(526c) Intracellular Delivery of Antibodies for Selective Cell Signaling Interference
AIChE Annual Meeting
Wednesday, November 10, 2021 - 4:42pm to 5:00pm
Accessing the intracellular space with biomolecules that disrupt intracellular processes continues to be a significant challenge in drug delivery. Antibodies and other biomolecules are critical tools for investigating intracellular signaling cascades because they are capable of interfering specifically with protein binding, unlike common small molecule drugs and gene knockdown or knockout strategies. While there are many partial solutions to the delivery problem, we are not aware of general strategies for intracellular delivery of antibodies that allow for probing of signaling pathways. Here, we use cationic lipid nanoparticle technology to devise strategies for accessing the intracellular space with antibodies. We use a series of recombinant antibody constructs that vary in total negative charge to investigate the role of electrostatic interactions between proteins and lipids in enhancing cytosolic access. Previously, an scFv-Fc antibody construct (termed scFv-Fc17) was reported to interfere specifically with the JAK/STAT pathway, by binding exclusively to STAT3 phosphorylated at tyrosine 705 (pYSTAT3). scFv-Fc17 had been introduced to the cytosol via transfection, and we sought to use this antibody construct to explore intracellular delivery of scFv-Fcs with a STAT3 luciferase reporter cell line and the HepG2 cell line. We started by delivering FITC-labelled constructs with 0, 5, or 15 glutamic acid residues genetically encoded at the C-terminus of the scFv-Fcs (termed scFv-Fc17, scFv-Fc17-5E, and scFv-Fc17-15E). To control our experimental design, we included delivery experiments with a fibroblast activation protein (FAP) binder, which has no intracellular target, to determine if observed signaling interference is a result of scFv-Fc17 binding with pYSTAT3. The best delivery conditions were observed using a 10 nM final concentration of scFv-Fcs complexed with the lipid PBA-Q76-O16B in a 1:8 mass ratio of antibody to lipid. In flow cytometry analysis, we observed 70.8%, 71.5%, and 71.2% FITC-positive cells for the STAT3 luciferase cell line, and 48.9%, 58.4%, and 60.5% FITC positive HepG2 cells for 0, 5, and 15 additional negative charges, respectively. Though there were only modest changes in observed delivery with constructs possessing different charges, we moved on to use scFv-Fc17-5E and scFv-Fc17-15E to assess changes in signaling following delivery. Luminescent assays with the STAT3 luciferase cells showed a 37.5% and 43.4% decrease in luciferase activity following 24 hours of delivery using scFv-Fc17-5E and scFv-Fc17-15E, respectively. Additionally, RT-qPCR assays of three genes known to be downstream of activated pYSTAT3 in HepG2 cells were observed to be transcribed at significantly lower levels following 24-hour delivery of scFv-Fc17-5E and scFv-Fc17-15E. Haptoglobin, serum amyloid A 1, and JunB all showed fold decreases in gene expression ranging from 1.5 to 4.5. Signaling assays with FAP-binding scFv-Fcs showed no significant changes in the functional readouts for pYSTAT3 inhibition, demonstrating that the delivery of scFv-Fc17 conjugates selectively inhibit the intracellular pYSTAT3 pathway. Studies involving characterization of nanoparticle size, encapsulation efficiency, and toxicity are ongoing. This work is an important step in showing that we can investigate the impact of intracellular antibody delivery on signaling interference using functional readouts. This approach will allow us to pursue new strategies for probing the roles of individual protein-protein interactions and post-translational modifications in cell signaling cascades to better understand intracellular processes and identify potential disease targets.