Grossman Brian D, Sanford Jack D, Zhu Yuyi, Zeller Cynthia B, Weldon John E
Department of Biological Sciences, The Jess and Mildred Fisher College of Science and Mathematics, Towson University, 8000 York Rd, Towson, MD 21252, USA.
Department of Chemistry, The Jess and Mildred Fisher College of Science and Mathematics, Towson University, 8000 York Rd, Towson, MD 21252, USA.
Biol Open. 2025 Jun 15;14(6). doi: 10.1242/bio.061792. Epub 2025 Jun 20.
Furin is a mammalian serine protease with important roles in cellular homeostasis and disease. It cleaves and activates numerous endogenous and exogenous substrates, including the SARS-CoV-2 viral spike protein and protein toxins such as diphtheria toxin and Pseudomonas exotoxin A (PE). Recombinant immunotoxins (RITs) are toxin conjugates used as cancer therapeutics that connect tumor-directed antibodies with toxins for targeted cell killing. RITs based on PE have shown success in treating a variety of cancers, but often suffer from safety and efficacy concerns when used clinically. We have explored furin as a potential limiting factor in the intoxication pathway of PE-based RITs. Although the furin has widely recognized importance in RIT intoxication, its role is incompletely understood. Circumstantial evidence suggests that furin may act as a transporter for RITs in addition to its role of activation by cleavage. Here, we describe the creation of a CRISPR-engineered furin-deficient HEK293 cell line, ΔFur293. Using ΔFur293 and derivatives that express mutant forms of furin, we confirm the importance of furin in the PE RIT intoxication pathway and show that furin trafficking has a significant impact on RIT efficacy. Our data support the hypothesis that furin acts as a transporter during RIT intoxication and suggest furin as a target for modification to improve the effectiveness of RITs.
弗林蛋白酶是一种哺乳动物丝氨酸蛋白酶，在细胞内稳态和疾病中发挥重要作用。它能切割并激活多种内源性和外源性底物，包括严重急性呼吸综合征冠状病毒2（SARS-CoV-2）的病毒刺突蛋白以及诸如白喉毒素和铜绿假单胞菌外毒素A（PE）等蛋白质毒素。重组免疫毒素（RITs）是用作癌症治疗药物的毒素偶联物，它将肿瘤靶向抗体与毒素连接起来以实现靶向细胞杀伤。基于PE的RITs在治疗多种癌症方面已取得成功，但在临床使用时常常存在安全性和有效性方面的问题。我们探究了弗林蛋白酶作为基于PE的RITs中毒途径中的一个潜在限制因素。尽管弗林蛋白酶在RIT中毒过程中的重要性已得到广泛认可，但其作用尚未完全明确。间接证据表明，弗林蛋白酶除了具有通过切割进行激活的作用外，还可能作为RITs的转运体。在此，我们描述了通过CRISPR技术构建的弗林蛋白酶缺陷型HEK293细胞系ΔFur293的创建过程。利用ΔFur293及其表达弗林蛋白酶突变形式的衍生物，我们证实了弗林蛋白酶在PE RIT中毒途径中的重要性，并表明弗林蛋白酶的转运对RIT的疗效有显著影响。我们的数据支持了弗林蛋白酶在RIT中毒过程中作为转运体的假说，并表明弗林蛋白酶可作为一个修饰靶点以提高RITs的有效性。
