Results: 5

1.
FIGURE 5.

FIGURE 5. From: Convergent Potency of Internalized Gelonin Immunotoxins across Varied Cell Lines, Antigens, and Targeting Moieties.

Aggregated data from experiments using different binding scaffolds, antigen targets, affinities, and cell lines all converge to the same TN50 curve. Shown are the accumulated internalized cytotoxicity data for rGel on all cell lines and immunotoxins on high and low antigen-expressing cells. The cumulative data set was fit using an exposure-response curve with variable slopes giving a TN50 of 4.7 × 106.

Christopher M. Pirie, et al. J Biol Chem. 2011 February 11;286(6):4165-4172.
2.
FIGURE 3.

FIGURE 3. From: Convergent Potency of Internalized Gelonin Immunotoxins across Varied Cell Lines, Antigens, and Targeting Moieties.

Correlated internalization and cytotoxicity measurements indicate that a precise number of rGel molecules must be internalized by a single cell before cytotoxicity is observed. A, time and concentration dependence of rGel internalization by HT-1080 cells using the described quantitative internalization flow cytometry assay. B, concentration- and exposure-matched cytotoxicity was measured using the WST-1 assay. C, data from A and B were combined and plotted to show the dependence of cytotoxicity on the number of internalized gelonin molecules, resulting in the determination of the TN50 near 5 × 106.

Christopher M. Pirie, et al. J Biol Chem. 2011 February 11;286(6):4165-4172.
3.
FIGURE 1.

FIGURE 1. From: Convergent Potency of Internalized Gelonin Immunotoxins across Varied Cell Lines, Antigens, and Targeting Moieties.

Antigen affinity of binding fragments is retained in their respective immunotoxin constructs. The four immunotoxins target either CEA or EGFR via either scFv or Fn3. The 3E and FE scFv clones targeting CEA, the C7 fibronectin clone targeting CEA, and the E4 fibronectin clone targeting EGFR were each fused to rGel and titrated for binding affinity on HT-1080(CEA) or A431 cells for CEA or EGFR, respectively. Binding on fixed cells was detected with goat anti-biotin-FITC antibody by flow cytometry. Data were fitted using least-squares regression with a binding isotherm, giving Kd values of 8 nm for 3ErGel, 15 nm for FErGel, 10 nm for C7rGel, and 13 nm for E4rGel.

Christopher M. Pirie, et al. J Biol Chem. 2011 February 11;286(6):4165-4172.
4.
FIGURE 4.

FIGURE 4. From: Convergent Potency of Internalized Gelonin Immunotoxins across Varied Cell Lines, Antigens, and Targeting Moieties.

Rate-limiting toxicity step measured in rGel is similarly observed in scFv- and Fn3-targeted immunotoxins using antigen-dependent internalization. A, the internalization of immunotoxins by antigen-positive cells was measured by the quantitative internalization flow cytometry assay at varying times and concentrations. All HT-29 cells treatments were made at 30 nm, as were the C7rGel and E4rGel treatments on HT-1080(CEA) and A431 cells, respectively, whereas the 3ErGel and FErGel treatments on HT-1080(CEA) cells were made at 10 nm. For all treatments, strictly antigen-dependent internalization is reported by subtracting signal from cells blocked with an unlabeled competitor. B, concentration- and exposure-matched cytotoxicity was measured using the WST-1 assay. C, data from A and B were combined and plotted to show the dependence of cytotoxicity on the number of internal immunotoxins, resulting in the determination of the TN50 near 3 × 106 for all species.

Christopher M. Pirie, et al. J Biol Chem. 2011 February 11;286(6):4165-4172.
5.
FIGURE 2.

FIGURE 2. From: Convergent Potency of Internalized Gelonin Immunotoxins across Varied Cell Lines, Antigens, and Targeting Moieties.

Fusion of scFv and Fn3 binding domains to rGel leads to enhanced cytotoxicity specific for antigen-positive cells. A, using the WST-1 assay, the cytotoxicity of soluble rGel was tested on all four cell lines used in the study. Across all cell lines, rGel showed an IC50 of ∼500 nm. B, antigen-negative cells (HT-1080) were treated with the four different immunotoxins that displayed roughly equivalent potency to soluble toxin. C, immunotoxins were also tested for cytotoxicity on the double-positive, low-antigen density HT-29 cell line. Surprisingly, none of the immunotoxins showed enhanced cytotoxicity compared with the IC50 of rGel. D, on high antigen-expressing cells (HT-1080(CEA) and A431), significantly greater potency was observed for the immunotoxins compared with the soluble toxin. Against cells expressing their respective antigen targets, they had IC50 values of 250 pm for 3ErGel, 1.5 nm for FErGel, 8 nm for C7rGel, and 30 nm for E4rGel.

Christopher M. Pirie, et al. J Biol Chem. 2011 February 11;286(6):4165-4172.

Supplemental Content

Recent activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...
Write to the Help Desk