Stabilization of an 211At-Labeled Antibody with Sodium Ascorbate

Abstract

²¹¹At, an α-particle emitter, has recently attracted attention for radioimmunotherapy of intractable cancers. However, our sodium dodecyl sulfate polyacrylamide gel electrophoresis and flow cytometry analyses revealed that ²¹¹At-labeled immunoconjugates are easily disrupted. Luminol assay revealed that reactive oxygen species generated from radiolysis of water caused the disruption of ²¹¹At-labeled immunoconjugates. To retain their functions, we explored methods to protect ²¹¹At-immunoconjugates from oxidation and enhance their stability. Among several other reducing agents, sodium ascorbate most safely and successfully protected ²¹¹At-labeled trastuzumab from oxidative stress and retained the stability of the ²¹¹At-labeled antibody and its cytotoxicity against antigen-expressing cells for several days.

Figure 1

Sodium dodecyl sulfate–polyacrylamide gel (SDS–PAGE) and autoradiography for ²¹¹At-labeled trastuzumab. SA concentrations (mg/mL) are indicated. SDS–PAGE and autoradiography were performed to determine the effects of SA on the stability of the immunoconjugate. (a) Polyacrylamide gel on day 0 and (b) autoradiograph of ²¹¹At-labeled trastuzumab on day 0 and (c) polyacrylamide gel after 1 d and (d) ²¹¹At-labeled trastuzumab after 1 d.

Figure 2

Flow cytometry analysis of the binding activity of ²¹¹At-labeled trastuzumab to human epidermal growth factor receptor 2 (HER2)-expressing cells. ²¹¹At-labeled trastuzumab with or without SA at the indicated concentrations facilitated binding with breast cancer cell lines having different HER2 expression levels. SK-BR-3: high HER2 expression; MCF-7: low HER2 expression. Sn-trastuzumab is N-[2-(maleimido)ethyl]-3-(trimethylstannyl)benzamide-conjugated trastuzumab. Negative control is a sample incubated with only the secondary antibody. The flow cytometry analysis was performed 6 d after ²¹¹At labeling.

Figure 3

Cytotoxic effects of ²¹¹At-labeled trastuzumab in breast cancer cell lines. The cytotoxic effects of ²¹¹At-labeled trastuzumab with and without SA on breast cancer cell lines with different expression levels of human epidermal growth factor receptor 2 (HER2) were determined using the WST-8 cell count assay. SK-BR-3: high HER2 expression; MCF-7: low HER2 expression. N = 4. Data are presented as mean ± standard deviation (SD) values.

Figure 4

Detection of ROS using luminol assay. (a) SA was added at different concentrations to ²¹¹At in PBS. The lower (boxed) graph is an expansion of the 6 mg/mL SA addition protocol. (b) SA was added at different concentrations to ²¹¹At-labeled trastuzumab in PBS. The lower (boxed) graphs are expansions of 6 × 10^–2 and 6 mg/mL SA addition protocols. RLU = relative luminescence units. Data are presented as mean ± SD values.

Figure 5

Quenching potential of different reducing agents for ROS in ²¹¹At or ²¹¹At-labeled trastuzumab solutions, as assessed using the luminol assay. (a) Reducing agents (6 × 10^–2 mg/mL) were added to ²¹¹At in PBS. (b) Reducing agents (6 × 10^–2 mg/mL) were added to ²¹¹At-labeled trastuzumab in PBS.

Figure 6

(a) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) of Sn- and ²¹¹At-labeled trastuzumab (IgG) in the presence of various reducing agents. Concentration of each reducing agent is 6 × 10^–2 mg/mL. SDS–PAGE analysis was performed 4 d after ²¹¹At labeling. (b) Flow cytometry analysis of the binding activity of ²¹¹At-labeled trastuzumab to different breast cancer cell lines in the presence of various reducing agents. Cell lines with high (SK-BR-3) and low (MCF-7) human epidermal growth factor receptor 2 (HER2) expression levels were treated with ²¹¹At-labeled trastuzumab in the presence of various reducing agents. Flow cytometry analysis was performed 6 d after ²¹¹At labeling. SA: sodium ascorbate; Cys: l-cysteine; SHS: sodium hydrosulfite; Mal: maltose. Concentrations of reducing agents are 6 × 10^–2 mg/mL. RLU = relative luminescence units.

Figure 7

Cytotoxic effects of ²¹¹At-labeled trastuzumab in breast cancer cell lines. The cytotoxic effects of ²¹¹At-labeled trastuzumab in the presence of reducing agents,SA and l-cysteine, and on breast cancer cell lines with different expression levels of human epidermal growth factor receptor 2 (HER2) were determined using the WST-8 assay. SK-BR-3: high HER2 expression; MCF-7: low HER2 expression. SA: sodium ascorbate; Cys: l-cysteine; SHS: sodium hydrosulfite; Mal: maltose. N = 4. Data are presented as mean ± SD values.