Delivery of antibodies to the cytosol: debunking the myths

Abstract

The use of antibodies to target their antigens in living cells is a powerful analytical tool for cell biology research. Not only can molecules be localized and visualized in living cells, but interference with cellular processes by antibodies may allow functional analysis down to the level of individual post-translational modifications and splice variants, which is not possible with genetic or RNA-based methods. To utilize the vast resource of available antibodies, an efficient system to deliver them into the cytosol from the outside is needed. Numerous strategies have been proposed, but the most robust and widely applicable procedure still remains to be identified, since a quantitative ranking of the efficiencies has not yet been done. To achieve this, we developed a novel efficiency evaluation method for antibody delivery based on a fusion protein consisting of a human IgG 1 Fc and the recombination enzyme Cre (Fc-Cre). Applied to suitable GFP reporter cells, it allows the important distinction between proteins trapped in endosomes and those delivered to the cytosol. Further, it ensures viability of positive cells and is unsusceptible to fixation artifacts and misinterpretation of cellular localization in microscopy and flow cytometry. Very low cytoplasmic delivery efficiencies were found for various profection reagents and membrane penetrating peptides, leaving electroporation as the only practically useful delivery method for antibodies. This was further verified by the successful application of this method to bind antibodies to cytosolic components in living cells.

Keywords: CPP; PTD; cell penetrating peptides; electroporation; electrotransfer; profection; protein delivery; protein transduction domain; transbody; transfection; yumab.

Figure 1. Comparison of DNA-transfection (GFP encoding plasmid) and Protein-transfection (SA-FITC) by biotinylated PEI. (A) cells were analyzed by flow cytometry. (B) flow cytometric analysis of life death staining by propidium iodide.

Figure 2. Cre-hIgG₁-Fc fusion recombinase activity in vitro and in vivo. (A) schematic representation of Cre-Fc and Fc-Cre fusions and the reporter cell system based on the cell line SC1 REW22. (B) the plasmid LoxP2+ was incubated with Cre-Fc or Fc-Cre fusion proteins and analyzed for recombination by agarose gel electrophoresis. Cre recombinase served as a positive control (p.c.), the linearized plasmid LoxP2+ alone as a negative control (n.c.). (C) electroporation of Fc-Cre fusion protein and controls into reporter cell line SC1 REW22. 2x10⁶ cells per sample were used for electroporation. Cells were analyzed by flow cytometry. The electroporation efficiency indicated by YFP⁺ cells reached up to more than 90%. The efficiency of electroporation and recombination together, indicated by GFP⁺ cells in samples electroporated with NLS-Cre DNA, reached up to more than 60%. Electroporation with Fc-Cre resulted in a maximum of almost 30% at the highest concentration of Fc-Cre protein. (D) effect of electroporation of DNA and proteins or empty electroporation on cell viability. Cells were analyzed by flow cytometry after life/death staining with propidium iodide. Electroporation of a plasmid encoding NLS-Cre was more toxic than electroporation of hIgG₁-Fc-Cre fusion proteins. The average percentage of living cells from all timepoints is shown.

Figure 3. Protein delivery into SC1 REW22 reporter cells by electroporation or profection with ProteoJuice, Bioporter, Ab-DeliverIN or Pulsin. 4.4x10⁵ cells per sample were used for electroporation and cells were analyzed by flow cytometry. (A) electrotransfer of NLS-Cre DNA, Fc-Cre protein and controls analyzed after 72h. Electrotransfer of NLS-Cre DNA resulted in two clearly separated populations while electrotransfer of Fc-Cre resulted in GFP⁺ cells with lower fluorescence intensity. Controls electropulsed in PBS (“empty”) or cells incubated with Fc-Cre without electropulse were not fluorescent. The electroporation efficiency indicated by YFP-DNA electroporated cells was above 90%. (B) electrotransfer: percentage of GFP⁺ cells over all timepoints. (C) negative controls for profection analysis. (D) profection of IgG-FITC and PE resulted in IgG-FITC or PE positive cells for all reagents, reaching maximal values of up to more than 90%. Incubation with only IgG-FITC resulted in a low percentage of IgG-FITC positive cells while cells incubated with PE alone were PE-negative. (E) profection of Fc-Cre. Fc-Cre was delivered as indicated by GFP⁺ cells, but for all reagents only in less than 10% of cells. If viability was so low it only allowed analysis of markedly less cells than for other samples this is indicated by an asterisk.

Figure 4. Viability of cells 48 h after treatment by profection or electroporation. Cells were analyzed by flow cytometry after life death staining with propidium iodide. At high concentrations of profection reagents viability decreased (Pulsin) or cells died so numerously leaving not enough cells for measurement (Ab-DeliverIN, ProteoJuice). For profection, cells had been incubated for 48 h with profection reagent and protein, the supernatant was substituted after 48 h by cell culture medium. Electroporation with proteins was performed using 0.9 mg/ml Fc-Cre.

Figure 5. Size exclusion chromatography of anti-Myosin (SF9) and anti-Tubulin (F2C) scFv-Fc antibodies.100 µg protein in a volume of 200 µl were loaded to the column for each sample. The calculated molecular mass of the analyzed scFv-Fc fusions is 102 kDa and the mobility determined by SEC corresponds to a size of 122 kDa (anti-Myosin, 14.08 mL retention volume) and 118 kDa (anti-Tubulin, 14.16 mL retention volume).

Figure 6. Electroporation of HeLa cells with different concentrations of anti-Myosin (SF9) scFv-Fc antibody. Cells were fixed, permeabilized and stained by a FITC-conjugated secondary antibody to detect the anti-Myosin scFv-Fc antibody. If not otherwise indicated, the exposure time for all images shown in this figure was 2s.

Figure 7. In vivo delivery of anti-Tubulin and anti-Myosin antibodies. HeLa cells were electroporated in the presence of the anti-Tubulin scFv-Fc antibody F2C (0.6 mg/mL) or the anti-Myosin scFv-Fc antibody SF9 (0.3 mg/mL). As a control, HeLa cells had been incubated with anti-Tubulin (1.1 mg/mL) or anti-Myosin (0.3 mg/mL). Cells were analyzed 24 h after electroporation or incubation, respectively.

Figure 8. Intracellular stability of anti-Myosin (SF9) and anti-Tubulin (F2C) in HeLa cells. HeLa cells were electroporated in the presence of anti-Tubulin (0.6 mg/mL) or anti-Myosin (0.3 mg/mL) scFv-Fc antibodies and after electroporation, permeabilized and stained with a detection antibody. The exposure time was 10s for all images shown.