Trastuzumab complexed to near-infrared fluorophore indocyanine green

[Tra-ICG]

Background

[PubMed]

The technique of non-invasive optical imaging is based on the principle that when a dye is exposed to light it absorbs energy and is elevated from the steady state to a high-energy state. To return to its normal, low-energy state, the molecule releases energy in the form of photons with wavelengths that are either in the visible (400–700 nm) or the near infrared (NIR; 700–900 nm) range (1). Although both types of dyes can be used for imaging, in general, NIR agents are favored over those functional in the visible range because fluorescence from NIR probes can be detected in deep tissue, have low autofluorescence, and generate low background signals because tissues do not absorb in the NIR wavelength range (2). Among the various NIR agents, only one, indocyanine green (ICG; also known as IR-125 or Cardiogreen), with absorption at ~780 nm and emission at ~820 nm, is approved by the United States Food and Drug Administration (US FDA) for clinical applications (3) and is under evaluation in several clinical trials for other applications. ICG is a tricarbocyanine dye that is fluorescent only in the free state and easily binds to proteins through non-covalent linkages (such as ionic, hydrophobic, electrostatic, or hydrogen bonds). When bound to proteins ICG is non-fluorescent, but on dissociation from the protein complex it reverts to the fluorescent state and the signal has been shown to be suitable for in vivo imaging in small animals (4).

The human epidermal growth factor receptor (HER; it has several isoforms: HER1, HER2, HER3, and HER4) plays an important role in the development and progression of cancers (5-7). The HER mediates its activity through a tyrosine kinase signaling pathway, and on binding to a ligand the receptor signaling pathway is activated, which promotes cell growth and division. Once activated the receptor-ligand complex is internalized by the cell for enzymatic digestion in the lysosome (8). Because of its role in the cancer process, several commercially available humanized monoclonal antibodies (MAb) directed toward HER, including trastuzumab (Tra), are approved by the US FDA to treat cancers. Tra targets the HER2 receptor and is used as the primary agent or as an adjuvant for the treatment of breast cancer in the clinical setting (9). It is also being investigated in several clinical trials to treat other neoplasms.

The characteristic properties of ICG (fluorescence in an unbound state) and the HER (internalization by the cell) prompted Ogawa et al. to develop an imaging probe for cancer by complexing ICG to Tra (Tra-ICG) (4). The investigators used the Tra-ICG complex to image cells that express the HER2 receptor under in vitro conditions and showed that the complex could also be used for the imaging of HER2-expressing xenograft tumors in mice.

Synthesis

[PubMed]

The synthesis of Tra-ICG was described by Ogawa et al. (4). ICG-N-hydroxysulfosuccinimide ester (ICG-sulfo-OSu) and Tra were obtained from commercial sources. Tra was incubated with ICG-sulfo-OSu (Tra:ICG ratio was either 1:1 or 1:5) in phosphate buffer (pH 8.5) for 30 min at room temperature. The Tra-ICG complex was purified on a PD-10 Sephadex G50 column with phosphate-buffered saline (PBS; pH not reported) as the elution buffer. With the reaction conditions used to generate the MAb-ICG complex, either one or five ICG molecules (Tra-ICG(1:1) or Tra-ICG(1:5), respectively) were reported to be bound to each Tra molecule, respectively. To determine specificity of the Tra-ICG complex, another MAb, panitumumab (Pan), which specifically binds to HER1, was complexed with ICG (Pan-ICG) and purified as described above.

For use as a control, a human polyclonal IgG-Cy5.5 complex was also synthesized in the manner described above. The storage conditions and stability of the MAb-ICG complexes were not reported.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Fluorescence imaging with the Tra-ICG complex was investigated in HER2 gene-transfected NIH3T3 (3T3/HER2+) cells (4). The cells were grown on a glass-bottomed tissue culture well and exposed to the Tra-ICG(1:1) or Tra-ICG(1:5) complex for 1 and 8 h at 37^oC. The cells were then washed with PBS and examined under a fluorescent microscope. No fluorescence was observed in cells exposed to Tra-ICG(1:1) at either time point. Fluorescence was apparent with both complexes at 8 h, indicating that the signal was obtained only after internalization of the MAb-ICG complex followed by degradation of the MAb and release of the ICG.

Animal Studies

Human Studies

[PubMed]

No references are currently available.