Chaung Wayne, Zhang Fangming, Ma Gaifeng, Yen HaoTing, Jacob Asha, Brenner Max, Wang Ping
TheraSource LLC, 350 Community Drive, Manhasset, NY.
Center for Immunology and Inflammation, Feinstein Institutes for Medical Research, Manhasset, NY.
Res Sq. 2023 May 15:rs.3.rs-2809755. doi: 10.21203/rs.3.rs-2809755/v1.
Human milk fat globule epidermal growth factor-factor VIII (MFG-E8) functions as a bridging molecule to promote the removal of dying cells by professional phagocytes. -expressed histidine-tagged recombinant human MFG-E8 (rhMFG-E8) is protective in various disease conditions. However, due to improper recombinant protein glycosylation, misfolding and possible antigenicity, -expressed histidine-tagged rhMFG-E8 is unsuitable for human therapy. Therefore, we hypothesize that human cell-expressed, tag-free rhMFG-E8 can be developed as a safe and effective novel biologic to treat inflammatory diseases such as radiation injury and acute kidney injury (AKI).
We produced a new tag-free rhMFG-E8 protein by cloning the human MFG-E8 full-length coding sequence without any fusion tag into a mammalian vector and expressed it in HEK293-derived cells. The construct includes the leader sequence of cystatin S to maximize secretion of rhMFG-E8 into the culture medium. After purification and confirmation of the protein identity, we first evaluated its biological activity . We then determined its efficacy utilizing two experimental rodent models of organ injury: partial body irradiation (PBI) and ischemia/reperfusion-induced AKI.
HEK293 cell supernatant containing tag-free rhMFG-E8 protein was concentrated, purified, and rhMFG-E8 was verified by SDS-PAGE analysis and mass spectrometry. The biological activity of human cell-expressed tag-free rhMFG-E8 was superior to that of -expressed His-tagged rhMFG-E8. Toxicity, stability, and pharmacokinetic studies indicate that tag-free rhMFG-E8 is safe, highly stable after lyophilization and long-term storage, and with an adequate half-life for therapeutic applications. In the PBI model, a dose-dependent improvement of the 30-day survival rate was observed after tag-free rhMFG-E8 treatment with a 30-day survival of 89%, which was significantly higher than the 25% survival in the vehicle group. The dose modification factor (DMF) of tag-free rhMFG-E8 was 1.073. Tag-free rhMFG-E8 also attenuated gastrointestinal damage after PBI. In the model of AKI, tag-free rhMFG-E8 treatment significantly attenuated kidney injury and inflammation, and improved the 10-day survival.
Our new human cell-expressed tag-free rhMFG-E8 can be further developed as a safe and effective therapy to treat victims of severe acute radiation injury and patients with acute kidney injury.
人乳脂肪球表皮生长因子 - 因子VIII（MFG - E8）作为一种桥接分子，可促进专职吞噬细胞清除死亡细胞。表达的带组氨酸标签的重组人MFG - E8（rhMFG - E8）在各种疾病状态下具有保护作用。然而，由于重组蛋白糖基化不当、错误折叠以及可能的抗原性，表达的带组氨酸标签的rhMFG - E8不适合用于人体治疗。因此，我们推测人细胞表达的无标签rhMFG - E8可被开发为一种安全有效的新型生物制剂，用于治疗诸如辐射损伤和急性肾损伤（AKI）等炎症性疾病。
我们通过将无任何融合标签的人MFG - E8全长编码序列克隆到哺乳动物载体中，并在HEK293衍生细胞中表达，制备了一种新的无标签rhMFG - E8蛋白。构建体包含胱抑素S的前导序列，以最大限度地将rhMFG - E8分泌到培养基中。在纯化并确认蛋白身份后，我们首先评估了其生物学活性。然后利用两种实验性啮齿动物器官损伤模型：局部身体照射（PBI）和缺血/再灌注诱导的AKI，确定了其疗效。
含有无标签rhMFG - E8蛋白的HEK293细胞上清液经过浓缩、纯化，rhMFG - E8通过SDS - PAGE分析和质谱进行了验证。人细胞表达的无标签rhMFG - E8的生物学活性优于表达的带组氨酸标签的rhMFG - E8。毒性、稳定性和药代动力学研究表明，无标签rhMFG - E8是安全的，冻干和长期储存后高度稳定，并且具有适合治疗应用的半衰期。在PBI模型中，无标签rhMFG - E8治疗后观察到30天生存率呈剂量依赖性提高，30天生存率为89%，显著高于载体组的25%生存率。无标签rhMFG - E8的剂量修正因子（DMF）为1.073。无标签rhMFG - E8还减轻了PBI后的胃肠道损伤。在AKI模型中，无标签rhMFG - E8治疗显著减轻了肾损伤和炎症，并提高了10天生存率。
我们新的人细胞表达的无标签rhMFG - E8可进一步开发为一种安全有效的疗法，用于治疗严重急性辐射损伤的受害者和急性肾损伤患者。
