Volume 140, Issue 3, Pages 935-946 (March 2011) An Immunoconjugate of Anti-CD24 and Pseudomonas Exotoxin Selectively Kills Human Colorectal Tumors in Mice  Shiran Shapira, Assaf Shapira, Alex Starr, Dina Kazanov, Sarah Kraus, Itai Benhar, Nadir Arber  Gastroenterology  Volume 140, Issue 3, Pages 935-946 (March 2011) DOI: 10.1053/j.gastro.2010.12.004 Copyright © 2011 AGA Institute Terms and Conditions

Figure 1 Purification of SWA11 monoclonal antibody (mAb) and soluble ZZ-PE38 fusion protein. (A) 5 μg purified ZZ-PE38 protein (50 kDa) was loaded on 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie Blue. (B) 5 μg purified SWA11 IgG2a antibody, at a yield of 14 mg/L of culture media, was separated on 12% SDS-PAGE under reducing and nonreducing conditions and stained with Coomassie Blue. Lanes 1 and 2 represent purified proteins from 2 different purifications. (C) HT-29 (CD24-positive) and HCT116 (CD24-negative) cells were seeded in 96-well plates, and enzyme-linked immunosorbent assay was performed using the purified mAb. (D) 20 μg total cell lysates were subjected to Western blotting for CD24 using 1 μg/mL purified SWA11. The membrane was then reprobed with goat anti-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA) as loading control. (E) Schematic representation of IgG2a-ZZ-PE38 immunoconjugate consisting of a murine IgG conjugated to ZZ-PE38 fusion protein by an affinity linkage; ZZ-Fc domain. Gastroenterology 2011 140, 935-946DOI: (10.1053/j.gastro.2010.12.004) Copyright © 2011 AGA Institute Terms and Conditions

Figure 2 In vitro ADP-ribosylation enzymatic activity of ZZ-PE38 and SWA11-ZZ-PE38. 50 ng of ZZ-PE38 protein and the equivalent amount of the immunotoxin were assayed in the absence (A) or presence (B) of urea and dithiothreitol. Counts per minute (CPM) of quadruplicates was measured on a β-counter. Data are expressed as mean counts vs the various molecules. Gastroenterology 2011 140, 935-946DOI: (10.1053/j.gastro.2010.12.004) Copyright © 2011 AGA Institute Terms and Conditions

Figure 3 Analysis of cellular CD24 binding by SWA11-ZZ-PE38 and SWA11 in human CRC cell lines. (A) Cellular CD24 binding of the immunotoxin and the unconjugated monoclonal antibody (mAb) was evaluated and compared in HT-29 and HCT116 cells. The error bars represent standard deviations of triplicate measurements. (B) HT-29 (1 × 105) cells were seeded in 24-well plates and treated with 0, 1, and 10 μg/mL SWA11-ZZ-PE38. Forty-eight hours later, protein extracts were prepared and 10 μg from each sample was subjected to Western blotting for CD24. Actin served as loading control. (C−F) Cells were incubated with 10 μg/mL SWA11 or SWA11-ZZ-PE38 for 30 minutes at room temperature. Fluorescein isothiocyanate−labeled goat anti-mouse antibody was used for the detection of bound antibody and immunotoxins; negative control (secondary antibody alone) is represented in red (C), binding of SWA11 represented in black (D), and binding of SWA11-ZZ-PE38 is represented in green (E). Overlapping staining intensities of both SWA11 and immunotoxin are shown in panel F. Gastroenterology 2011 140, 935-946DOI: (10.1053/j.gastro.2010.12.004) Copyright © 2011 AGA Institute Terms and Conditions

Figure 4 Cytotoxicity of SWA11-ZZ-PE38, wt PE and control immunocomplex in CD24-positive and negative cells and induction of apoptosis. (A) 1 × 104 cells were seeded in 96-well plates and serial dilutions of immunotoxins or the highest concentration of the individual components were added in triplicates for 48 hours. The relative number of viable cells compared to cells grown in the absence of the fusion toxin was determined using the MTT assay. Each point represents the mean of a set of data determined in 3 independent experiments. The error bars represent standard deviations. The IC50 values were determined as the concentration needed to kill half of the cells and were indicated by broken lines. The bidirectional arrows indicate the “therapeutic window” of the immunocomplex treatment. (B−D) 1 × 105 HT-29 cells were seeded in 12-well plates. Untreated cells (B, left panel), SWA11 (B, middle panel), ZZ-PE38 (B, right panel), and 0.01 to 10 μg/mL SWA11-ZZ-PE38 (C, left panel; 10 μg/mL) or IgG-ZZ-PE38 immunocomplexes (C, right panel; 10 μg/mL) were added for 48 hours. Apoptosis was detected and quantified by Annexin V-fluorescein isothiocyanate/Propidium iodide fluorescence-activated cell sorting analysis. The dotplots are representative results of the highest concentrations. The apoptotic fractions were quantified and expressed as percentage of apoptotic cells (D). Gastroenterology 2011 140, 935-946DOI: (10.1053/j.gastro.2010.12.004) Copyright © 2011 AGA Institute Terms and Conditions

Figure 5 Altered C-terminus of PE38 and its binding activity. (A) PE38 derivative, PE38-QQΔ, does not contain lysines. The 6XHIS sequence was added followed by alternation of the REDL sequence by the KDEL, C-terminus ER retention tetrapeptide sequence Lys-Asp-Glu-Leu, resulting in the pET22b-ZZ-PE38-QQ-HIS-KDEL vector. (B) 20 μg purified protein was loaded in duplicates on a 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. The membrane was probed with horseradish peroxidase−conjugated goat anti-mouse antibody (right panel) for the detection of the antibody components (heavy and light chains) or with mouse anti-HIS monoclonal antibody (mAb) for detection of PE38 (left panel). (C) HT-29 cells were incubated with serial dilutions of immunotoxin (diamond) and native mAb (squares). Error bars represent standard deviations of 4 different measurements. The binding affinity was estimated as the IgG or immunocomplex concentration that generates 50% of the maximal signal. Gastroenterology 2011 140, 935-946DOI: (10.1053/j.gastro.2010.12.004) Copyright © 2011 AGA Institute Terms and Conditions

Figure 6 Evaluation of the immunotoxin cytotoxicity by protein synthesis inhibition assay. (A) The cytotoxic activities of the PE38-based immunotoxins were evaluated by inhibition of protein synthesis as measured by the [3H]-Leucine incorporation on target (HT-29) and nontarget (HCT116) cells. The assay was carried out twice in tetraplicates. The IC50 values are indicated by broken lines and the “therapeutic window” is indicated by bidirectional arrows. (B) Examination of the immunocomplex components. Gastroenterology 2011 140, 935-946DOI: (10.1053/j.gastro.2010.12.004) Copyright © 2011 AGA Institute Terms and Conditions

Figure 7 Animal toxicity of the immunocomplexes and inhibition of tumor growth in mice. (A) Various concentrations of immunotoxins were diluted in phosphate-buffered saline and 0.2 mL aliquots were injected intraperitoneally (ip) to female BALB/c mice (n = 3). Animals were monitored for 12 days for death and weight changes. The weight curve represents the average weight of the mice in each group. (B, C) Tumors were formed in nude mice by subcutaneous injection of 5 × 106 HT-29 cells on day 0 and were treated with SWA11-ZZ-PE38 (0.75 mg/kg), ip, twice weekly. (B) Tumor size was measured at the indicated time points and tumor volumes were calculated. The mean values for each group are shown and the standard deviation is represented by error bars at each measurement. P values are indicated here. (C) Representative pictures of mice from control (left panel) and immunotoxin treatment (right panel) groups. Gastroenterology 2011 140, 935-946DOI: (10.1053/j.gastro.2010.12.004) Copyright © 2011 AGA Institute Terms and Conditions