scholarly journals Intracellular Delivery of Antibodies for Selective Cell Signaling Interference

ChemMedChem ◽  
2021 ◽  
Author(s):  
Rebecca L. Hershman ◽  
Yamin Li ◽  
Feihe Ma ◽  
Qioabing Xu ◽  
James Van Deventer
Keyword(s):  
Cell Signaling ◽  
Intracellular Delivery

Cationic Lipid‐based Intracellular Delivery of Bacterial Effectors for Rewiring Malignant Cell Signaling

2020 ◽  
Vol 59 (41) ◽  
pp. 18087-18094 ◽  
Author(s):  
Shaojun Yang ◽  
Qiao Tang ◽  
Long Chen ◽  
Jin Chang ◽  
Tian Jiang ◽  
...  
Keyword(s):  
Cell Signaling ◽  
Cationic Lipid ◽  
Intracellular Delivery ◽  
Malignant Cell ◽  
Bacterial Effectors

Cationic Lipid‐based Intracellular Delivery of Bacterial Effectors for Rewiring Malignant Cell Signaling

2020 ◽  
Vol 132 (41) ◽  
pp. 18243-18250
Author(s):  
Shaojun Yang ◽  
Qiao Tang ◽  
Long Chen ◽  
Jin Chang ◽  
Tian Jiang ◽  
...  
Keyword(s):  
Cell Signaling ◽  
Cationic Lipid ◽  
Intracellular Delivery ◽  
Malignant Cell ◽  
Bacterial Effectors

scholarly journals Intracellular Delivery of Antibodies for Selective Cell Signaling Interference

2021 ◽  
Author(s):  
Rebecca L. Hershman ◽  
Yamin Li ◽  
Feihe Ma ◽  
Qioabing Xu ◽  
James A. Van Deventer
Keyword(s):  
Cell Signaling ◽  
Intracellular Signaling ◽  
Hepg2 Cells ◽  
Protein Delivery ◽  
Intracellular Delivery ◽  
Cationic Lipids ◽  
Luciferase Reporter ◽  
Reporter Cell ◽  
Intracellular Space ◽  
Signaling Events

AbstractMany 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.

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