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bioRxiv · 10.1101/2024.03.28.587257

Comparing cell penetration of biotherapeutics across human cell lines

Batistatou, N.·Kritzer, J.
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Abstract

A major obstacle in biotherapeutics development is maximizing cell penetration. Ideally, assays would allow for optimization of cell penetration in the cell type of interest early in the drug development process. However, few assays exist to compare cell penetration across different cell types, independent of drug function. In this work, we applied the chloroalkane penetration assay (CAPA) in seven mammalian cell lines as well as primary cells. Careful controls were used to ensure data could be compared across cell lines. We compared the penetration of several peptides and drug-like oligonucleotides and saw significant differences among the cell lines. To help explain these differences, we quantified the relative activities of endocytosis pathways in these cell lines and correlated them with the penetration data. Based on these results, we knocked down clathrin in a cell line with an efficient permeability profile and observed reduced penetration of peptides, but not oligonucleotides. Finally, we used small-molecule endosomal escape enhancers and observed enhancement of cell penetration of some oligonucleotides, but only in some of the cell lines tested. CAPA data provide valuable points of comparison among different cell lines, including primary cells, for evaluating the cell penetration of various classes of peptides and oligonucleotides. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/587257v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@9e75e9org.highwire.dtl.DTLVardef@14f209forg.highwire.dtl.DTLVardef@1e6cd59org.highwire.dtl.DTLVardef@1b3aabb_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Batistatou, N., Kritzer, J.. 2024-03-31. Comparing cell penetration of biotherapeutics across human cell lines. https://doi.org/10.1101/2024.03.28.587257

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