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1 SUPPLEMENTAL MATERIALS AND METHODS Western Blotting Tumor tissues or cells were homogenized and proteins were extracted using T-PER tissue protein extraction buffer. Protein concentrations were determined using a microbca protein assay kit. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transfer, polyvinylidene fluoride (PVDF) membranes were blocked using 5% nonfat milk blocking buffer, and incubated overnight at 4 C with anti-egfr primary antibody (1:1000; Bioworld). Membranes were then incubated at room temperature for 1 h with horseradish peroxidase-conjugated secondary antibodies (GE Healthcare). β-actin was used as the loading control, and bands were detected using enhanced chemiluminescent western blotting detection system. Immunoreactivity Comparison The EGFR immunoreactivity of PaniF(ab') 2 was compared with that of panitumumab by competition binding assay using 125 I-PaniF(ab') 2 as the radioligand. 125 I-PaniF(ab') 2 was generated by radiolabeling PaniF(ab') 2 with Na 125 I using the Iodogen method described previously (1). HT-29 cells were incubated with 125 I-PaniF(ab') 2 in the presence of increasing concentrations of panitumumab and PaniF(ab') 2 in 96-well filter plates. After a 2 h incubation at 4 C, the plates were washed using phosphate-buffered saline (PBS), and the PVDF filters were collected and measured in a γ-counter. Data were fitted with nonlinear regression using
2 GraphPad Prism 4.0 (GraphPad Software, San Diego, CA) (n = 4). Preparation of DyLight680-PaniF(ab') 2 Conjugate PaniF(ab') 2 was mixed with DyLight680-NHS (10 mg/ml in dimethylformamide) in bicarbonate buffer (ph = 9.0) at a 7:1 molar ratio of DyLight680-NHS:PaniF(ab') 2. After incubation at room temperature for 1 h, the DyLight680-NHS-PaniF(ab') 2 conjugate, named Dye-PaniF(ab') 2, was purified using a PD-10 desalting column (GE Healthcare). The degree of labeling (dye:protein ratio) for Dye-PaniF(ab') 2 was calculated to be ~3.6 based on UV measurements of A 280 and A 684. Preparation of NOTA-PaniF(ab') 2 Conjugate and 68 Ga Radiolabeling p-scn-bn-nota (Macrocyclics) was added to PaniF(ab') 2 at the molar ratio of p-scn-bn-nota/panif(ab') 2 = 200 : 1 in bicarbonate butter (ph = 9.0). After incubating overnight at 4 C, the NOTA-PaniF(ab') 2 conjugate was purified using a PD-10 column, and concentrated with a Centricon filter (Millipore). The number of NOTA per PaniF(ab') 2 was determined using a previously reported method (2), and it was calculated to be ~6. For 68 Ga radiolabeling, 370 MBq of 68 Ga ( 68 Ge/ 68 Ga generator; Obninsk) was diluted in 300 μl of 0.2 M sodium acetate buffer (ph 5.5) and added to 20 μg of NOTA-PaniF(ab') 2. The reaction mixture was incubated for 10 min at 39 C with constant shaking. 68 Ga-PaniF(ab') 2 was then purified on a PD-10 column using PBS as the mobile phase.
3 Near-infrared Optical Imaging and Blocking Studies The in vivo tumor targeting specificity of Dye-PaniF(ab') 2 was determined using a small-animal near-infrared imaging study. HT-29 tumor-bearing nude mice were injected intravenously (i.v.) with 0.5 nmol of Dye-PaniF(ab') 2 with or without 1 mg of unlabeled panitumumab for blocking (4 h prior to Dye-PaniF(ab') 2 injection). At 4 h postinjection of Dye-PaniF(ab') 2, optical imaging was performed using the same procedure described in the main text (n = 4 per group). Immunofluorescence Staining The EGFR expression levels in HT-29 tumor tissues in the presence or absence of trastuzumab treatment were determined using fluorescence staining. A fluorescein isothiocyanate (FITC)-panitumumab conjugate was generated by conjugating the EGFR-specific antibody panitumumab to FITC-NHS (Pierce), as described previously (3). The tumor sections were incubated with FITC-panitumumab (2 μg/ml) with or without a blocking dose of unlabeled panitumumab (20 μg) for 1 h at 37 C. After washing with PBS, the samples were visualized using confocal microscopy. Cross-reaction of Panitumumab and Trastuzumab A cell competition binding assay was performed to determine whether panitumumab and trastuzumab cross-reacted. Panitumumab and trastuzumab were radiolabeled with Na 125 I using the Iodogen method described previously (1). The immunoreactivities of panitumumab and trastuzumab to EGFR and HER2 were
4 evaluated using a whole-cell displacement assay as described above. Briefly, HT-29 cells were incubated with 125 I-panitumumab or 125 I-trastuzumab in the presence of increasing concentrations of panitumumab and trastuzumab in 96-well filter plates. After a 2 h incubation at 4 C, the plates were washed, and the PVDF filters were collected and measured in a γ-counter. Data were fitted with nonlinear regression using GraphPad Prism 4.0 (GraphPad Software, San Diego, CA) (n = 4). Biodistribution of 125 I-Panitumumab The A549 tumor-bearing nude mouse model was established by inoculating tumor cells subcutaneously into the right front flanks of female BALB/c nude mice. The animals were used for biodistribution study when the tumor size reached mm 3. Each mouse was injected intraperitoneally daily with trastuzumab (200 μg per mouse) or PBS for 5 days (n = 4 per group). After treatment, each mouse was injected i.v. with 370 kbq of 125 I-panitumumab. At 24 h postinjection, animals were sacrificed, and tissues and organs were harvested, weighed, and counted in a γ-counter. The results were presented as percentage of injected dose per gram of tissue (%ID/g). REFERENCES 1. Liu Z, Yu Z, He W, Ma S, Sun L, Wang F. In-vitro internalization and in-vivo tumor uptake of anti-egfr monoclonal antibody LA22 in A549 lung cancer cells and animal model. Cancer Biother Radiopharm. 2009;24:15-24.
5 2. Liu Z, Liu Y, Jia B, et al. Epidermal growth factor receptor-targeted radioimmunotherapy of human head and neck cancer xenografts using 90 Y-labeled fully human antibody panitumumab. Mol Cancer Ther. 2010;9: Liu Z, Jia B, Zhao H, Chen X, Wang F. Specific targeting of human integrin alphavbeta3 with 111 In-labeled Abegrin in nude mouse models. Mol Imaging Biol. 2011;13:
6 Supplemental Figure 1. SDS-PAGE analysis of panitumumab (Pani) and PaniF(ab') 2.
7 Supplemental Figure 2. The EGFR immunoreactivity of PaniF(ab') 2 was compared with that of panitumumab (Pani) by competition binding assay using 125 I-PaniF(ab') 2 as the radioligand. The IC 50 values for Pani and PaniF(ab') 2 were (0.13 ± 0.03) nm and (0.20 ± 0.01) nm, respectively.
8 Supplemental Figure 3. Representative optical images (A) and quantified tumor uptake (B) in HT-29 tumor-bearing mice at 4 h postinjection of 0.5 nmol Dye-PaniF(ab') 2 (with or without panitumumab blocking). **, P < 0.01.
9 Supplemental Figure 4. EGFR staining (with or without blocking with panitumumab) of control and trastuzumab-treated HT-29 tumor tissues.
10 Supplemental Figure 5. Competition binding assays. (A) The inhibition of 125 I-panitumumab ( 125 I-Pani) binding to EGFR in HT-29 cells by panitumumab (Pani) and trastuzumab (Tras). (B) Inhibition of 125 I-trastuzumab ( 125 I-Tras) binding to HER2 in HT-29 cells by panitumumab (Pani) and trastuzumab (Tras).
11 Supplemental Figure 6. Biodistribution of 125 I-panitumumab (24 h postinjection) in A549 tumor-bearing nude mice after daily treatment with trastuzumab (200 μg per mouse) or PBS for 5 days. **, P < 0.01.
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