Cancer Biology & Therapy Molecular imaging of gefitinib activity in an ...

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Molecular imaging of gefitinib activity in an epidermal growth factor receptor (EGFR)-bearing xenograft model Jing Gong, David J. Yang, Saady Kohanim, Laura S. Angelo & Razelle Kurzrock Published online: 01 Dec 2009.

To cite this article: Jing Gong, David J. Yang, Saady Kohanim, Laura S. Angelo & Razelle Kurzrock (2009) Molecular imaging of gefitinib activity in an epidermal growth factor receptor (EGFR)-bearing xenograft model, Cancer Biology & Therapy, 8:23, 2237-2245, DOI: 10.4161/cbt.8.23.9986 To link to this article: http://dx.doi.org/10.4161/cbt.8.23.9986

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Research Paper

Research paper

Cancer Biology & Therapy 8:23, 2237-2245; December 1, 2009; © 2009 Landes Bioscience

Molecular imaging of gefitinib activity in an epidermal growth factor receptor (EGFR)-bearing xenograft model Jing Gong,1 David J. Yang,2 Saady Kohanim,2 Laura S. Angelo1 and Razelle Kurzrock1,* Department of Investigational Cancer Therapeutics (Phase I Program); Division of Cancer Medicine; and 2Department of Experimental Diagnostic Imaging; U.T.M.D. Anderson Cancer Center; Houston, TX USA

1

Downloaded by [Mohaghegh Ardebili University] at 07:56 02 September 2015

Key words: EGFR, anti-phospho-tyrosine antibody, gefitinib, imaging Abbreviations: DMSO, dimethyl sulfoxide; EC, ethylenedicysteine; EGFR, epidermal growth factor receptor; 111In-EC-P-Tyr, indium-labeled phospho-tyrosine antibody; PET, positron emission tomography; SPECT, single photon emission-computed tomography; T/M, tumor/muscle ratio

Finding noninvasive methods to discern which patients’ tumors bear a specific target molecule, and are presumably more likely to respond, remains a critical challenge. An anti-phospho-tyrosine antibody was labeled with indium (111In) using ethylenedicysteine (EC) as a chelator (111In-EC-P-Tyr). We hypothesized that tumor phosphokinase activity would be discernible by imaging with 111In-EC-P-Tyr. A xenograft of A431 cells, a human epithelial carcinoma cell line overexpressing epidermal growth factor receptor (EGFR), was employed. Biodistribution studies confirmed increased tumor/muscle ratios of 111In-EC-P-Tyr in the A431 model. Imaging demonstrated that a marked decrease in tumor uptake of 111In-ECP-Tyr occurred after 3 d of gefitinib therapy in A431 cells (gefitinib-sensitive), but not in H441 cells (gefitinb-resistant). Our results indicate that 111In-EC-P-Tyr can detect tumor phospho-tyrosine kinase activity in animal models. This type of agent merits investigation in the clinic to determine if it can predict patient responses to kinase inhibitors based on phosphokinase imaging.

Introduction Tyrosine kinase inhibitors have recently enjoyed remarkable success in the clinic.1 For instance, blockade of EGFR tyrosine kinase activity results in responses in several different tumor types including lung, colorectal and breast cancer.1-7 Mutations that activate EGFR tyrosine phosphorylation in lung cancer correlate well with the clinical response to kinase inhibitors, especially to the EGFR inhibitor gefitinib.7 Therefore, a key challenge in the development of EGFR and other tyrosine kinase inhibitors is identifying, as noninvasively as possible, which patients bear the target molecule and would therefore be the most likely to respond. Positron emission tomography (PET) and single photon emission-computed tomography (SPECT) use radiotracers to image, map and measure tumor-related activities, such as angiogenesis, metabolism, apoptosis and proliferation. They are considered to be targeted molecular imaging modalities.8 To assess clin ical endpoints adequately, a specific target assessment marker is needed for the precise measurement of tumor targets on a wholebody image following the administration of a functional agent. Reliable molecular imaging agents that rapidly assess the presence of the target and/or its modulation, could potentially predict

therapeutic response, and would therefore be extremely valuable. In addition, if these agents were linked to a radio-ablative molecule, they could be therapeutic. We hypothesized that it is feasible to discern the level of phosphokinase activity by imaging with a radio-labeled phosphokinase antibody. We developed a novel, indium (111In)-labeled, anti-phospho-tyrosine antibody, 111In-EC-P-Tyr, by linking the radiolabel to the antibody via ethylenedicysteine (EC) (a chel ator). Here we demonstrate that tumor tyrosine phosphorylation of EGFR can be imaged with 111In-EC-P-Tyr. Further, attenuation of phosphorylation in tumor xenografts after a brief course of treatment with the EGFR inhibitor gefitinib was observed, and was associated with tumor regression after a more prolonged course of therapy. These results suggest that further investigation of this technology is warranted in order to determine if it might be capable of predicting the clinical response to tyrosine kinase inhibitors. Results Detection of EGFR phosphorylation by immuno-precipitation and western blot analysis. Immuno-precipitation and western blot analysis was performed to investigate the effect of gefitinib

*Correspondence to: Razelle Kurzrock; Email: [email protected] Submitted: 07/08/09; Revised: 08/24/09; Accepted: 09/02/09 Previously published online: www.landesbioscience.com/journals/cbt/article/9986 www.landesbioscience.com

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of the blot is immunoglobulin heavy chain left over from the immuno-precipitation portion of the experiment. Densitometry performed on the western blots demonstrated a dose-dependent decrease of phospho-EGFR after 6 h of gefitinib treatment in A431, MDA-MB-231 and H3255 cells, but not in H441 cells (Fig. 1E). The effect was pronounced in H3255 and A431 cells. When compared to the DMSO treated control group, phosphorylation of EGFR was inhibited by 58, 73 and 75% in MDA-MB-231, A431 and H3255 cells, respectively, at 20 μM gefitinib. No phospho-EGFR inhibition was observed in H441 cells, even at the highest dose of gefitinib (20 μM). In vitro analysis of apoptosis. Results of Annexin-V-Fluos staining followed by fluorescence-activated cell sorting analysis after 72 h of treatment with 10 μM gefitinib is shown in Figure 2. Apoptosis was induced differentially in the four cell lines depending on each cell’s sensitivity to the drug. After subtracting the percent of apoptotic cells in the DMSO control group for each cell line, the increase in the percentage of cells undergoing apoptosis was 29.8% for H3255 and 29.7% for A431. In H441 and MDA-MB-231 cells, the percent of apoptotic cells increased only slightly (6 and 4%, respectively) (Fig. 2). Radiosynthesis of 111In-EC-IgG1 and 111InEC-P-Tyr. Radiochemical purity for 111In-ECIgG1 and 111In-EC-P-Tyr was greater than 95% as determined by radio-TLC elution with saline or acetone (Rf = 0.1) (data not shown). HPLC Figure 1. Inhibition of EGFR phosphorylation following treatment with gefitinib. 1 x analysis revealed that the ultraviolet (UV) peak 6 10 cells were treated with 1, 5, 10 or 20 μM of gefitinib without serum for 6 h and then corresponded to the sodium iodide radioactive stimulated with 20% serum for 30 min. Immuno-precipitation was performed with murine peak (Fig. 3). The concentration used was 10 μg anti-phospho-tyrosine antibody followed by western blot with anti-EGFR rabbit polyclonal antibody to detect the level of phospho-EGFR. (A) A431 epidermoid carcinoma cells (EGFR of 111In-EC-P-Tyr in 20 μCi. The specific activity amplification); (B) MDA-MB-231 breast carcinoma cells (high expressor of EGFR); (C) was 2 μCi/μg. There were no marked new peaks H3255 lung adenocarcinoma cells (EGFR mutant); (D) H441 lung papillary adenocarcinoma from 111In-EC-P-Tyr, which indicates the stabilcells (wild-type EGFR). (E) densitometry results of western blots from (A–D). Results ity of 111In-EC-P-Tyr. demonstrate a dose-dependent decrease in phospho-EGFR after gefitinib treatment for 6 h Scintigraphic imaging studies. Represen in A431, MDA-MB-231 and H3255 cells, but not in H441 cells. The effect was pronounced in H3255 cells (EGFR mutant) and A431 cells (EGFR amplification). No inhibition of phosphotative scintigraphic imaging of 111In-labeled EGFR was observed for H441 cells, even at the highest gefitinib dose level (20 mM). Equal compounds in A431 xenografts are shown in amounts of protein were immuno-precipitated with 2 μM of antibody. The lower band Figure 4. The animals received either 2.5% (IgG) is immunoglobulin heavy chain left over from the immuno-precipitation portion of the DMSO alone or 100 mg/kg/day gefitinib in 2.5% experiment. DMSO for three consecutive days. 111In-ECantibodies were injected 24 h after the final treattreatment on phosphorylated EGFR expression in our cell lines. ment. The numbers indicate tumor/muscle (T/M) uptake 48 h Phosphorylation of EGFR was inhibited in three of the four cell after 111In-EC-antibody injection. 111In-EC-P-Tyr injection resulted lines tested: A431 and MDA-MB-231 (both express high lev- in higher T/M ratios and therefore greater uptake by the tumor els of EGFR) and H3255 (contains EGFR mutation). In the than the isotypic control labeled antibody (111In-EC-IgG1) (comMDA-MB-231 cells, concentrations of 20 μM were needed to pare DMSO-treated groups, Fig. 4 and Table 3). achieve significant inhibition, whereas in A431 or H3225 cells, Region of interest analysis generated from A431 planar images treatment with only 1 μM of gefitinib resulted in inhibition. showed that 111In-EC-P-Tyr had 18%-40% higher T/M ratios than Phospho-EGFR was not inhibited in the gefitinib-resistant H441 111In-EC-IgG1 in the untreated group (baseline, Figs. 4 and 5A). cell line (wild-type EGFR) (Fig. 1A–D). The band at the bottom Quantitation of the uptake of 111In-EC-P-Tyr by A431 xenografts

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Figure 2. A431 and H3225 show increased apoptosis after treatment with gefitinib as determined by Annexin-V-Fluos staining followed by flow cytometry. Cells were cultured for 72 h with 10 μM of gefitinib, and then harvested. Apoptosis was quantified using Annexin-V-Fluos staining followed by fluorescence-activated cell sorter (FACS) analysis. Results are expressed as the percent of cells undergoing apoptosis. DMSO treatment was used as a control. Gefitinib-induced apoptosis of A431, H3225, H441 and MDA-MB-231 cells is shown. The percent of cells undergoing apoptosis increased the most in the H3255 cell line (mutant EGFR-bearing) (26% increase over DMSO alone) followed by the A431 cell line (25% increase over DMSO alone) (EGFR amplification).

demonstrates a significant increase in T/M ratios as compared to the uptake of 111In-EC-IgG1 isotypic control at 24 and 48 h (Fig. 5A, compare untreated group injected with 111In-EC-IgG1 with untreated group injected with 111 In-EC-P-Tyr, p < 0.05). Significant uptake of radiolabeled antibodies was not seen at 2 h post-injection (Fig. 5A, open column). There were significant decreases in T/M ratios between untreated and gefitinib-treated xenografts. Decreased T/M ratios (51–20%) could be measured by 24–48 h of labeling with 111In-EC-P-Tyr but not with 111In-EC-IgG1 after 3 d of geftinib treatment (Fig. 5B). The greatest decrease occurred at 24 h (p < 0.05) (Fig. 5A and B). The percentage of change in the T/M ratio between untreated and gefitinib-treated xenografts was minimal in the gefitinib-resistant H441 animal model (Fig. 5B and C). In our western blots, the level of expression of phospho-EGFR in the A431 cell line could be inhibited by gefitinib (Fig. 1A). In the A431 xenograft model, tumor uptake of 111In-EC-P-Tyr was decreased after gefitinib treatment (Fig. 5A and B). Therefore, the in vitro and in vivo findings were well correlated. These findings show that gefitinib reduced the level of expression of phosphoEGFR in the A431 animal model. Tissue distribution of 111In-EC-P-Tyr in A431 and H441 tumor-bearing nude mice. Bio-distribution studies confirmed tumor uptake at 24 and 48 h after injection of 111In-EC-P-Tyr in both A431 and H441 xenografts (Tables 1 and 2), although the tumor/muscle ratios were greater in the H441 model. Discussion Several techniques have been suggested for predicting which patients will respond to treatment with kinase

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Figure 3. High pressure liquid chromatography (HPLC) analysis of 111In-EC-P-Tyr. The ultraviolet (UV) (A) peak corresponds to sodium iodide radioactive peak (B). The concentration used was 10 μg of 111In-EC-P-Tyr in 20 μCi. The specific activity was 2 μCi/μg. There were no marked new peaks from 111In-EC-P-Tyr, pointing to its stability.


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Figure 4. Planar scintigraphy of 111In-EC-Antibodies in A431 xenograft. The animals received either 2.5% DMSO alone or 100 mg/kg/day gefitinib in 2.5% DMSO for three consecutive days. 111In-EC-antibody was injected a day after the final treatment. The numbers indicate T/M uptake 48 h after 111 In-EC-antibody injection. A standard of 27 μCi was used to help quantify the data. Tumor location is indicated by arrows. Results show that the A431 xenograft assessed with 111In-EC-P-Tyr has a higher T/M ratio (3.1 vs. 1.7) than the xenograft assessed with 111In-EC-IgG1. The effect of gefitinib treatment on EGFR tyrosine phosphorylation can be seen clearly in the tumors assessed with 111In-EC-P-Tyr (T/M 3.1 for DMSO versus T/M 1.6 for gefitinib treated) versus those assessed with 111In-EC-IgG1.

inhibitors. Although biopsies can be used to detect the expression of specific kinases in tumor tissue, their use is problematic. First, biopsies are invasive, and can be painful and dangerous. Second, the data that they yield have been questioned because of variable results, both within and among patients. Further, patients are reluctant to undergo biopsies both before and after treatment. The optimal approach would enable clinicians to assess both the target and effect of a given kinase inhibitor on the target of interest in a noninvasive way. Our novel technique uses an indium-labeled anti-phospho-tyrosine antibody, 111In-EC-P-Tyr, to measure tumor phospho-EGFR activity before and after gefitinib treatment. Covalent and coordination chemistries are frequently utilized to develop clinically useful tracers.21-25 The radioisotope can either be linked to the molecule by a covalent bond or by a coordination bond using a chelator. In covalent chemistry, displacement and/or addition reactions are used to place an isotope in the molecule. The labeled product provides minimal structural alteration; however, the procedure may be lengthy, tedious, costly and may produce low yields. Isotopes commonly used in covalent chemistry include 18F, 123I, 131I, 75Br, 77Br and 11C.21-26 In coordination chemistry, a chelator is required to trap metal isotopes ionically. This type of chemistry is simple and has a high yield. The metallic isotopes can be obtained from generators and cyclotrons. Though coordination chemistry is attractive, chemical properties may be altered due to the addition of a chelator. Thus, chelation chemistry is commonly utilized in conjunction with high molecular weight compounds, such as peptides and proteins. Several chelators have been reported to trap metallic isotopes.26-32 EC is the most recent and successful example of N2S2 chelates. EC can be labeled with metallic isotopes easily and efficiently with high radiochemical purity and stability.33,34 Though EC conjugates may alter the structure of the homing agent, a series of EC agent conjugates with low molecular weight

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were reported to bind the tumor targets and to be involved in cell nuclei activity.35-39 The low molecular weight EC conjugates entered the cells either through transporters or lipophilic characters. It is likely that SH bonds of EC conjugates bind to cytosolic and transmembrane enzymes or membrane-associated proteins that form S-S (protein) linkages and support the translocation of EC-conjugates into the cell nucleus.38,39 It is well established that antibodies target tumors through cell surface antigens. Certain antibodies, such as EGFR antibody, may internalize into the cell after surface receptor binding.40 We have previously reported that EC (a stable metallic chelator) interacts with intracellular proteins and provides a docking effect. 111 In-EC-P-Tyr may have a similar internalization effect as the EGFR antibody. Once an anti-phospho-tyrosine antibody is internalized, it induces tyrosine phosphorylation. Combining the tumor-specific monoclonal antibody with the gamma emitter 111Indium would be an attractive approach for imaging by trapping the radionuclide in the tumor cells after monoclonal antibody intracellular processing. We used the EC chelator in our experiments to detect phospho-tyrosine with an 111In-labeled anti-P-Tyr antibody. EC was covalently linked to the lysine residue of an anti-phosphotyrosine antibody and 111In was trapped by EC via coordination bonds. 111In has a long half-life (67 h), which is suitable for antibody imaging. We have previously used this methodology to successfully image apoptosis by EC-annexin V,41 angiogenesis by EC-endostatin,42 EGFR expression in head and neck cancer by EC-C225 antibody,43 and TRAIL (death) receptor by EC-ETR1 and EC-ETR2 antibodies.44 Several reports have suggested that elevated baseline levels of phospho-EGFR may be associated with the sensitivity of the tumor to an EGFR inhibitor.7,45-47 Furthermore, with EGFR and other kinase inhibitors, it is plausible that downregulation of


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Figure 5. Effect of gefitinib treatment on tumor/ muscle ratios as determined by imaging with 111InEC-Antibody in A431 and H441 xenografts. The animals received either 2.5% DMSO alone or 100 mg/kg/day gefitinib in 2.5% DMSO for three consecutive days. The 111 In-EC-antibody was injected one day after the final treatment. The numbers indicate T/M uptake at 2, 24 and 48 h after 111In-EC-antibody injection. A standard of 27 μCi was placed to help to quantify the data. (A) T/M ratios were higher as a function of time with 111InEC-P-Tyr compared to 111In-EC-IgG1 at 24 and 48 h in the untreated A431 group (Student’s t-test, *p < 0.05 between untreated groups at the corresponding time). After 3 d of geftinib treatment, the A431 xenograft showed decreased T/M ratios by 111In-EC-P-Tyr imaging at 24 hours post-administration (Student’s t-test, **p < 0.05 between untreated and treated groups at the corresponding time). (B) Region of interest analysis generated from A431 planar images showed that 111InEC-P-Tyr had 18–40% higher T/M ratios than 111In-ECIgG1 in the untreated group (baseline). Decreased T/M ratios (51–20%) could be measured by 24–48 h of labeling with 111In-EC-P-Tyr but not with 111In-EC-IgG1 after 3 d of geftinib treatment. The percentage of change in the T/M ratio between untreated and gefitinib-treated xenografts was minimal in H441 animal models. The physical amount of antibody used was 5 μg/mouse (250 μg/kg). (C) There were no marked changes in T/M ratios between untreated and gefitinib treated groups in the H441 animal model.

phosphorylation is necessary for tumor response. Using the methodology described above, we showed that an anti-phospho-tyrosine antibody can be labeled with 111In (111In-EC-P-Tyr) and then used to assess EGFR-expressing xenografts. Furthermore, bio-distribution studies confirmed increased tumor/muscle ratios of 111In-EC-P-Tyr uptake both in A431 and H441 xenografts. Since the bio-distribution of 111In-EC-P-Tyr and 111In-ECIgG1 are different, the muscle did not have uniform uptake. Computer outlined region of interest (ROI) (counts per pixel) of tumor and muscle at symmetric sites were used to determine the specific uptake difference between pre- and post-geftinib treatment using the same compound, not different compounds (Table 3). Planar single-photon imaging is not generally an accurate method of quantitation and may likely vary greater than 20% between observers or between imaging the same subject multiple times. Thus, tumor/blood and tumor/muscle ratios on post-necropsy animals are given in the bio-distribution tables. In imaging and bio-distribution (post necropsy) of this antibody, the liver uptake is high and goes as high as 12.9%. This is true for almost all other antibodies (large proteins). The imaging findings correlate well with biodistribution findings. Region of interest analysis generated from A431 planar images showed that 111In-EC-P-Tyr had 18–40% higher T/M ratios than 111 In-EC-IgG1 in the untreated group (baseline). Decreased T/M ratios (51–20%) could be measured by 24–48 h of labeling with


In-EC-P-Tyr but not with 111In-EC-IgG1 after 3 d of geftinib treatment (Fig. 5B). The findings suggest that 111In-EC-P-Tyr is specific for measuring tumor uptake changes after geftinib treatment. Importantly, imaging demonstrated that a marked decrease in tumor uptake of 111In-EC-P-Tyr occurred after only three days of gefitinib therapy in A431 (gefitinib-sensitive) xenografts but not in H441 (gefitinib-resistant) xenografts. These findings correlate with the observation that gefitinib causes A431 cells to undergo apoptosis in vitro (Fig. 2) and growth inhibition in vivo. In contrast, H441 (gefitinib-resistant) cells demonstrated little apoptosis after geftinib treatment (Fig. 2). Interestingly, at the doses used, tumor regression, as measured by tumor volume 111


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Table 1. Bio-distribution of 111In-EC-P-Tyr in A431 tumor-bearing nude mice* 0.5 h

2h

4h

24 h

48 h

BLOOD

16.876 ± 3.763**

13.861 ± 1.532

HEART

4.760 ± 1.043

3.355 ± 0.452

9.644 ± 0.456

1.630 ± 0.137

0.787 ± 0.042

3.325 ± 0.139

1.696 ± 0.128

LUNG

7.809 ± 1.587

1.638 ± 0.136

4.646 ± 0.551

5.082 ± 0.370

2.604 ± 0.186

2.624 ± 0.207

THYROID

6.249 ± 1.708

4.724 ± 0.542

5.056 ± 0.268

2.252 ± 0.141

2.360 ± 0.178

PANCREAS

3.234 ± 1.205

1.622 ± 0.170

1.807 ± 0.202

1.624 ± 0.187

1.902 ± 0.154

LIVER

3.808 ± 0.598

3.890 ± 0.597

4.537 ± 0.187

5.795 ± 0.444

6.637 ± 0.494

SPLEEN

3.230 ± 0.678

3.996 ± 0.565

4.468 ± 0.514

4.980 ± 0.714

5.066 ± 0.479

KIDNEY

24.154 ± 2.015

17.524 ± 2.507

15.864 ± 1.496

20.925 ± 2.337

18.675 ± 2.403

STOMACH

2.461 ± 0.353

2.421 ± 0.221

2.278 ± 0.077

2.206 ± 0.048

2.347 ± 0.056

INTESTINE

2.159 ± 0.567

2.349 ± 0.254

3.059 ± 0.112

4.379 ± 0.188

4.222 ± 0.471

TUMOR

3.328 ± 0.386

2.905 ± 0.382

4.152 ± 0.196

2.909 ± 0.208

3.148 ± 0.397

MUSCLE

1.592 ± 0.217

1.379 ± 0.069

1.525 ± 0.048

1.032 ± 0.127

1.036 ± 0.146

BRAIN

0.487 ± 0.121

0.348 ± 0.043

0.341 ± 0.030

0.303 ± 0.124

0.206 ± 0.012

T/BLOOD

0.211 ± 0.033

0.207 ± 0.025

0.432 ± 0.028

1.790 ± 0.079

3.980 ± 0.367

T/MUSCLE

2.118 ± 0.160

2.092 ± 0.188

2.732 ± 0.184

2.889 ± 0.368

3.096 ± 0.379

*% of injected dose per gram of tissue weight (n = 3/time interval, iv). **Values shown represent the mean ± standard deviation of data from three animals.

Table 2. Bio-distribution of 111In-EC-P-Tyr in H441 tumor-bearing nude mice* 0.5 h

2h

4h

24 h

48 h

BLOOD

22.099 ± 0.905**

14.050 ± 0.642

11.542 ± 0.370

2.307 ± 0.040

0.819 ± 0.105

HEART

4.570 ± 0.306

4.081 ± 0.124

3.709 ± 0.100

2.877 ± 0.162

2.517 ± 0.335

LUNG

9.507 ± 0.849

7.181 ± 0.193

6.163 ± 0.389

3.938 ± 0.041

3.250 ± 0.094

THYROID

5.661 ± 0.879

4.274 ± 0.621

5.331 ± 0.408

3.479 ± 0.173

3.295 ± 0.081

PANCREAS

1.970 ± 0.368

2.166 ± 0.186

3.303 ± 1.086

2.695 ± 0.068

2.463 ± 0.256

LIVER

8.370 ± 0.866

8.637 ± 0.404

7.183 ± 3.051

12.372 ± 0.704

12.910 ± 0.360

SPLEEN

5.697 ± 0.733

5.102 ± 0.198

4.532 ± 1.184

7.856 ± 0.429

6.631 ± 0.595

KIDNEY

19.324 ± 2.494

25.011 ± 1.910

28.225 ± 0.659

25.462 ± 0.764

25.021 ± 1.853

STOMACH

2.772 ± 0.190

2.651 ± 0.230

3.141 ± 0.126

3.411 ± 0.147

2.804 ± 0.306

INTESTINE

2.665 ± 0.345

2.953 ± 0.163

3.697 ± 0.260

6.261 ± 1.168

4.229 ± 0.507

TUMOR

3.646 ± 0.333

4.824 ± 0.551

6.785 ± 1.540

7.582 ± 0.409

7.363 ± 0.928

MUSCLE

1.680 ± 0.111

1.464 ± 0.054

1.917 ± 0.127

1.313 ± 0.087

1.247 ± 0.055

BRAIN

0.691 ± 0.092

0.529 ± 0.023

0.400 ± 0.068

0.299 ± 0.008

0.267 ± 0.033

T/BLOOD

0.167 ± 0.004

0.331 ± 0.047

0.582 ± 0.117

3.295 ± 0.236

9.378 ± 1.992

T/MUSCLE

2.142 ± 0.040

3.227 ± 0.193

3.473 ± 0.558

5.786 ± 0.074

5.988 ± 1.019

*% of injected dose per gram of tissue weight (n = 3/time interval, iv). **Values shown represent the mean ± standard deviation of data from three animals.

Table 3. Raw data comparing tumor uptake with symmetrical muscle uptake 48 h after injection (images shown in fig. 4)* TUMOR

MUSCLE

TREATMENT

Sum

Number

Sum/number

Sum

Number

Sum/number

IgG-DMSO

1574.00

61

25.8032

916.00

61.00

15.0163934

1.7183

IgG-gefitinib

1500.00

88

17.0454

897.00

88.00

10.1932

1.6722

T/M ratio

APT-DMSO

1960.00

93

21.0753

624.00

93.00

6.7097

3.1410

APT-gefitinib

2000.00

72

27.7778

1250.00

72.00

17.3611

1.6

*In both the bio-distribution and imaging experiments, the tumor was in the proximal part of the right posterior limb and was compared with a symmetrical area in the proximal left posterior limb to avoid possible uptake variation in different muscle groups (central part of the tumor uptake compared to a mirrored area of muscle on the opposite limb). 2242


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in mice bearing A431 xenografts, generally becomes significant after approximately two weeks of therapy. Using this imaging technique, the decrease in p-EGFR could be seen after only three days of gefitinib treatment, much earlier than with conventional tumor measurements. In addition, the results of early post treatment 111In-EC-P-Tyr imaging correlated with EGFR inhibitor sensitivity in the A431 xenograft model.


Materials and Methods Antibodies. Anti-phospho-tyrosine mouse monoclonal antibody (p-Tyr), used for immuno-precipitation and imaging, and EGFR polyclonal antibody (used for western blot analysis) were purchased from Cell Signaling Technology Inc., (Danvers, MA). Mouse IgG1 (Clone 15H6), an isotypic control for imaging, was purchased from Southern Biotech (Birmingham, AL). Horseradish peroxidase-conjugated goat anti-rabbit secondary antibody was obtained from Amersham Pharmacia Biotech (Freiburg, Germany). Cell culture. A431 human epidermoid carcinoma cells expressing amplified EGFR,9 MDA-MB-231 human breast carcinoma cells expressing high levels of wild-type EGFR,10 and human lung papillary H441 adenocarcinoma cells (wild-type EGFR) were obtained from American Type Culture Collection (ATCC) (Rockville, MD). The H3255 human lung adenocarcinoma cell line bearing an EGFR gene mutation (L 858 R)11-13 was a gift from Dr. Matthew Meyerson (Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA). A431 and MDA-MB-231 cells were cultured in Dulbecco’s modified Eagle’s medium and Leibovitz’s L-15 medium (ATCC) containing 10% heat-inactived fetal bovine serum (FBS) (Invitrogen Corporation, Carlsbad, CA). RPMI 1640 (Gemini Bio-Products, Woodland, CA) with 10% FBS was used to maintain the H441 cell line. H3255 cells were grown in ACL-4 medium (Invitrogen Corporation) with 5% FBS. All cells were grown in a 37°C incubator with 5% CO2. Immuno-precipitation and western blot. Immuno-preci pitation and western blot analysis was performed to investigate the effect of gefitinib treatment on phosphorylated EGFR expression in our cell lines. 1 x 106 cells (A431, MDA-MB-231, H3255 or H441) were grown to 85% confluence in a 10-cm2 dish, serumstarved for 24 h, then treated with 1, 5, 10 or 20 μM of gefitinib (AstraZeneca, Wilmington, DE) without serum for 6 h; and then stimulated with 20% serum for 30 min. Cells were rinsed twice in ice-cold phosphate-buffered saline (PBS) and scraped into 0.5 ml lysis buffer (Pierce Chemical Co., Rockford, IL). Lysates were rotated for 10 min prior to centrifugation at 14,000 RPM for 10 min at 4°C. Determination of total protein was performed using the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA). Cell lysates containing 0.5 mg protein were incubated with anti-p-Tyr antibody at 4°C for 2 h. Protein G-agarose beads were then added and the samples rotated overnight at 4°C. Beads were collected by brief centrifugation and washed three times, after which the beads were boiled for 5 min in 2x Laemmli sample buffer. Denatured samples were electrophoresed on 8% sodium dodecyl sulfate-polyacrylamide gels. Proteins were transferred to


nitrocellulose (Bio-Rad, Hercules, CA) by electroblotting. After transfer, the membrane was blocked with 1X Tris-buffered saline plus 0.2% Tween-20 plus 5% nonfat dry milk for 1 h at room temperature and then probed with anti-EGFR antibody at 4°C overnight. The membrane was washed, and then incubated for 1 h at room temperature with anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody. The membrane was developed using enhanced chemiluminescence (ECL) (Amersham, Little Chalfont, Buckinghamshire, UK) according to the manufacturer’s instructions, and exposed to film. In vitro analysis of apoptosis. Following treatment with 10 μM gefitinib for 72 h, adherent cells were collected and combined with nonadherent cells. The cells were washed in PBS, then stained with Annexin-V-Fluos (Roche Diagnostics, Mannheim, Germany) for 30 min before labeled cells were quantitated by flow cytometry (EpicsXL; Beckman Coulter, Miami, FL). Propidium iodide is generally excluded from viable cells; it can be used to stain DNA in dead cells. Annexin-V-Fluos can identify both apoptotic and necrotic cells by binding to phosphatidylserine exposed to the outer leaflet of the membrane during the apoptotic process.14 Radiosynthesis of 111In-EC-IgG1 and 111In-EC-P-Tyr. Ethylene dicysteine (EC) was selected as a chelator because EC drug conjugates can be labeled with 111In easily and efficiently with high radiochemical purity and stability.15-17 Synthesis of EC was performed in two-steps according to a previously described method.18,19 Sulfo-N-hydroxysuccinimide (sulfo-NHS) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl (EDC) were purchased from Pierce Chemical Co., (Rockford, IL). 111 In was purchased from DuPont NEN (Boston, MA). EC was conjugated to IgG and p-Tyr antibodies using sulfo-NHS and EDC as coupling agents. Briefly, 1 N sodium bicarbonate was added to a stirred solution of EC (0.019 mmol). Sulfo-NHS (0.019 mmol) and EDC (0.019 mmol) were added, followed by the antibody. The mixture was stirred at room temperature for 24 h, and then dialyzed for 48 h with a molecular weight cutoff of 10,000. After dialysis, the product was freeze-dried. 111 In was added into a vial containing EC antibodies (0.1 mg) to yield 111In-EC-IgG1 and 111In-EC-P-Tyr. 111In-EC-P-Tyr represents the anti-p-Tyr mouse antibody linked to the 111In label. Radiochemical purity for 111In-EC-IgG1 and 111In-EC-P-Tyr was determined by using radio-TLC (Bioscan, Inc., Washington, DC) eluted with saline or acetone. HPLC analysis of 111In-ECP-Tyr was performed to demonstrate purity and specific activity. HPLC, equipped with two detectors and Bio Sep-SEL-S 3000 reverse phase column (7.8 x 300 mm) and eluted with 0.1% trifluoroacetic acid in water as the mobile phase, was used to analyze 111In-EC-P-Tyr. Growth of tumors in nude mice after treatment with gefitinib. The animal experiments were approved by The University of Texas M.D. Anderson Cancer Center Institutional Animal Care and Use Committee (IACUC). 6–8 w-old female nude mice (National Cancer Institute, Bethesda, MD) were inoculated intramuscularly into the hind legs with 0.1 ml of either A431 or H441 tumor-cell suspensions (3 x 106 cells/mouse) and allowed to form tumors. When tumor size reached 1 cm (greatest diameter), the mice were gavaged daily with 100 mg/kg gefitinib


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dissolved in 2.5% dimethyl sulfoxide (DMSO)20 or DMSO alone for three consecutive days. Scintigraphic imaging studies. We examined A431 and H441 xenograft models, which are sensitive and resistant to EGFR kinase inhibition, respectively. H3255 was not examined in vivo, despite its in vitro sensitivity to EGFR kinase inhibition because we found that it was very difficult to create a xenograft model using this cell line. MDA-MB-231 was not examined because it was not sensitive to in vitro kinase inhibition at levels of exposure to gefitinib below 20 μM. Three animals were used in each experimental group and the experiments were repeated twice. Animals were divided into two groups: group I, control (gavaged with 2.5% DMSO) and group II, treatment (gavaged with 100 mg/kg gefitinib). The antibodies (0.1 mg) were labeled with 111 In with an activity of 2 mCi/2 ml saline. Group I was subdivided into two groups: group IA, 111In-EC-IgG1 and group IB, 111 In-EC-P-Tyr. Group II was also subdivided into two groups: group IIA, 111In-EC-IgG1 and group IIB, 111In-EC-P-Tyr. The imaging studies were performed after three consecutive days and during this time, 100 mg/kg gefitinib or DMSO alone was administered orally. Each animal was injected intravenously (tail vein) with 100 μCi of 111In-labeled antibody (physical amount 5 μg per mouse) as described above. At 2, 24 and 48 h following administration of the radiotracers, scintigraphic images were obtained using a γ-camera (M-camera, Siemens Medical Systems, Hoffman, IL) equipped with a medium energy parallel-hole collimator. The field of view is 53.3 cm x 38.7 cm. The intrinsic spatial resolution is 3.2 mm and the pixel size is 19.18 mm (32 x 32, zoom = 1) to 0.187 mm (1024 x 1024, zoom = 3.2). Computer outlined regions of interest (ROI) (counts per pixel) of tumor lesion sites and symmetric normal muscle sites were used to determine tumor-to-muscle count density ratios. The ratios were used to compare p-Tyr activity before and after gefitinib treatment. When determining the percent change in tumor to muscle ratios, the following formulae were used (Fig. 5C). I B vs. I A = [Untreated ( In-EC-P-Tyr) - Untreated ( In-ECIgG1)]/[Untreated (111In-EC-IgG1)] x 100% 111

111

II B vs. I B = [Post-gefitinib (111In-EC-P-Tyr) - Untreated ( In-EC-P-Tyr)]/[Untreated (111In-EC-P-Tyr)] x 100% 111

Tissue distribution of 111In-EC-P-Tyr in A431 and H441 tumor-bearing nude mice. Female nude mice were inoculated with human A431 and H441 cancer cells (3 x 106 cells/ mouse, intramuscularly) in the mid-dorsal region. After the tumor reached 10 mm, separate bio-distribution studies of two References 1.

2.

3.

Tibes R, Trent J, Kurzrock R. Tyrosine kinase inhibitors and the dawn of molecular cancer therapeutics. Ann Rev Pharamacol Toxicol 2005; 45:357-84. Mendelsohn J, Baselga J. The EGF receptor family as targets for cancer therapy. Oncogene 2000; 19:655065. Karunagaran D, Tzahar E, Beerli RR, Chen X, GrausPorta D, Ratzkin BJ, et al. ErbB-2 is a common auxiliary subunit of NDF and EGF receptors: implications for breast cancer. EMBO J 1996; 15:254-64.

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5.

animal tumor models using 111In-EC-P-Tyr were conducted. Each mouse received 111In-EC-P-Tyr intravenously (n = 3/time point). The injection activity was 5 μCi/mouse. The injected mass was 1 μg/mouse. The mice were divided into five groups, each group representing a time interval (0.5, 2, 4, 24 and 48 h) and containing three mice. Following administration of the radiotracers, the rodents were sacrificed and the selected tissues were excised, weighed and counted for radioactivity. The bio-distribution of tracer in each sample was calculated as a percentage of the injected dose per gram of tissue wet weight (%ID/g). Tumor/ nontarget tissue count density ratios were calculated from the corresponding %ID/g values. Statistical analysis. The Student’s t-test was used to compare differences in percentage of T/M ratios. p < 0.05 indicated a statistically significant difference. All statistical computations were processed by the Excel software program (Microsoft, Redmond, WA). Conclusion In conclusion, we demonstrated that a radio-labeled antiphospho-tyrosine antibody (111In-EC-P-Tyr) detected tumor phosphorylation before and after treatment with gefitinib in vivo in a noninvasive manner. Thus, this technology and similar ones may be applicable for use in the clinic by helping to select patients for treatment with a specific kinase inhibitor. Pal and colleagues48 also successfully performed preclinical molecular imaging using a 124 I-labeled small molecular tracer (binds to the adenosine-triphosphate binding site of the activated EGFR kinase) and imaging with positron emission tomography. Information gleaned from these new technologies may be beneficial in assessing and predicting the effectiveness of EGFR as well as other kinase inhibitors. In addition, antibodies such as these may also, if labeled with radioablative motifs, be exploitable for therapeutic purposes.49,50 Acknowledgements

The animal research was supported in part by the M.D. Anderson Cancer Center Support Grant NIH CA-16672. This publication was made possible by Grant Number RR024148 from the National Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH), and NIH Roadmap for Medical Research. Its contents are solely the responsibility of the authors and do not necessarily represent the official view of NCRR or NIH. Information on NCRR is available at www.ncrr. nih.gov/. Information on Re-engineering the Clinical Research Enterprise can be obtained from nihroadmap.nih.gov/clinicalresearch/overview-translational.asp.

Sarup JC, Johnson RM, King KL, Fendly BM, Lipari MT, Napier MA, et al. Characterization of an antip185HER2 monoclonal antibody that stimulates receptor function and inhibits tumor cell growth. Growth Regul 1991; 1:72-82. Montemurro F, Balabrega G, Aglietta M. Lapatinib: a dual inhibitor of EGFR and HER2 tyrosine kinase activity. Expert Opin Biol Ther 2007; 2:257-68.

6.

7.

8.


Ono M, Kuwano M. Molecular mechanisms of epidermal growth factor receptor (EGFR) activation and response to gefitinib and other EGFR-targeting drugs. Clin. Cancer Res 2006; 12:7242-51. Paez JG, Janne PA, Lee JC, Tracy S, Greulich H, Gabriel S, et al. EGFR mutations in lung cancer: Correlation with clinical response to gefitinib therapy. Science 2004; 304:1497-500. Yang DJ, Kim EE. Tracer development and hybrid imaging. Eur J Med Mol Imaging 2005; 32:1001-2.

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9.

10.

11.


12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

Merlino GT, Ishii S, Whang-Peng J, Knutsen T, Xu YH, Clark AJ, et al. Structure and localization of genes encoding aberrant and normal epidermal growth factor receptor RNAs from A431 human carcinoma cells. Mol Cell Biol 1985; 5:1722-34. Takabatake D, Fujita T, Shien T, Kawasaki K, Taira N, Yoshitomi S, et al. Tumor inhibitory effect of gefitinib (ZD1839, Iressa) and taxane combination therapy in EGFR overexpressing breast cancer cell lines (MCF7/ ADR, MDA-MB-231). Int J Cancer 2007; 120:1818. Heimberger AB, Learn CA, Archer GE, McLendon RE, Chewning TA, Tuck FL, et al. Brain tumors in mice are susceptible to blockade of epidermal growth factor receptor (EGFR) with the oral, specific, EGFRtyrosine kinase inhibitor ZD1839 (IRESSA). Clin Cancer Res 2002; 8:3496-502. Anderson NG, Ahmad T, Chan K, Dobson R, Bundred NJ. ZD1839 (IRESSA), a novel epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, potently inhibits the growth of EGFR-positive cancer cell lines with or without erbB2 overexpression. Int J Cancer 2001; 94:774-82. Cragg MS, Kuroda J, Puthalakath H, Huang DCS, Strasser A. Gefitinib-induced killing of NSCLC cell lines expressing mutant EGFR requires BIM and can be enhanced by BH3 mimetics. PLoS Medicine 2007; 4:1681-90. Vermes I, Haanen C, Steffens-Nakken K, Reutelingsperger C. A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J Immunol Methods 1995; 184:39-51. Blondeau P, Berse C, Grave ID. Dimerization of an intermediate during the sodium in liquid ammonia reduction of 1-thiazolidine-4-caroxylic acid. Can J Chem 1967; 45:49-52. Van Nerom CG, Bormans GM, De Roo MJ, Verbruggen AM. First experience in healthy volunteers with Tc-99m-L-ethylenedicysteine, a new renal imaging agent. Eur J Nuc Med 1993; 20:738-46. Surma MJ, Wiewiora J, Liniecki J. Usefulness of Tc-99m-N,N-ethylene-1-dicysteine complex for dynamic kidney investigations. Nucl Med Commun 1994; 15:628-35. Ilgan S, Yang DJ, Higuchi T, Zareneyrizi F, Bayhan H, Yu D, et al. 99mTc-ethylenedicysteine-folate: a new tumor imaging agent. Synthesis, labeling and evaluation in animals. Cancer Biother Radiopharm 1998; 13:427-35. Zareneyrizi F, Yang DJ, Oh CS, Ilgan S, Yu DF, Tansey W, et al. Synthesis of 99mTc-ethylenedicysteine-colchicine for evaluation of antiangiogenic effects. Anticancer Drugs 1999; 10:685-92. Adam MJ, Ruth TJ, Grierson JR, Abeysekera B, Pate BD. Routine synthesis of L-[18F]6-fluorodopa with fluorine-18 acetyl hypofluorite. J Nucl Med 1986; 27:1462-6. Semnani ES, Wang K, Adelstein SJ, Kassis AI. 5-[123I/125I]iodo-2'-deoxyuridine in metastatic lung cancer: radiopharmaceutical formulation affects targeting. J Nucl Med 2005; 46:800-6. Bettio A, Honer M, Muller C, Brühlmeier M, Müller U, Schibli R, et al. Synthesis and preclinical evaluation of a folic acid derivative labeled with 18F for PET imaging of folate receptor-positive tumors. J Nucl Med 2006; 47:1153-60.


23. Fei X, Wang JQ, Miller KD, Sledge GW, Hutchins GD, Zheng QH. Synthesis of [18F] Xeloda as a novel potential PET radiotracer for imaging enzymes in cancers. Nucl Med Biol 2004; 31:1033-41. 24. Rosen MA, Jones RM, Yano Y, Budinger TF. Carbon-11 choline: synthesis, purification and brain uptake inhibition by 2-dimethylaminoethanol. J Nucl Med 1985; 26:1424-8. 25. Ohtsuki K, Akashi K, Aoka Y, Blankenberg FG, Kopiwoda S, Tait JF, et al. Technetium-99m HYNICannexin V: a potential radiopharmaceutical for the in-vivo detection of apoptosis. Eur J Nucl Med 1999; 26:1251-8. 26. Saha GB, MacIntyre WJ, Go RT. Cyclotrons and positron emission tomography radiopharmaceuticals for clinical imaging. Semin Nucl Med 1992; 22:150-61. 27. Vriens PW, Blankenberg FG, Stoot JH, Ohtsuki K, Berry GJ, Tait JF, et al. The use of technetium 99mTc annexin V for in vivo imaging of apoptosis during cardiac allograft rejection. J Thorac Cardiovasc Surg 1998; 116:844-53. 28. Van Nerom CG, Bormans GM, De Roo MJ, Verbruggen AM. First experience in healthy volunteers with technetium-99m L,L-ethylenedicysteine, a new renal imaging agent. Eur J Nucl Med 1993; 20:73846. 29. Canet EP, Casalic C, Desenfant A, An MY, Corot C, Obadia JF, et al. Kinetic characterization of CMDA2-Gd-DOTA as an intravascular contrast agent for myocardial perfusion measurement with MRI. Magn Reson Med 2000; 43:403-9. 30. Laissy JP, Faraggi M, Lebtahi R, Soyer P, Brillet G, Méry JP, et al. Functional evaluation of normal and ischemic kidney by means of gadolinium-DOTA enhanced TurboFLASH MR imaging: a preliminary comparison with 99Tc-MAG3 dynamic scintigraphy. Magn Reson Imaging 1994; 12:413-9. 31. Kao CH, ChangLai SP, Chieng PU, Yen TC. Technetium-99m methoxyisobutylisonitrile chest imaging of small cell lung carcinoma: relation to patient prognosis and chemotherapy response—a preliminary report. Cancer 1998; 83:64-8. 32. Wu HC, Chang CH, Lai MM, Lin CC, Lee CC, Kao A. Using Tc-99m DMSA renal cortex scan to detect renal damage in women with type 2 diabetes. J Diabetes Complications 2003; 17:297-300. 33. Blondeau P, Berse C, Gravel D. Dimerization of an intermediate during the sodium in liquid ammonia reduction of L-thiazolidine-4-carboxylic acid. Can J Chem 1967; 45:49-52. 34. Ratner S, Clarke HT. The action of formaldehyde upon cysteine. J Am Chem Soc 1937; 59:200-6. 35. Yang DJ, Ozaki K, Oh CS, Azhdarinia A, Yang T, Ito M, et al. 99mTc-EC-guanine: synthesis, biodistribution and tumor imaging in animals. Pharm Res 2005; 22:1471-9. 36. Yang DJ, Bryant J, Chang JY, Mendez R, Oh CS, Yu DF, et al. Assessment of COX-2 expression with 99mTclabeled celebrex. Anti-Cancer Drugs 2004; 15:255-63. 37. Song HC, Bom HS, Cho KH, Kim BC, Seo JJ, Kim CG, et al. Prognostication of recovery in patients with acute ischemic stroke using brain spect with 99mTcmetronidazole. Stroke 2003; 34:982-6.


38. Yang DJ, Kim CG, Schechter NR, Azhdarinia A, Yu DF, Oh CS, et al. Imaging with 99mTc ECDG targeted at the multifunctional glucose transport system: feasibility study with rodents. Radiology 2003; 226:46573. 39. Yang DJ, Yukihiro M, Oh CS, Kohanim S, Azhdarinia A, Yu DF, et al. Assessment of therapeutic tumor response using 99mTc-ethylenedicysteine-glucosamine. Cancer Biother Radiopharm 2004; 19:443-58. 40. Hens M, Vaidyanathan G, Welsh P, Zalutsky MR. Labeling internalizing anti-epidermal growth factor receptor variant III monoclonal antibody with (177) Lu: in vitro comparison of acyclic and macrocyclic ligands. Nucl Med Biol 2009; 36:117-28. 41. Yang DJ, Azharinia A, Wu P, Yu DF, Tansey W, Kalimi SK, et al. In vivo and in vitro measurement of apoptosis in breast cancer cells using 99mTc-EC-annexin V. Cancer Biother Radiopharm 2001; 16:73-83. 42. Yang DJ, Kim KD, Schecter NR, Yu DF, Wu P, Azhdarinia A, et al. Assessment of anti-angiogenic effect using 99mTc-EC-endostatin. Cancer Biother Radiopharm 2002; 17:233-45. 43. Schechter NR, Yang DJ, Azhdarinia A, Kohanim S, Wendt R, 3rd, Oh CS, et al. Assessment of epidermal growth factor receptor with 99mTc-ethylenedicysteineC225 monoclonal antibody. Anticancer Drugs 2003; 14:49-56. 44. Gong J, Yang D, Kohanim S, Humphreys R, Broemeling L, Kurzrock R. Novel in vivo imaging shows upregulation of death receptors by paclitaxel and correlates with enhanced antitumor effects of receptor agonist antibodies. Mol Cancer Ther 2006; 5:2991-3000. 45. Wakeling AE, Guy SP, Woodburn JR, Ashton SE, Curry BJ, Barker AJ, et al. ZD1839 (Iressa): An orally active inhibitor of epidermal growth factor signaling with potential for cancer therapy. Cancer Res 2002; 62:5749-54. 46. Uramoto H, Sugio K, Oyama T, Sugaya M, Hanagiri T, Yasumoto K. Resistance to gefitinib. Int J Clin Oncol 2006; 11:487-91. 47. Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto RA, Brannigan BW, et al. Activating mutations in lung cancer: correlation with clinical response to gefitinib therapy. N Engl J Med 2004; 350:212939. 48. Pal A, Glekas A, Doubrovin M, Balatoni J, Namavari M, Beresten T, et al. Molecular imaging of EGFR kinase activity in tumors with 124I-labeled small molecular tracer and positron emission tomography. Mol Imaging Biol 2006; 8:262-77. 49. Crow DM, Williams L, Colcher D, Wong JY, Raubitschek A, Shively JE. Combined radioimmunotherpy and chemotherapy of breast tumor with Y-90-labeled anti-HER2 and anti-CEA antibodies with taxol. Bioconjugate Chem 2005; 16:1117-25. 50. Blend M, Stastny J, Swanson SM, Brechbiel MW. Labeling anti-HER2/neu monoclonal antibodies with 111 In and 90Y using a bifunctional DTPA chelating agent. Cancer Biother Radiopharm 2003; 18:355-63.

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