Anti-c-Met monoclonal antibody linked to biotinylated bovine serum albumin and gadolinium

[Anti-c-Met-Gd-albumin]

Background

[PubMed]

Gliomas and glioblastoma multiforme are characterized as highly infiltrative and invasive forms of brain cancer that may be treated with either surgical resection, chemotherapy, radiotherapy, or a combination of these modalities (1). However, with a median survival time of less than 18 months, the prognosis for the patient is poor regardless of the option used to treat this cancer (2). The tyrosine kinase receptor c-Met plays an important role in invasive cell growth and motility during embryogenesis and is a tumor marker for several cancers including gliomas (3, 4). c-Met is a transmembrane glycoprotein (Mw = 190 kD) that consists of a β subunit and an extracellular α subunit that binds and is activated by the hepatocyte growth factor (HGF) (5). For individuals with malignant meningiomas, overexpression of HGF correlates with a poor prognosis, and this factor has also been used to predict tumor recurrence (6). Because of their role in the development of brain tumors, both c-Met and HGF are considered important markers to predict angiogenesis and tumor growth (7). Several clinical trials are in progress to develop suitable treatment(s) for these brain tumors.

Towner et al. envisioned that early detection of glioma tumors would perhaps improve treatment outcomes for the patients (8). They used an anti-c-Met monoclonal antibody (MAb) linked to gadolinium (through diethylenetriaminepentaacetic acid (DTPA)) and biotinylated bovine serum albumin (anti-c-Met-Gd-biotinylated BSA) to specifically target c-Met. The MAb linked to the contrast agent (CA) was then used to visualize the glioma tumors with magnetic resonance imaging (MRI) in rats bearing C6 cell glioma tumors in the brain. Biotinylated BSA was also used to facilitate detection of the CA with fluorescence staining of cryosections of brain tissue (8).

Synthesis

[PubMed]

The commercially available MAb used for this study was directed toward the extracellular β chain of the c-Met antigen (8).

Synthesis of the MRI CA was performed as described by Towner et al. (8). Biotinylation of the BSA was performed using a modification of the method of Dafni et al. (9). The conjugation of DTPA to biotinylated BSA was done in Hepes buffer (pH 8.8) by the addition of small amounts of DTPA suspended in dimethyl sulfoxide at room temperature, followed by adjustment of the reaction mixture with sodium hydroxide to pH 8.5. The mixture was stirred for 2 h at 4°C. The product was dialyzed against several changes of citrate buffer (pH 6.5), and gadolinium(III) chloride in acetate buffer (pH 6.0) was gradually added to the dialyzed compound. The mixture was incubated for 24 h at 4°C, and the resultant product, biotin-BSA-Gd-DTPA, was dialyzed against citrate buffer (pH 6.5) and double-distilled water. Biotin-BSA-Gd-DTPA was then freeze-dried and stored until required (storage conditions were not provided).

The anti-c-Met MAb was linked to biotin-BSA-Gd-DTPA through an N-hydroxysulfosuccinimide (sulfo-NHS) and 1-ethyl-3-[3-dimethylamino-propyl]carbodiimide hydrochloride (EDC) link between the MAb and the biotinylated BSA using the Hermanson's method (10). To obtain anti-c-Met-biotin-BSA-Gd-DTPA, the sulfo-NHS was reacted with EDC for 15 min at room temperature, and a solution of the anti-Met MAb was added to the mixture. The reaction proceeded for 2 h at room temperature in the dark. The final product, anti-c-Met-biotin-BSA-Gd-DTPA, was freeze-dried and stored at 4°C until required. The number of biotin and Gd-DTPA molecules linked to the BSA was not reported (8).

For use as a control, commercially available normal rat IgG was linked to biotin-BSA-Gd-DTPA as detailed above (8). The purity and stability of the various CA prepared and used in the reported studies was not reported (8).

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Towner et al. used indirect immunofluorescence to confirm the overexpression of c-Met in rat glioma tissue compared with normal brain tissue (8). Cryosections of the brain tissue were incubated with a rabbit anti-c-Met antibody, followed by treatment with a donkey anti-rabbit IgI-Cy3 antibody in phosphate-buffered saline. Glioma and normal tissue were also obtained from animals treated in vivo with anti-c-Met-biotin-BSA-Gd-DTPA, IgG-biotin-BSA-Gd-DTPA, or biotin-BSA-Gd-DTPA. The various tissues were then stained with streptavidin-labeled IgG linked to the Cy3 fluorescent dye. The stained sections were viewed under a confocal laser-scanning microscope. Compared with the normal tissue immunofluorescence, an enhanced signal was detected only in the glioma tissue, indicating an overexpression of c-Met in these cells. This observation was confirmed with histological immunofluorescence of brain tissue obtained after MRI of animals 3 h after treatment with the anti-c-Met-biotin-BSA-Gd-DTPA or anti-IgG-biotin-BSA-Gd-DTPA antibodies, or with biotin-BSA-Gd-DTPA, respectively, as described above (8).

Presence of higher levels of c-Met in the glioma tissues, compared with normal tissues, was confirmed with Western blot analysis (8).

Animal Studies

Human Studies

[PubMed]

No references are currently available.

Supplemental Information

[Disclaimers]

NIH Support

Supported by National Institutes of Health grant number 5R03CA121359-2.