Intracellular Delivery of Antibodies for Selective Cell Signaling Interference
Intracellular Delivery of Antibodies for Selective Cell Signaling Interference
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DOI：
10.1002/cmdc.202100678
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发表时间：
2021-10
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影响因子：
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通讯作者：
Rebecca L. Hershman;Yamin Li;Feihe Ma;Qioabing Xu;J. V. Van Deventer
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作者：
Rebecca L. Hershman;Yamin Li;Feihe Ma;Qioabing Xu;J. V. Van Deventer
由于普遍缺乏干扰“不可药物”靶标的工具，许多细胞内信号转导事件的特征仍然很差。抗体具有通过靶向破坏细胞信号级联来阐明细胞内机制的潜力，因为它们能够以高特异性和亲和力与靶标结合。然而，由于它们的大小和化学组成，抗体不能天生穿过细胞膜，因此这些大分子进入细胞质受到限制。在这里，我们描述了利用阳离子脂质纳米颗粒介导的重组抗体进入细胞内空间的策略，以选择性地破坏细胞内信号转导事件。为了进行这样的研究，我们首先制作了一系列抗体构建体，称为scFv-Fcs，在构建体的c末端含有额外的遗传编码负电荷。制备带负电荷基序的蛋白质先前已被证明可以增强带阳离子脂质的细胞内蛋白质递送，但通常用于基因组编辑或靶向细胞死亡。我们首先生成了scFv-Fc17的衍生物，scFv-Fc17是一种抗体结构，以前报道过特异性结合Tyr705磷酸化的信号传感器和转录激活子3 （STAT3）。我们用流式细胞术从我们的组合脂质库中筛选了少量脂质，发现在测试条件下，PBA-Q76-O16B促进了scFv-Fcs的最有效传递。在HepG2细胞中，我们观察到高达60.5%的递送效率，而在stat3 -荧光素酶报告细胞系中，我们观察到高达71.5%的递送效率。这些结果证明了scFv-Fcs进入细胞内空间的可行性。然而，我们也注意到，在传递过程中，在改变这些构式的负电荷数量时，观察到的变化并不大。用PBA-Q76-O16B对scFv-Fcs的细胞毒性、大小和包封效率进行了表征，结果表明该结构总体上表现良好，添加不同数量的负电荷导致最温和的影响。重要的是，监测荧光素酶报告细胞系和HepG2细胞转录活性的功能分析显示，在递送scFv-Fc17构建物后，pYSTAT3下游的基因表达显著降低。总之，我们的研究结果建立了使用重组产生的抗体来选择性地干扰由单个翻译后修饰驱动的细胞信号事件。有效的细胞内递送工程抗体开辟了以前“不可药物”靶点的调节可能性，包括潜在的治疗应用。
Many intracellular signaling events remain poorly characterized due to a general lack of tools to interfere with “undruggable” targets. Antibodies have the potential to elucidate intracellular mechanisms via targeted disruption of cell signaling cascades because of their ability to bind to a target with high specificity and affinity. However, due to their size and chemical composition, antibodies cannot innately cross the cell membrane, and thus access to the cytosol with these macromolecules has been limited. Here, we describe strategies for accessing the intracellular space with recombinant antibodies mediated by cationic lipid nanoparticles to selectively disrupt intracellular signaling events. To enable such investigations, we first produced a series of antibody constructs, known as scFv-Fcs, containing additional, genetically encoded negative charges located at the C-termini of the constructs. Preparing proteins with negatively charged motifs has previously been shown to enhance intracellular protein delivery with cationic lipids, but usually for the purpose of genome editing or targeted cell death. We started by generating derivatives of scFv-Fc17, an antibody construct previously reported to bind specifically to signal transducer and activator of transcription 3 (STAT3) phosphorylated at Tyr705 (pYSTAT3). We screened a small number of lipids from our combinatorial lipid library with flow cytometry and found that PBA-Q76-O16B facilitated the most efficient delivery of scFv-Fcs under the conditions tested. In HepG2 cells, we observed up to 60.5% delivery efficacy, while in a STAT3-luciferase reporter cell line up to 71.5% delivery efficacy was observed. These results demonstrated the feasibility of accessing the intracellular space with scFv-Fcs. However, we also note that no more than modest changes were observed upon changing the numbers of negative charges in these constructs during delivery. Characterization of the cytotoxicity, size, and encapsulation efficiency of scFv-Fcs with PBA-Q76-O16B revealed that the constructs were generally well-behaved, with addition of differing quantities of negative charge resulting in at most modest effects. Importantly, functional assays monitoring transcriptional activity in luciferase reporter cell lines and HepG2 cells demonstrated significant reduction of gene expression downstream of pYSTAT3 following delivery of scFv-Fc17 constructs. Together, our results establish the use of recombinantly produced antibodies to selectively interfere with cell signaling events driven by a single posttranslational modification. Efficient intracellular delivery of engineered antibodies opens up possibilities for modulation of previously “undruggable” targets, including for potential therapeutic applications.