα拮抗剂已成功用于治疗这些疾病。在此，公开了rhTNFα及其拮抗剂阿达木单抗和依那西普的新缔合模式。通过IMAC从转化的大肠杆菌的可溶性级分中纯化活性rhTNFα。蛋白质检测通过SDS-PAGE和Western blot进行评估。rhTNFα与其拮抗剂的相互作用的KD值通过非竞争性ELISA和微量热泳（MST）获得。通过尺寸排阻色谱法-高效液相色谱法（SEC-HPLC）评价复合物的分子大小。令人惊讶的是，两种拮抗剂在还原和非还原条件下均能识别rhTNFα的单体形式，表明拮抗剂与线性表位和rhTNFα单体的意外结合。通过MST首次评估了rhTNFα与Adalimumab和Etanercept的相互作用，该方法可以评估浓度范围很宽的溶液中的分子相互作用。在热泳实验中观察到了具有低和高KD值（<10-9 M和> 10-8 M）的双相结合曲线，表明生成了具有不同化学计量的复合物，这已通过SEC-HPLC证实。我们的结果证明了TNFα拮抗剂与rhTNFα单体和线性表位的结合。另外，观察到高分子量的复合物。这项开创性的研究为将来研究TNFα及其拮抗剂的相互作用机制提供了有价值的数据。