The Characterization of Anti-HER-2/neu Monoclonal Antibody using Different in vivo Imaging Techniques

Abstract

Recently, specific antibodies have been used extensively to diagnose and treat various diseases. It is essential to assess the efficacy and specificity of antibodies, especially the in vivo environment. Anti-HER-2/neu mAb was evaluated as a possible transporting agent for radioimmunotherapy. The monoclonal antibody was successfully radio-labeled with 131 I. In vitro binding assays were performed to confirm its targeting ability using another radio-iodine, 125 I. Binding percentage of 125 I labeled anti-HER-2/neu mAb in HER-2/neu expressing CT-26 cells was found to be 4.5%, whereas the binding percentage of 125 I labeled anti-HER-2/neu mAb in wild-type CT-26 was only 0.45%. In vivo images were obtained and analyzed through γ-camera and an optical fluorescent modality, IVIS-200. γ-camera images showed that 131 I labeled anti-HER-2/neu mAb accumulated in HER-2/neu CT-26 tumors. Optical imaging based on near infrared fluorescence labeled anti-HER-2/neu mAb showed higher fluorescence intensities in HER-2/neu CT-26 tumors than in wild-type CT-26 tumors. Anti-HER-2/neu mAb was found to specifically bind to its receptor expressing tumor. Our study demonstrates that in vivo imaging technique is a useful method for the evaluation of an antibody's therapeutic and diagnostic potentials.

Efficient and Site-Specific 125I-Radioiodination of Bioactive Molecules Using Oxidative Condensation Reaction

“…The crude product was purified using a silica gel column (ethyl acetate/hexane = 1:2) to give di-tert-butyl-(4hydroxy-1,2-phenylene)dicarbamate (2.2 g, 6.79 mmol, 52%) as a white solid. 1 To the mixture of intermediate precursor 1 (2.0 g, 6.17 mmol) and methyl 5bromovalerate (1.7 g, 9.2 mmol) in 20 mL of DMF, potassium carbonate (K 2 CO 3 ) (2.5 g, 18.51 mmol) was added gradually. The reaction was carried out at 25 °C for 1 day.…”

“…The crude product was then dried using anhydrous sodium sulfate (Na 2 SO 4 ), concentrated under a reduced pressure, and purified by using a silica gel column (ethyl acetate/hexane = 1:4) to give 2 as a brown oil (1.9 g, 4.33 mmol, 70%). 1 H NMR (500 MHz, CDCl 3 ): δ 7.137 (br, 1H), 6.904 (br, 1H), 6.574 (dd, J 1 = 8.5 Hz, J 2 = 2.5 Hz, 1H), 3.928 (t, J = 6.6 Hz, 2H), 3.65 (s, 3H), 2.369 (t, J = 7.3 Hz, 2H), 1.70−1.78 (m, 6H), 1.495 (s, 9H) and 1.482 (s, 9H); 13 2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-5-oxopentyl)oxy)-1,2-phenylene)dicarbamate 3. To the water/dioxane mixture (1:3, 4.8 mL), intermediate precursor 2 (1.5 g, 3.4 mmol) and lithium hydroxide (287 mg, 11.98 mmol) were added at 0 °C.…”

“…The crude mixture was purified using a silica gel column (ethyl acetate/hexane 2.5:1) to give compound 3 as a yellow oil (1 g, 1.8 mmol, 52.94%). 1 H NMR (500 MHz, CDCl 3 ): δ 7.138 (br, 1H), 6.929 (br, 1H), 6.687 (s, 2H), 5.951 (dd, J 1 = 8.5 Hz, J 2 = 2.5 Hz, 1H), 3.928 (t, J = 6.6 Hz, 2H), 3.669 (t, J = 6.5 Hz, 2H), 3.446 (m, 2H), 2.169 (t, J = 7.3 Hz, 2H), 1.668−1.862 (m, 6H), 1.498 (s, 9H) and 1.488 (s, 9H); 13 Synthesis of 5-(3,4-Diaminophenoxy)-N-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)pentanamide 4. Intermediate compound 3 (100 mg, 0.18 mmol) was stirred in the mixture of trifluoroacetic acid (2 mL) and dichloromethane (2 mL) for 3 h at an ambient temperature.…”

“…Among the various available radioisotopes, radioiodine is the most extensively used for radiolabeling of biologically active macro and micromolecules. 1 − 3 Radioiodine can be used for positron emission tomography (PET) imaging ( 124 I), 4 singlephoton emission computed tomography (SPECT) imaging ( 123/125 I), 5 , 6 treatment of various cancers ( 131 I), 7 and pharmacokinetics and biodistribution studies of newly designed therapeutic drugs ( 125 I). 8 − 10 Several methodologies have been reported in the literature for the efficient and chemoselective radioiodination of biomolecules.…”

“…Among the various available radioisotopes, radioiodine is the most extensively used for radiolabeling of biologically active macro and micromolecules. − Radioiodine can be used for positron emission tomography (PET) imaging ( 124 I), singlephoton emission computed tomography (SPECT) imaging ( 123/125 I), , treatment of various cancers ( 131 I), and pharmacokinetics and biodistribution studies of newly designed therapeutic drugs ( 125 I). − Several methodologies have been reported in the literature for the efficient and chemoselective radioiodination of biomolecules. , The most frequently used strategy is direct radioiodination via the tyrosine group in the selected peptides and proteins by using some strong oxidizing agents . The use of an electrophilic aromatic substitution reaction on the tyrosine group provides a high radiochemical yield within a short time .…”