Synthesis and Preliminary Biological Evaluations of Fluorescent or 149Promethium Labeled Trastuzumab-Polyethylenimine

Abstract

Background: Radioimmunotherapy utilize a targeting antibody coupled to a therapeutic isotope to target and treat a tumor or disease. In this study we examine the synthesis and cell binding of a polymer scaffold containing a radiotherapeutic isotope and a targeting antibody.

Methods: The multistep synthesis of a fluorescent or ¹⁴⁹Promethium-labeled Trastuzumab-polyethyleneimine (PEI), Trastuzumab, or PEI is described. In vitro uptake, internalization and/or the binding affinity to the Her2/neu expressing human breast adenocarcinoma SKBr3 cells was investigated with the labeled compounds.

Results: Fluorescent-labeled Trastuzumab-PEI was internalized more into cells at 2 and 18 h than fluorescent-labeled Trastuzumab or PEI. The fluorescent-labeled Trastuzumab was concentrated on the cell surface at 2 and 18 h and the labeled PEI had minimal uptake. DOTA-PEI was prepared and contained an average of 16 chelates per PEI; the compound was radio-labeled with ¹⁴⁹Promethium and conjugated to Trastuzumab. The purified ¹⁴⁹Pm-DOTA-PEI-Trastuzumab had a radiochemical purity of 96.7% and a specific activity of 0.118 TBq/g. The compound demonstrated a dissociation constant for the Her2/neu receptor of 20.30 ± 6.91 nM.

Conclusion: The results indicate the DOTA-PEI-Trastuzumab compound has potential as a targeted therapeutic carrier, and future in vivo studies should be performed.

Keywords: Actinium-225; Promethium-149; Trastuzumab; polyethylenimine; radiotherapy; targeted therapy.

Figure 1

Synthesis scheme used to make PEI-OG, Trastuzumab-OG, and Trastuzumab-PEI-OG. (1) and (2) NaHCO₃ pH = 8.3, (3) PBS pH = 7.4, 5 h, (4) PBS/0.005 M EDTA (pH = 8.0) 30 min, and (5) PBS/0.005 M EDTA (pH = 7.4) 6 h.

Figure 2

Synthetic scheme used to make ¹⁴⁹Pm-DOTA-PEI-Trastuzumab. Reaction conditions: (1) NaHCO₃ pH = 8.5, (2) PBS, RT, 1 h, (3) NH₄Ac pH = 6.0, 80 °C, 1 h, (4) NaHCO₃ pH = 8.5, EDTA, RT, 1 h, and (5) PBS, 3 h, RT.

Figure 3

Confocal microscope images of SKBr3 cells incubated for 2 and 18 h with (A,D) Trastuzumab-PEI-OG, (B,E) Trastuzumab-OG, and (C) PEI-OG. The Trastuzumab-PEI-OG was visible inside the cell (A and D arrows), and Trastuzumab-OG was visible around the cells (B and E arrows).

Figure 4

Dissociation constant data (Kd) for ¹⁴⁹Pm-DOTA-PEI-Trastuzumab with human breast adenocarcinoma SkBr-3 cells (n = 3).

Figure 5

Total, nonspecific and specific binding for ¹⁴⁹Pm-DOTA-PEI-Trastuzumab with human breast adenocarcinoma SkBr-3 cells (n = 3).