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Modified trastuzumab and cetuximab mediate efficient toxin delivery while retaining antibody-dependent cell-mediated cytotoxicity in target cells Roger Gilabert-Oriol, Mayank Thakur, Benedicta von Mallinckrodt, Thomas Hug, Burkhard Wiesner, Jenny Eichhorst, Matthias F. Melzig, Hendrik Fuchs, and Alexander Weng Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/mp400444q • Publication Date (Web): 19 Sep 2013 Downloaded from http://pubs.acs.org on September 26, 2013

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Molecular Pharmaceutics

Modified trastuzumab and cetuximab mediate efficient toxin delivery while retaining antibody-dependent cell-mediated cytotoxicity in target cells Roger Gilabert-Oriol a, Mayank Thakur a, Benedicta von Mallinckrodt a, Thomas Hug a, Burkhard Wiesner b, Jenny Eichhorst b, Matthias F. Melzig c, Hendrik Fuchs a, Alexander Weng a,d*

a

Institut für Laboratoriumsmedizin, Klinische Chemie und Pathobiochemie, Charité –

Universitätsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, D-12200 Berlin, Germany b

Leibnizinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany

c

Institut für Pharmazie , Freie Universität Berlin, Königin-Luise-Straße 2+4, D-14195 Berlin,

Germany d

Present address: Wolfson Centre for Gene Therapy of Childhood Disease, University

College London - Institute of Child Health, London, 30 Guilford Street, London, WC 1N 1EH, United Kingdom

* Corresponding author: Institut für Laboratoriumsmedizin, Klinische Chemie und Pathobiochemie Charité – Universitätsmedizin Berlin, Germany Campus Benjamin Franklin Hindenburgdamm 30 D-12200 Berlin Germany Tel.: +49-30-8445-2507 Fax: +49-30-8445-4152 Email: [email protected], [email protected]

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Abstract Monoclonal antibody-based therapy is one of the most successful strategies for treatment of cancer. However, the insufficient cell killing activity of monoclonal antibodies limits their therapeutic potential. These limitations can be overcome by the application of immunotoxins, which consist of a monoclonal antibody that specifically delivers a toxin into the cancer cell. An ideal immunotoxin combines the functionality of the monoclonal antibody (antagonistic binding to targeted receptors and interaction with the innate immune system) with the cellkilling activity of the toxic moiety. In addition, it should be sensitive for certain triterpenoid saponins that are known to lead to a tremendous augmentation of the anti-tumoral efficacy of the immunotoxin. In this study, the monoclonal antibodies trastuzumab (Herceptin®) and cetuximab (Erbitux®) were conjugated via cleavable disulfide bonds to the plant derived toxin saporin. The ability of the modified tumor-specific therapeutic antibodies to deliver their toxic payload into the target cells was investigated by impedance-based real-time viability assays and confocal live cell imaging. We further provide evidence that the immunotoxins retained their ability to trigger antibody-dependent cell-mediated cytotoxicity. They specifically bound to their target cell receptor and their cell-killing activity was drastically augmented in the presence of triterpenoid saponins. Further mechanistic studies indicated a specific saponinmediated endo/lysosoaml release of the toxin moiety. These results open a promising avenue to overcome the present limitations of therapeutic antibodies and to achieve a higher antitumoral efficacy in cancer therapy.

Keywords Immunotoxin, cell-mediated cytotoxicity, Herceptin®, Erbitux®, saporin, triterpenoid saponin.


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Abbreviations Alexa

ST: saporin-trastuzumab-Alexa Fluor 488

ADCC: antibody-dependent cell-mediated cytotoxicity EGFR: human epidermal growth factor receptor 1 FBS: fetal bovine serum HER2: human epidermal growth factor receptor 2 IDCC: immunotoxin-dependent cell-mediated cytotoxicity NCI: normalized cell index NK: natural killer cells NTA: nitrilotriacetic acid PBMC: peripheral blood mononuclear cell PS: Penicillin/Streptomycin RD: response difference RU: response units RIP: ribosome inactivating protein SC: saporin-cetuximab ST: saporin-trastuzumab



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Introduction Cancer therapy with monoclonal antibodies is considered as one of the most successful strategies for treatment of solid tumors and hematologic malignancies. In this approach, the therapeutic antibodies are directed to cancer cells by targeting proteins that are specifically expressed on the cellular surface of tumor cells. The most common differentiation antigens that are overexpressed in solid tumors are growth factor receptors e.g. epidermal growth factor receptor (EGFR) and epidermal growth factor receptor 2 (HER2). In case of lymphoma and leukemia, most of the differentiation antigens include glycoproteins usually referred to as CD (cluster of differentiation), such as CD20, CD33 and CD52 1. Various modes of action of monoclonal antibodies have been characterized in vitro. Binding of the antibody to a target receptor displayed on the cellular surface blocks the interaction of the receptor with a native ligand, interferes with a multimerization process and triggers the internalization of the receptor, thus inhibiting signal transduction pathways and cell cycle progression or causing apoptosis of the target cells 2. Examples of FDA-approved therapeutic monoclonal antibodies include cetuximab (anti-EGFR, Erbitux®), panitumumab (anti-EGFR, Vectibix®), trastuzumab (anti-HER2, Herceptin®), rituximab (anti-CD20, Rituxan®, MabThera®) and ofatumumab (anti-CD20, Arzerra®) 3. In addition, monoclonal antibodies may also interact in several ways with the innate immune system of the recipient. One of the most important modalities of therapeutic antibodies in vivo is the antibody-dependent cell-mediated cytotoxicity (ADCC), mainly effectuated by natural killer (NK) cells 4. When NK cells are recruited and activated through the interaction between the Fc region of the monoclonal antibody and the Fc gamma receptor III (FcγRIII, CD16) of the NK cells, they release perforin/granzyme, leading to apoptosis of the target cells 5. Unfortunately monoclonal antibody-based therapy encompasses certain limitations and may result in the failure of the treatment due to certain mechanisms, which include dysfunctions in 4 ACS Paragon Plus Environment

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triggering the ADCC

6

or other factors such as the compensatory activation of parallel

signaling pathways 7, 8. Alternatively solid tumors and hematologic cancer may be treated with targeted protein toxins. This group of therapeutics consists of two functional parts: a ligand (antibody, antibody fragment, growth factor or cytokine) that specifically directs the conjugate to the target cell and a toxin (bacterial toxins such as Pseudomonas exotoxin A and diphtheria toxin having ADP-ribosyltransferase activity or ribosome inactivating proteins such as saporin, gelonin and ricin A-chain possessing N-glycosidase activity) 9. Because targeted toxins (referred to as immunotoxins in case an antibody serves as the targeting ligand) carry a toxin capable to cause cell death, the application of these toxins is a promising strategy to circumvent some of the aspects that can lead to the failure of the monoclonal antibody-based therapy. Nevertheless, one of the general believes is that when monoclonal antibodies are conjugated to a toxin, which results in a more effective immunotoxin, the ability of triggering ADCC by the immunotoxin is lost

10

. Furthermore, despite numerous successful preclinical

data, immunotoxins are still causing immunogenic responses and may therefore be rapidly blocked and neutralized by the immune system of recipients, thus leading to a failure of the therapy 11. A relevant characteristic of certain triterpenoid saponins is their ability to specifically augment the cytotoxicity of particular ribosome inactivating proteins (RIPs)

12

. This

synergistic principle has been reported for a definite subset of isolated triterpenoid saponins from Gypsophila paniculata L.

13

and from Saponaria officinalis L.

mechanism has been recently elucidated EGFR-directed targeted toxin

15

14

. The molecular

and its efficacy in vivo has been studied using an

16

. It is envisaged that through a process of saponin-mediated

sensitization, the immunotoxins achieve a higher efficiency and their dosage can be reduced resulting in a drastically lower immunogenicity but preserving the cell-mediated interactions 5 ACS Paragon Plus Environment


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with the immune system. It is therefore a great step forward if it will be possible to put together the functionalities of monoclonal antibodies and immunotoxins. In this study, two therapeutic antibodies (trastuzumab and cetuximab) were modified by chemical addition of saporin thus creating two immunotoxins. We demonstrate that the immunotoxins deliver their toxic payload into the target cells and most importantly trigger the ADCC. In addition, we show that the saponin-sensitized immunotoxins preserved the advantages of the naked monoclonal antibodies while their direct toxicity was drastically augmented in combination with a triterpenoid saponin.


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Materials and methods Protein expression and purification The plasmid coding for saporin (hissaporin-pET11d 17) was transformed into Escherichia coli Rosetta™ 2(DE3) pLysS competent cells (Novagen, San Diego, CA, USA). The bacteria were propagated until A600nm = 0.9 in a volume of 2 L and expression of saporin was induced by adding β-D-1-thiogalactopyranoside (IPTG, final conc. 1 mM) and incubation at 37 °C and 200 rpm for 3 h. After expression, bacteria were lysed (Branson Sonifier 250, G. Heinemann, Schwäbisch Gmünd, Germany) and purified by Ni-nitrilotriacetic acid (NTA) agarose affinity chromatography (Protino® Ni-NTA agarose, Macherey-Nagel, Düren, Germany). Proteins were separated from bacteria debris by centrifugation at 15800g and 4 °C for 30 min and the supernatant was poured to the column. Saporin was eluted by increasing concentrations of imidazole (31, 65, 125 and 250 mM) and eluates were analyzed by SDS-PAGE [12% (w/v) gel]. Fractions with purified proteins were dialysed against 3 L PBS overnight at 4 °C. The protein concentration was determined by a bicinchoninic acid assay (Pierce BCA Protein Assay, Thermo Scientific, Rockford, IL, USA).

Production of immunotoxins Chemical cross-linking of antibodies to saporin was performed via N-succinimidyl 3-(2pyridyldithio) propionate (SPDP) (Thermo Scientific). Trastuzumab (5 mg) (Herceptin®, Roche Applied Science, Mannheim, Germany), cetuximab (5 mg) (Erbitux®, Merck, Darmstadt, Germany) and saporin (1 mg) (molar ratio of 1 : 1 for trastuzumab : saporin and for cetuximab : saporin) were dialyzed overnight against PBS supplemented with 1 mM EDTA, 0.02 % sodium azide, pH 7.5 (PBS-EDTA). All proteins were modified with 20 mM SPDP for 60 min at 25 °C and were dialyzed against PBS-EDTA. The modified saporin was 7 ACS Paragon Plus Environment


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chemically reduced by 150 mM dithiothreitol (DTT) for 30 min at 25 °C and thereafter DTT was removed using a PD-10 desalting column (GE Healthcare, Uppsala, Sweden). SPDPmodified trastuzumab and cetuximab were mixed with the modified saporin and incubated for 18 h at 25 °C. Saporin-trastuzumab (ST) and saporin-cetuximab (SC) were purified by sizeexclusion chromatography (Bio-Gel P-30 Medium, Bio-Rad, Hercules, CA, USA) and NiNTA chromatography. The purified immunotoxins were analyzed by SDS-PAGE [7.5% (w/v) gel] under reducing and non-reducing conditions.

Surface plasmon resonance spectroscopy Carboxymethylated C1 sensor chips (GE Healthcare, Freiburg, Germany) were activated by EDC/NHS chemistry following the instructions of the manufacturer (GE Healthcare). ST or SC was immobilized in 10 mM sodium acetate buffer, pH 5.0 at 5 µL/min using HBS-EP running buffer (GE Healthcare). ST was immobilized to a level of 270 response units (RU) and SC to 355 RU. To delineate specific from unspecific binding, a control surface treated chemically with EDC/NHS but without immobilized protein was used. Running buffer in this experiment was Dulbecco's PBS buffer without Ca and Mg supplementation (PAA Laboratories, Coelbe, Germany). The flow rate was adjusted to 30 µL/min and 100 µl of the soluble extracellular domain of each receptor was injected for a potential interaction with the immobilized immunotoxins. Soluble HER2 (“ErbB2/HER2 Protein (His Tag)” #10004-H08H, Sino Biological Inc., Beijing, P. R. China) was diluted with the running buffer and injected at final concentrations of 1, 2 and 4 µg/mL to test binding to ST. Soluble recombinant EGFR (“EGFR Human Sf9” #PKA-344, ProSpec-Tany Technogene Ltd., East Brunswick, NJ, USA) was also diluted with running buffer and injected at final concentrations of 1.3, 2.6 and 5.2 µg/mL to investigate binding to SC. Both association and dissociation phases lasted for 200 s.


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Sensorgrams were referred to the control surface and analyzed by BIAevaluation software 4.1.1. (GE Healthcare).

Cell culture BT-474 cells (ACC-64, human breast ductal carcinoma) were cultured in RPMI-1640 (PAA Laboratories) supplemented with 10% fetal bovine serum (FBS) (BioChrom KG, Berlin, Germany) and 1% Penicillin/Streptomycin (PS) (Gibco/Invitrogen, Karlsruhe, Germany) at 37 °C and 5% CO2. ECV-304 cells (ACC-310, human urinary bladder carcinoma) were cultured in DMEM (PAA Laboratories, Pasching, Austria) supplemented with 10% FBS and 1% PS under the same conditions.

Isolation of natural killer cells Blood (60 ml) was collected from donor I and II. Blood was diluted (1:1) with PBS and peripheral blood mononuclear cell (PBMC) fraction was isolated using Lymphocyte Separation Media (PromoCell, Heidelberg, Germany). After separation of PBMCs, the cell fraction was washed with PBS containing 0.1% BSA and centrifuged at 400g and 25 °C for 20 min. The washing step was repeated twice. PBMCs were filtered through a CellTrics® 30 µm filter device (Partec GmbH, Görlitz, Germany) to remove cell clusters and were washed again under the same conditions as before. NK cells were isolated from the PBMC fraction by a negative selection by magnetic-activated cell sorting (MACS). Human NK Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) was used for this purpose. After collection of purified NK cells, they were centrifuged at 500g and 25 °C for 30 min. Cells were resuspended in Mononuclear Cell 9 ACS Paragon Plus Environment


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Medium (PromoCell) and immediately used in the subsequent experiment of cell-mediated cytotoxicity.

Real-time monitoring of cell-mediated cytotoxicity Real-time monitoring of cell-mediated cytotoxicity was conducted using the xCELLigence System RTCA (Roche Applied Science). Firstly, 50 µL/well RPMI-1640 medium supplemented with 10% FBS and 1% PS was pipetted in a 96-well e-plate (Roche Applied Science) and after setting the impedance to zero, 10,000 BT-474 cells were seeded (50 µL/well) and allowed to proliferate at 37 °C and 5% CO2 For competitive assays of trastuzumab/ST and cetuximab/SC either of the following compounds were added after 13 h: (1) 100 µL medium or 100 µL medium supplemented with (2) 2.5 µg/mL SO1861 (triterpenoid saponin purified from Saponaria officinalis L. 14); (3) ST (final concentration of 1 nM) and SO1861; (4) SC (final concentration of 0.001 nM) and SO1861; (5) ST, SO1861 and 1000 nM trastuzumab; or (6) SC, SO1861 and 1000 nM cetuximab. After addition of the compounds, cells were monitored fur further 57 h. Results were evaluated with the RTCA Software 1.2.1.1002 (Roche Applied Science). For non-competitive assays in the presence and absence of NK cells the following compounds were added after 23 h: (1) 100 µL medium or 100 µL medium supplemented with (2) 10 nM trastuzumab; (3) 10 nM ST; (4) 1:2 NK cells (ratio between BT-474 and NK cells); (5) 10 nM trastuzumab and 1:2 NK cells or (6) 10 nM ST and 1:2 NK cells. Cetuximab and SC were tested analogously. Both NK cells from donor I and donor II were tested for each of the conditions. Cell viability was continuously monitored for further 48 h and then medium supplemented with SO1861 (50 µL/well) was added reaching a final concentration of 2.5


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µg/mL. Cells were again monitored for 39 h (i.e. in total 110 h) and results were analyzed with the RTCA Software 1.2.1.1002.

Stability in cell culture medium Proteins (ST, SC and saporin) were incubated at 37 °C for 6 or 24 h with conditioned RPMI1640 medium supplemented with 10% FBS and 1% PS (medium was consumed for one week by BT-474 cells at 37 °C and 5% CO2 and centrifuged at 800g for 5 min before addition to cells). After the incubation period, proteins in the medium were separated by SDS-PAGE [7.5% (w/v) gel] and then blotted at 50 V and 15 °C for 60 min to a nitrocellulose membrane (Hybond-C Extra, GE Healthcare). The Membrane was blocked for 30 min and incubated with a self-raised rabbit polyclonal antibody against saporin for further 30 min. Membrane was washed 4 times with PBSB0.2 [PBS with 0.2% (w/v) Brij®58, pH 7.4] for 10 min each time and thereafter treated with polyclonal goat anti-rabbit immunoglobulins/horse radish peroxidase (Dako Cytomation, Hamburg, Germany) for 30 min. The membrane was washed furthermore for 4 times with PBSB0.2. The binding of the secondary antibody was detected by enhanced chemi-luminescence reaction. After exposure, the photographic paper (Amersham Hyperfilm™ ECL, GE Healthcare) was developed by an OPTIMAX® X-Ray Film Processor (Protec Medizintechnik, Oberstenfeld, Germany).

Fluorescence labeling ST was conjugated to Alexa Fluor® 488 5-TFP (Molecular Probes, Eugene, USA) by adding 1 M hydrogen carbonate buffer (100 µL), pH 9.0 and 108 µL of the fluorescent dye (5 mg/mL in DMSO) to a solution of 1 mL of ST (0.186 mg/ml in PBS). Labeling reaction was allowed for 1 h at room temperature and thereafter ST-Alexa Fluor® 488 (AlexaST) was purified from 11 ACS Paragon Plus Environment


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the unconjugated fluorophore overnight by dialysis against PBS. Saporin was conjugated to Alexa Fluor® 488 5-TFP following the same procedure and after the dialysis against PBS a purified product (Alexasaporin) was obtained.

Confocal live cell imaging BT-474 cells (50,000 cells/dish) were seeded in cell culture dishes (µ-Dish 35 mm, low, IBIDI, Martiensried, Germany) and grown in RPMI-1640 medium supplemented with 10% FBS and 1% PS at 37 °C and 5% CO2 for 24 h. Medium was removed and 800 µL medium supplemented with 100 nM AlexaST was added to the cells. In the case of the competitive assay with free antibody, cells were covered with medium containing 100 nM AlexaST and 1000 nM unconjugated trastuzumab. Cells were incubated for 24 h. Then cells were treated with 8 µL (1 mg/mL) Hoechst 33342 for 2 h. Five minutes before finishing the incubation time, 1 µL CellMask™ (Invitrogen, Darmstadt, Germany) was added to the cells. For the co-localization studies, 8 µg pHrodo™ Red Dextran, 10 kDa (Invitrogen, Darmstadt, Germany) and Hoechst 33342 were added 2 h before the end of the incubation time while no CellMask™ was administered. Cells were washed three times with live cell imaging solution (Invitrogen) supplemented with 5 mM D(+)glucose and thereafter covered with 800 µL of the same solution. Cell culture dishes were fixed in a heating chamber and maintained at 37 °C during the microscopy process. For the visualization of the endo/lysosomal escape of AlexaST, a final concentration of 2.5 µg/mL SO1861 was added 140 s after the start of the experiment and images were taken every 20 s. For the experiment with

Alexa

saporin, ECV-304 cells (50,000 cells/dish) were seeded in cell

culture dishes and grown in DMEM medium supplemented with 10% FBS and 1% PS at 37 °C and 5% CO2 for 24 h. Medium was removed and 800 µL medium supplemented with 1000 12 ACS Paragon Plus Environment

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nM

Alexa

saporin was added to the cells. Cells were incubated for 6 h and two hours before

finishing the incubation time, 8 µg pHrodo™ Red Dextran, 10 kDa was added. Cells were washed three times with live cell imaging solution supplemented with 5 mM D(+)glucose and covered with 800 µL of the same solution. Similarly to the already described set set up, cell culture dishes were fixed in a heating chamber and maintained at 37 °C during the microscopy process. For the visualization of the endo/lysosomal escape, a final concentration of 10 µg/mL SA1641 was added at the start of the experiment and images were captured every 30 s. In both cases, cells were observed by laser scanning microscopy (LSM780, Axio Observer Z1, Carl Zeiss, MicroImaging, Jena, Germany) equipped with a Plan-Apochromat 63×/1.40 Oil objective. Focus stability was insured by Definite Focus (Carl Zeiss, Jena, Germany). Acquisition of images was done via the software ZEN 2010 (Carl Zeiss).



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Results Conjugation and purification of saporin-trastuzumab and saporin-cetuximab Saporin was cross-linked to trastuzumab and cetuximab via covalent linkage. After conjugation and purification, an SDS-PAGE analysis under non-reducing conditions (Figure 1a) for both ST and SC showed two bands corresponding to one molecule of antibody coupled to either one or two molecules of toxin. In the case of ST, a third weak band, probably a diffusion of ST (one molecule of antibody coupled to one molecule of toxin) due to a different glycosylation pattern of the antibody moiety (similarly to the diffusion observed by single trastuzumab) was identified. Neither free antibodies nor free saporin was observed in saporintrastuzumab and saporin-cetuximab solutions, displaying the purity of the immunotoxin solutions after purification. Under reducing conditions (Figure 1b) the disulfide bonds introduced by the cross-linker in the case of the conjugates and the disulfide bonds of the antibodies were cleaved and the antibody heavy chains were separated from the antibody light chains and saporin. The bands that have approximately 25 kDa and are observed by trastuzumab and cetuximab represent the light chain of the respective monoclonal antibodies. The band that appears at approximately 25 kDa by saporin corresponds to the same saporin. Considering the information provided by the controls, it is concluded that the bands that have approximately 25 kDa and are observed by ST and SC correspond to both light chains of monoclonal antibodies and saporin. The final production level for the immunotoxins and the total yield of the process after chemical conjugation and the two-step purification (size-exclusion chromatography and NiNTA agarose affinity chromatography) are around 1.5 mg of immunotoxin and a 25% of protein (monoclonal antibody plus toxin) input.


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Binding of immunotoxins to cellular receptors Immunotoxins were immobilized on the carboxymethylated surface of a gold sensor chip and soluble truncated variants of either HER2 (Figure 2a) or EGFR (Figure 2b) were injected to determine binding of the receptors to ST or SC by surface plasmon resonance spectroscopy. During the association phase, HER2 (4 µg/mL) showed a binding to the immunotoxin with a response difference (RD) of 48.9. During the dissociation phase, HER2 remained strongly bound to ST and only a minimal amount of receptor was dissociated from the immunotoxins. At the end of the dissociation phase an RD of 45.8, which was similar to the value at the end of the association phase, was measured. The binding profile of HER2 to ST was similar at the two other concentrations tested. At 1 and 2 µg/mL, HER2 bound strongly to the immobilized ST with an RD of 26.0 and 45.0 at the end of the association phase and an RD of 9.7 and 29.7, respectively, at the end of the dissociation phase. Soluble EGFR bound strongly to SC during the association phase at a concentration of 5.2 µg/mL (RD of 95.9 at the end of association). Most of the EGFR remained bound to SC during the dissociation phase (RD of 78.1 at the end of dissociation) indicating a very strong interaction between the receptor and the immunotoxins. A very similar pattern was observed by lower concentrations of EGFR (1.3 and 2.6 µg/mL). At the end of the association phase, EGFR bound in a concentration-dependent manner. An RD of 62.9 was measured at a concentration of 1.3 µg/mL, while an RD of 75.7 was determined at a concentration of 2.6 µg/mL. At the end of the dissociation phase almost all EGFR was still bound to the immunotoxin (RD = 49.2 at 1.3 µg/mL and RD = 57.1 at 2.6 µg/mL).



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Competitive binding between immunotoxins and unconjugated antibodies Cells were treated with an excess of unconjugated antibody to study its competitive effect on the binding of the immunotoxins to the cellular receptor (Figure 3). The impedance measured by the xCELLigence system (representing the area of attached, i.e. living cells) was normalized at the time point of 13.5 h by assigning a normalized cell index (NCI) of 1.00. Cells had reached a plateau phase from 8 to 13.5 h, but as observed in the control, after addition of fresh medium, cells started to grow again until reaching another stable phase which would be maintained with slightly loss of signal until the end of the experiment (NCI = 1.60 at 70.0 h). All other samples were treated with SO1861, a purified triterpenoid saponin from the common soapwort Saponaria officinalis L., a member of the Caryophyllaceae family, which has been shown to substantially augment the efficacy of a targeted toxin composed of saporin and epidermal growth factor in mice

16

. The impedance of the cells

treated with SO1861 (2.5 µg/mL) alone increased only slightly more than control cells and then gradually decreased to the impedance of untreated cells until the end of the experiment (after 70.0 h), wherein an NCI of 1.51 was determined (Figure 3a and 3b). The NCI of cells treated with ST at 1 nM in combination with SO1861 (Figure 3a) increased tremendously to a maximum value of 4.01 after 23.5 h but then continuously decreased to very low values, representing almost a total cell death at the end of the experiment. The appearance of a peak directly after treatment can be attributed to cell swelling, which results in an increased impedance due to the larger area covered by the swollen cells. Repeating the same conditions (ST at 1 nM and SO1861 at 2.5 µg/mL) but with the addition of trastuzumab in excess (1000 nM), cells remained unaffected by the immunotoxin indicating target-specific competition. Cells treated with ST, SO1861 and trastuzumab grew similar to the controls. Cells were also incubated in the presence of SC (0.001 nM) and SO1861 (Figure 3b). NCI incremented to a maximum value of 3.54 at 24.5 h but decreased again rapidly to a NCI value 16 ACS Paragon Plus Environment

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of 0.95 (at 44.5 h). Cell viability further continuously decreased and at the end of the experiment almost all the cells were dead. However, cells treated with SC and SO1861 in the same concentrations as before but with the addition of cetuximab in excess (1000 nM), proliferated in a totally different way. The curve increased to a NCI of 3.04 at 34.0 h and decreased to control values at the end of the experiment (NCI = 1.29). In both cases, the presence of the unconjugated antibody (trastuzumab and cetuximab) blocked the toxicity of the immunotoxin (ST and SC, respectively).

Cell-mediated cytotoxicity and synergism between immunotoxins and SO1861 Cells were treated with unconjugated antibodies and immunotoxins in the presence of NK cells isolated from the blood of two donors in order to analyze the antibody-dependent cellmediated cytotoxicity (Figure 4a and 4b). BT-474 cells grew quickly within the first 2 h but during the next 21 h the cell growth only increased up to a NCI of 1.00 at the normalization time (23.0 h). At this moment, compounds were added to the cells. In the case of control cells, only fresh medium was pipetted and the NCI increased almost linearly until a value of 7.38 at 60.0 h. Trastuzumab was non-toxic at a concentration of 10 nM while ST was slightly toxic at the same concentration (NCI = 6.3 at 60.0 h). ADCC of trastuzumab and ST was measured with NK cells from donor I and II at a BT-474:NK cell ratio of 1:2. In the case of NK cells from donor I, the sole addition of NK cells already caused an effect on BT-474 cells as the cell viability diminished (NCI = 5.06). However, this effect was much more pronounced when NK cells were administered in the presence of trastuzumab (NCI = 1.33) and ST (NCI = 2.45) (Figure 4a). In the case of NK cells from donor II, the addition of NK cells also caused an effect on BT-474 cells and the cell viability was reduced to an NCI of 5.37. This effect was 17 ACS Paragon Plus Environment


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again much more pronounced when the NK cells were given in combination with trastuzumab (NCI = 3.07) and ST (NCI = 4.62). Cetuximab and SC were also tested at 10 nM and both compounds were non-toxic to cells as the cell viability at 60.0 h was similar to the control. Cell-mediated cytotoxicity of cetuximab and SC was determined with NK cells from donor I and II at the same cellular ratio as before. In the case of NK cells from donor I, cell-mediated cytotoxicity was observed when BT-474 cells were incubated with cetuximab (NCI = 1.92 at 60.0 h) and SC (NCI = 0.25) (Figure 4b). In the case of NK cells from donor II, this phenomenon was also observed (NCI = 1.59) and SC (NCI = 0.21). The complete experiment was then continued by adding 2.5 µg/ml SO1861 to monitor the synergistic effect between SO1861 and the immunotoxins (Figure 4c and 4d). Curves were normalized again at 70.5 h. The impedance signal of the control cells (treated only with medium) slowly decreased until the end of the experiment (NCI = 0.80 at 110.0 h). A noncytotoxic concentration of SO1861 (NCI = 0.87 at 110.0 h) was further used in the experiments in combination with the different compounds. The addition of SO1861 caused the death of all BT-474 cells, which were treated with ST or SC (10 nM) in the absence of natural killer cells. However, no effect was observed in the cells treated with unconjugated trastuzumab or cetuximab, indicating that the presence of saporin was necessary to elicit this effect by the immunotoxin for the synergistic augmentation of cytotoxicity. In the case of cells that were previously treated with immunotoxins and NK cells from donor I, the addition of SO1861 also resulted in cell death of all of the remaining BT474 cells treated either with ST (Figure 4c) or with SC (Figure 4d). In the case of cells previously treated with immunotoxins and NK cells from donor II, the addition of SO1861 resulted in cell death of almost all BT-474 cells treated with ST (Figure 4c) while the few


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cells remaining after SC-dependent ADCC finally completely died after the addition of the per se non-toxic concentration of SO1861 (Figure 4d).

Stability of saporin-trastuzumab and saporin-cetuximab To investigate the stability of the immunotoxins, ST and SC were incubated at 37 °C for 6 and 24 h with conditioned cell culture medium previously used to grow BT-474 cells. A western blot was performed to detect possible degradation (Figure 5). Similar to the results observed in Figure 1a, the two bands corresponding to ST and the two bands corresponding to SC were clearly detected for both incubation times. Most interestingly, no degradation products were observed after 6 h or 24 h of incubation as indicated by the absence of a band for free saporin. Saporin that served as a positive control was detected at the expected position.

Internalization, co-localization and endo/lysosomal escape The internalization of immunotoxins was studied by treating cells with AlexaST in the absence and presence of an excess of trastuzumab. The internalization of

Alexa

ST was visualized by

live cell imaging 24 h after addition of the compounds to cells (Figure 6a). The morphology of the cells was still intact indicating that the labeled immunotoxin was not toxic at the administered concentration.

Alexa

ST accumulated mostly in the organelles that surround the

cell nucleus. However, when cells were competitively treated with

Alexa

ST and an excess of

trastuzumab, the amount of internalized immunotoxins decreased dramatically (Figure 6b) indicating a specific receptor-dependent internalization of ST. In order to identify the organelles in which the accumulation of the immunotoxins took place after internalization, co-localization with red dextran (a compound incorporated in acidic 19 ACS Paragon Plus Environment


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vesicles) was investigated (Figure 6c). Cells were incubated for 24 h with the same concentration of

Alexa

ST as before. The amount of internalized immunotoxin was not affected

by this procedure. The labeled immunotoxin accumulated again in vesicles that surround the nucleus and co-localized with red dextran demonstrating that ST accumulated in acidic vesicles e.g. early endosomes, late endosomes and lysosomes. Further cells were treated with

Alexa

ST for 24 h to visualize the endo/lysosomal escape of the

immunotoxin (Figure 7). After the incubation time,

Alexa

ST accumulated again mainly in

acidic organelles surrounding the nucleus and no endosomal release was observed. Interestingly, a small amount of protein remained bound to the receptors in the cell membrane (Figure 7, see red arrows in the merged picture). During the first 140 s of the experiment, no release of

Alexa

ST from the vesicles was observed. At that moment, SO1861 was added at a

non-toxic concentration of 2.5 µg/mL and 300 s later the first indications of the endo/lysosomal escape appeared (Figure 7, see red arrows in the pictures with the green channel). After the formation of a bubble in the cellular vesicle, toxin diffused to the cytosol. Endo/lysosomal escape of AlexaST was observed in three time intervals (440–460 s, 520–560 s, 640–700 s). In these three cases, a perturbation in the vesicle membrane was formed leading to the release of saporin to the cytosol. Additional mechanistic studies were performed with

Alexa

saporin to observe the

endo/lysosomal escape of the toxin in the presence of triterpenoid saponin (Movie S1 available in the supplementary information). Release of

Alexa

saporin from the endo/lysosmes

into the cytosol was detected after the addition of saponin. A diffused fluorescence within the whole cell clearly indicated the release of the toxin. Cell membrane remained unaffected by saponin because no leakage of toxin out of the cell was observed. Endo/lysosmal membrane also remained unaffected by saponin since the pH-sensitive dye pHrodo™ Red Dextran, 10 kDa exhibited continuously fluorescence even after the toxin release. Endo/lysosomes 20 ACS Paragon Plus Environment

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preserved their integrity and thus their internal acidic environment until the end of the experiment.



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Discussion Site-specific chemical modification of an antibody or antibody fragment may result in better stability and full retention of antigen-binding properties. However, chemical modification may affect the potency of the conjugate. To check if there was any effect of the cross-linking reaction, the binding of the modified antibodies to the tumor-specific cellular receptors was verified by the interaction of ST to the soluble recombinant HER2 and SC to the soluble extracellular domain of EGFR in a surface plasmon resonance-based binding assay. Specificity of the binding is another therapeutic aspect that renders the antibodies to be highly successful in clinics. Specificity of binding was confirmed in the present study by inhibition of cytotoxic effects in the presence of unconjugated antibodies in excess. Saporin-modified antibodies retained their ability to trigger an ADCC response. ADCC is an important aspect already established for therapeutic monoclonal antibodies (e.g. trastuzumab, cetuximab, rituximab, alemtuzumab and ofatumumab)

18

. Notably, there was a clear ADCC

with the immunotoxins which was comparable to the effect of unconjugated antibodies. This immunotoxin mediated effect may be summarized as the immunotoxin-dependent cellmediated cytotoxicity (IDCC). A stability assay confirmed that the molecules responsible for triggering IDCC against tumor cells were indeed the immunotoxins and not antibodies released from the modified immunotoxins by proteolytic degradation or cleavage of the disulfide bond. It is a common understanding that immunotoxins do not present an immunotherapeutic mechanism of action such as the ability to trigger ADCC

10

. This assumption is expected to

hold true for an appreciable number of immunotoxins that were designed with antibody fragments, that lack the Fc portion and therefore the ability to interact with NK cells

19

.

Nevertheless, immunotoxins were also constructed with complete monoclonal antibodies that possess the Fc part

20

. In the past, a few immunotoxins consisting of trastuzumab or 22 ACS Paragon Plus Environment

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cetuximab as targeting moiety coupled with some plant or bacterial toxins as toxic moiety, such as a truncated version of Pseudomonas exotoxin A

21

, gelonin

22

or saporin

23, 24

, were

designed. It can be expected that these immunotoxins retain the reported ability of the integrated monoclonal antibodies to trigger ADCC after chemical conjugation to the toxin. The constructed immunotoxins showed a low cytotoxicity as ST and SC were slightly toxic or non-toxic to the tumor cells at a concentration of 10 nM (see Figure 4) and the incubation of cells for 24 h with a concentration of 100 nM also resulted in no toxicity (see Figure 6a). Therapeutic antibodies were used for the construction of immunotoxins. Since the mechanism of action of this kind of antibodies is the engagement with cell surface receptors (to either activate or inhibit signaling), or to interact with the innate immune system, it is generally desirable that the antigen-antibody complex should not be rapidly internalized 3. Therefore, the use of trastuzumab and cetuximab as immunotoxin ligands would cause a long internalization cycle. However, it has been shown that a substantially higher efficacy of the immunotoxins can be achieved by saponin-sensitization

25, 26

. This effect depends on the presence of saporin or

dianthin 13. Therefore, the saporin-containing immunotoxins were incubated in the presence of the purified saponin SO1861. A synergistic effect between saporin and SO1861 resulted in a tremendous increase of cytotoxicity of the immunotoxins. This aspect is very relevant for the perspective of immunotoxin applications, as these augmentation effects allow the decrease of effective doses in patients resulting in amelioration of side effects. Reduced dosages may concomitantly circumvent an expected immune reaction that can block the immunotoxins. As previously indicated, the functionality of monoclonal antibodies (binding to targeted receptors and triggering of ADCC) was combined with the functionality of immunotoxins (specific toxin delivery). A discussion about how these two functionalities act and how the toxicity of the conjugate is drastically augmented by triterpenoid saponin is included below. 23 ACS Paragon Plus Environment


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First, immunotoxins are expected to specifically bind to targeted tumor cells. Following the addition of NK cells, the Fc domains can interact with the FcγRIII of these cells, resulting in the initiation of the ADCC

27

. However, part of the immunotoxins will trigger their

internalization due to the binding of the antibody units to the cellular receptors (EGFR 28 and HER2

29

) and therefore the IDCC effect will decrease to the same extent as the amount of

internalized immunotoxin increases. After a certain time, internalized immunotoxins will be accumulated in endosomes and finally lysosomes. In these acidic organelles, the disulfide bond introduced during the conjugation of saporin to the monoclonal antibodies will be cleaved and free saporin will be released into the lysosomal environment

30

. Following the

addition of SO1861, an interaction between the toxin and SO1861 occurs and this will result in the endosomal escape of saporin

15

leading to the induction of apoptosis by the enzymatic

removal of a specific adenine residue at position 4324 of the 28S rRNA

31

. Therefore, the

addition of SO1861 will concomitantly enhance the cytotoxicity of the toxin moiety of the immunotoxin. The specific binding of immunotoxins to tumor-specific cellular receptors and the accumulation of immunotoxins in acidic organelles were further confirmed by live cell imaging using fluorescence labeled ST (AlexaST). Endo/lysosomal escape of delivered toxin (Alexasaporin) was visualized by the same technique in further mechanistic studies. Since the integrity of cell and endo/lysosomal membranes was preserved in the presence of triterpenoid saponin, the toxin was not delivered into the cytosol via unspecific membrane permeabilization but mediated by a specific interaction between the toxin and triterpenoid saponin. The results open a path for utilizing the saponin-sensitization to other monoclonal antibodies directed to cancer-specific cellular receptors that are already in the market such as rituximab, panitumumab and ofatumumab. Animal experiments to evaluate toxicity and anti-tumor efficacy, as well as to characterize the immunogenicity of the immunotoxins in mice are of 24 ACS Paragon Plus Environment

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great importance for the future of the targeted tumor therapy with modified antibodies described here. The IDCC together with the saponin-sensitization may further help to overcome present limitations of the development of clinical resistance for a number of antibody-based tumor therapies. This would further help to achieve a successful and efficient treatment of solid tumors and hematologic malignancies.



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Acknowledgements We acknowledge the financial support of the Deutsche Forschungsgemeinschaft (WE 4784/11, TH 1810/1-1) and the Wilhelm Sander-Stiftung (2011.121.1).

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- Figure legends Figure 1. SDS-PAGE analysis of saporin-trastuzumab, trastuzumab, saporin-cetuximab, cetuximab and saporin. Electrophoresis was performed under non-reducing conditions (a) and under reducing conditions (b) on a 7.5% (w/v) SDS-gel. Figure 2. Binding of immunotoxins to soluble receptors. (a) Saporin-trastuzumab was immobilized on a sensor chip and soluble human recombinant HER2 was injected at three different concentrations (1, 2 and 4 µg/mL). (b) Saporin-cetuximab was immobilized on the sensor chip and soluble human recombinant EGFR was injected at 1.3, 2.6 and 5.2 µg/mL. In both cases, the binding during the association phase was concentration-dependent and immunotoxin and receptor remain strongly bound during the dissociation phase. Figure 3. Competitive assay between immunotoxins and antibodies in excess. BT-474 (10,000 cells/well) were seeded and allowed to grow until the compounds were added at 13.5 h. Only medium was added to control cells while a second control was treated only with 2.5 µg/mL SO1861. (a) Cells were incubated with 1 nM saporin-trastuzumab and SO1861 (2.5 µg/mL) in the presence and absence of unconjugated trastuzumab (1000 nM). (b) Saporincetuximab was tested at a concentration of 0.001 nM in combination with SO1861 (2.5 µg/mL) in the presence and absence of unconjugated cetuximab (1000 nM). The curves representing control cells and cells treated with SO1861 alone are equally depicted in both panels for clarity. Figure 4. ADCC and synergistic augmentation of the cytotoxicity of immunotoxins and triterpenoid saponins. BT-474 (10,000 cells/well) were seeded and allowed to grow until the different compounds were added at 23.0 h. Cells treated with only cell culture medium served as control. To investigate ADCC, BT-474 cells were incubated with trastuzumab and ST in the presence or absence of NK cells from donor I and II (a). In addition, BT-474 cells were 29 ACS Paragon Plus Environment


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treated with cetuximab and SC in the presence or absence of NK cells from both donors (b). All unconjugated antibodies and immunotoxins were administered at a concentration of 10 nM. To study the synergism between the immunotoxins and SO1861, the experiment was continued and SO1861 (2.5 µg/mL) was added at 70.5 h to all previously treated cells. At this time, cells treated with cell culture medium and cells treated with only SO1861 served as control. SO1861 was added to BT-474 cells previously incubated with trastuzumab and ST in the presence or absence of NK cells from donor I and II (c). In the same way, SO1861 was added to BT-474 cells previously treated with cetuximab and SC in the presence or absence of NK cells from both donors (d). For better comparison, some samples (e.g. controls) are equally drawn in more than one panel. Figure 5. Western blot of immunotoxins after incubation with conditioned cell culture medium. ST and SC were incubated at 37 °C for 6 and 24 h with medium used to cultivate BT-474 cells in order to determine the stability of the conjugates under these conditions. Note that no degradation products of the conjugates were detected. Saporin served as positive control. Figure 6. Internalization and co-localization studies of immunotoxins. (a) Cells were visualized by live cell imaging 24 h after addition of AlexaST. The cell nucleus is visualized in cyan fluorescence (upper left quarter), the cell membrane in magenta (lower left quarter) and immunotoxins in green (upper right quarter). The lower right quarter shows the merger. (b) Cells were treated for 24 h with internalized

Alexa

Alexa

ST and an excess of trastuzumab. The amount of

ST is lower than in the absence of trastuzumab. (c) Cells were co-incubated

with red dextran, which accumulates in acidic vesicles. Co-localization of both compounds indicates that ST is enriched in acidic vesicles in the vicinity of the cellular nucleus.


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Figure 7. Endo/lysosomal escape of immunotoxins. After the incubation with

Alexa

ST for 24

h, most of the immunotoxin accumulated in acidic organelles surrounding the nucleus although a minor amount remained bound to the receptors in the cell membrane (red arrows in the merged picture). No endo/lysosomal escape was observed at the beginning of the experiment. However, after addition of 2.5 µg/mL SO1861, release of toxin was detected in three time intervals (440–460 s, 520–560 s, 640–700 s; red arrows in pictures with green channel).



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Figure 1. SDS-PAGE analysis of saporin-trastuzumab, trastuzumab, saporin-cetuximab, cetuximab and saporin. Electrophoresis was performed under non-reducing conditions (a) and under reducing conditions (b) on a 7.5% (w/v) SDS-gel. 103x72mm (300 x 300 DPI)

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Figure 2. Binding of immunotoxins to soluble receptors. (a) Saporin-trastuzumab was immobilized on a sensor chip and soluble human recombinant HER2 was injected at three different concentrations (1, 2 and 4 µg/mL). (b) Saporin-cetuximab was immobilized on the sensor chip and soluble human recombinant EGFR was injected at 1.3, 2.6 and 5.2 µg/mL. In both cases, the binding during the association phase was concentration-dependent and immunotoxin and receptor remain strongly bound during the dissociation phase. 95x85mm (300 x 300 DPI)



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Figure 3. Competitive assay between immunotoxins and antibodies in excess. BT-474 (10,000 cells/well) were seeded and allowed to grow until the compounds were added at 13.5 h. Only medium was added to control cells while a second control was treated only with 2.5 µg/mL SO1861. (a) Cells were incubated with 1 nM saporin-trastuzumab and SO1861 (2.5 µg/mL) in the presence and absence of unconjugated trastuzumab (1000 nM). (b) Saporin-cetuximab was tested at a concentration of 0.001 nM in combination with SO1861 (2.5 µg/mL) in the presence and absence of unconjugated cetuximab (1000 nM). The curves representing control cells and cells treated with SO1861 alone are equally depicted in both panels for clarity. 99x108mm (300 x 300 DPI)


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Figure 4. ADCC and synergistic augmentation of the cytotoxicity of immunotoxins and saponins. BT-474 (10,000 cells/well) were seeded and allowed to grow until the different compounds were added at 23.0 h. Cells treated with only cell culture medium served as control. To investigate ADCC, BT-474 cells were incubated with trastuzumab and ST in the presence or absence of NK cells from donor I and II (a). In addition, BT-474 cells were treated with cetuximab and SC in the presence or absence of NK cells from both donors (b). All unconjugated antibodies and immunotoxins were administered at a concentration of 10 nM. To study the synergism between the immunotoxins and SO1861, the experiment was continued and SO1861 (2.5 µg/mL) was added at 70.5 h to all previously treated cells. At this time, cells treated with cell culture medium and cells treated with only SO1861 served as control. SO1861 was added to BT-474 cells previously incubated with trastuzumab and ST in the presence or absence of NK cells from donor I and II (c). In the same way, SO1861 was added to BT-474 cells previously treated with cetuximab and SC in the presence or absence of NK cells from both donors (d). For better comparison, some samples (e.g. controls) are equally drawn in more than one panel. 174x108mm (300 x 300 DPI)



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Figure 5. Western blot of immunotoxins after incubation with conditioned cell culture medium. ST and SC were incubated at 37 °C for 6 and 24 h with medium used to cultivate BT-474 cells in order to determine the stability of the conjugates under these conditions. Note that no degradation products of the conjugates were detected. Saporin served as positive control. 84x118mm (300 x 300 DPI)


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Figure 6. Internalization and co-localization studies of immunotoxins. (a) Cells were visualized by live cell imaging 24 h after addition of AlexaST. The cell nucleus is visualized in cyan fluorescence (upper left quarter), the cell membrane in magenta (lower left quarter) and immunotoxins in green (upper right quarter). The lower right quarter shows the merger. (b) Cells were treated for 24 h with AlexaST and an excess of trastuzumab. The amount of internalized AlexaST is lower than in the absence of trastuzumab. (c) Cells were co-incubated with red dextran, which accumulates in acidic vesicles. Co-localization of both compounds indicates that ST is enriched in acidic vesicles in the vicinity of the cellular nucleus. 914x1006mm (72 x 72 DPI)



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Figure 7. Endo/lysosomal escape of immunotoxins. After the incubation with AlexaST for 24 h, most of the immunotoxin accumulated in acidic organelles surrounding the nucleus although a minor amount remained bound to the receptors in the cell membrane (red arrows in the merged picture). No endo/lysosomal escape was observed at the beginning of the experiment. However, after addition of 2.5 µg/mL SO1861, release of toxin was detected in three time intervals (440–460 s, 520–560 s, 640–700 s; red arrows in pictures with green channel). 209x264mm (300 x 300 DPI)


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Table of Contents Graphic 148x96mm (300 x 300 DPI)


Modified Trastuzumab and Cetuximab Mediate ... - ACS Publications

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