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Jan 28, 2014 - 177Lu-Labeled Antibodies for EGFR-Targeted SPECT/CT Imaging and Radioimmunotherapy in a Preclinical Head and Neck Carcinoma Model ... e...
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Lu-Labeled Antibodies for EGFR-Targeted SPECT/CT Imaging and Radioimmunotherapy in a Preclinical Head and Neck Carcinoma Model Zhaofei Liu,*,†,‡ Teng Ma,†,‡ Hao Liu,†,‡ Zhongxia Jin,†,‡ Xianlei Sun,†,‡ Huiyun Zhao,† Jiyun Shi,† Bing Jia,†,‡ Fang Li,§ and Fan Wang*,†,‡ †

Medical Isotopes Research Center and ‡Department of Radiation Medicine, School of Basic Medical Sciences, Peking University, Beijing 100191, China § Department of Nuclear Medicine, Peking Union Medical College Hospital, Beijing 100857, China S Supporting Information *

ABSTRACT: Epidermal growth factor receptor (EGFR) has been well characterized as an important target for cancer therapy. Immunotherapy based on EGFR-specific antibodies (e.g., panitumumab and cetuximab) has shown great clinical promise. However, increasing evidence has indicated that only a subgroup of patients receiving these antibodies will benefit from them, and even patients who do initially experience a major response may eventually develop therapeutic resistance. In this study, we investigated whether panitumumab and cetuximab can serve as delivery vehicles for tumor-targeted radionuclide therapy in a preclinical tumor model that did not respond to immunotherapy. The in vitro toxicity and cell binding properties of panitumumab and cetuximab were characterized. Both antibodies were conjugated with 1,4,7,10-tetraazadodecaneN,N′,N″,N‴-tetraacetic acid (DOTA) and radiolabeled with 177Lu. Small-animal SPECT/CT, biodistribution, and radioimmunotherapy (RIT) studies of 177Lu-DOTA−panitumumab (177Lu-Pan) and 177Lu-DOTA−cetuximab (177Lu-Cet) were performed in the UM-SCC-22B tumor model. Both 177Lu-Pan and 177Lu-Cet exhibited favorable tumor targeting efficacy. The tumor uptake was 20.92 ± 4.45, 26.10 ± 5.18, and 13.27 ± 1.94% ID/g for 177Lu-Pan, and 15.67 ± 3.84, 15.72 ± 3.49, and 7.82 ± 2.36% ID/g for 177Lu-Cet at 24, 72, and 120 h p.i., respectively. RIT with a single dose of 14.8 MBq of 177Lu-Pan or 177LuCet significantly delayed tumor growth. 177Lu-Pan induced more effective tumor growth inhibition due to a higher tumor uptake. Our results suggest that panitumumab and cetuximab can function as effective carriers for tumor-targeted delivery of radiation, and that RIT is promising for targeted therapy of EGFR-positive tumors, especially for those tumors that are resistant to antibody-based immunotherapy. KEYWORDS: epidermal growth factor receptor, antibody, small-animal imaging, targeted therapy

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of patients with refractory metastatic colon cancer.5 Cetuximab (Erbitux), a chimeric anti-EGFR IgG1, is approved by the FDA for the treatment of colorectal cancer and head and neck cancer.6 Similar to panitumumab, cetuximab also competitively inhibits the binding of EGF and other ligands to EGFR. Both cetuximab and panitumumab exhibited high EGFR-targeting specificity7−11 and clinical promise.12,13 However, antibodybased EGFR signaling inhibitors (e.g., panitumumab and cetuximab) have limitations for clinical cancer therapy. First, clinical trials have indicated that only a subgroup of patients receiving these agents will benefit from them, and the response rate is usually less than 40%.4,14 Second, EGFR levels determined by immunohistochemistry (IHC) or enzyme-linked

INTRODUCTION The epidermal growth factor (EGF) receptor (EGFR) is a transmembrane tyrosine kinase protein overexpressed in the majority of epithelial malignancies.1 EGFR-mediated signaling pathways play a pivotal role in the progression of tumor growth and metastasis, and EGFR overexpression is typically associated with an aggressive malignant phenotype and poor prognosis in epithelial cancers.2,3 In the past decades, EGFR has been one of the most comprehensively studied molecular targets in oncology therapeutics. Current clinically available EGFRtargeted inhibitors include antibodies that bind to the extracellular domain of the receptor and small molecule tyrosine kinase inhibitors (TKIs) that selectively inhibit the kinase activity of the receptor.4 Panitumumab (Vectibix) is a Food and Drug Administration (FDA)-approved fully human IgG2 monoclonal antibody (mAb) that targets EGFR and competitively inhibits EGF binding to EGFR. Panitumumab is indicated for the treatment © 2014 American Chemical Society

Received: Revised: Accepted: Published: 800

August 22, 2013 January 10, 2014 January 28, 2014 January 28, 2014 dx.doi.org/10.1021/mp4005047 | Mol. Pharmaceutics 2014, 11, 800−807

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mice and BALB/c nude mice (4−5 weeks of age) were obtained from Department of Experimental Animal, Peking University Health Science Center (Beijing, China). 177LuCl3 solution was obtained from Perkin-Elmer. Panitumumab was purchased from Amgen Inc. Cetuximab was purchased from ImClone Systems Inc. NHS-Rhodamine ester was purchased from Pierce. Cell Culture and Animal Model. UM-SCC-22B human head and neck squamous carcinoma cell line was obtained from the University of Michigan.10 Cells were grown in high glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C in humidified atmosphere containing 5% CO2. All animal experiments were performed in accordance with the Guidelines of Peking University Animal Care and Use Committee. For UM-SCC-22B tumor bearing animal model, UM-SCC-22B cells (5 × 106) were inoculated subcutaneously into the right thigh of female BALB/c nude mice. The animals were used for in vivo studies when the tumor size reached ∼200 mm3. Cell Proliferation Assay. The 3-(4,5-dimethylthiazolyl-2)2,5-diphenyltetrazolium bromide (MTT) assay was performed to measure the cell proliferation rate upon antibody treatment. UM-SCC-22B tumor cells (103 cells per well) were seeded in 96-well plates and incubated overnight to allow adherence. The cells were treated with various concentrations (ranging from 0.64 pmol/L to 0.67 μmol/L) of panitumumab or cetuximab for 144 h, and then 50 μL of sterile filtered MTT (1.0 mg/mL) was added to each well. After incubating at 37 °C for 4 h, 100 μL of DMSO was added to each well and the plate was left in the dark for 2 h at room temperature. The absorbance at 570 nm was then measured using a plate reader. All studies were performed with 7 parallel samples. Cell Binding Assays. Cell competitive binding assays were performed to compare the EGFR binding affinity of panitumumab and cetuximab. The radioligands 125I-panitumumab (125I-Pan) and 125I-cetuximab (125I-Cet) were prepared using a previously described method.22 The UM-SCC-22B cells were incubated with radioligand (125I-Pan or 125I-Cet) in the presence of increasing concentrations of panitumumab or cetuximab in a 96-well filter plate. After 2 h of reaction at 4 °C, the plates were washed, and the PVDF filters were collected and measured in a gamma counter. The best-fit 50% inhibitory concentration (IC50) values were calculated by fitting the data with nonlinear regression using Graph Pad Prism 4.0 (GraphPad Software, Inc.). Experiments were performed twice with quadruplicate samples. 177 Lu Radiolabeling. Panitumumab and cetuximab were first conjugated with DOTA to produce DOTA−panitumumab and DOTA−cetuximab, respectively.10 177LuCl3 (370 MBq) was diluted in 300 μL of 0.2 M sodium acetate buffer (pH 5.5) and added to 250 μg of DOTA−panitumumab or DOTA− cetuximab. The reaction mixture was incubated for 1 h at 42 °C with constant shaking. 177Lu-DOTA−panitumumab (177LuPan) or 177Lu-DOTA−cetuximab (177Lu-Cet) was then purified by PD-10 column using phosphate-buffered saline (PBS) as the mobile phase. 177Lu-labeled antibodies were generated at an average yield of ∼80%, and the radiochemical purity was >95% after PD-10 column purification. Small-Animal SPECT/CT Imaging. Small-animal SPECT/ CT scanning of UM-SCC-22B tumor-bearing nude mice was performed using a NanoSPECT/CT tomograph (Bioscan Inc.). Each mouse was injected via tail vein with 37 MBq of 177Lu-Pan

immunosorbent assay (ELISA) fail to serve as an outcome predictor of antibody therapy.15,16 Patients with high EGFR expression are not certain to benefit from anti-EGFR antibodybased immunotherapy. In fact, several patients with EGFRnegative tumors have been reported to demonstrate response to cetuximab therapy.15 Third, even patients who do initially experience a major response may eventually develop therapeutic resistance to EGFR antibodies after several rounds of therapy.4 In contrast to “naked” antibody-based immunotherapy, other strategies have also been extensively investigated for cancer therapy. For example, antibodies can function as carriers for cancer-targeted specific delivery of cytotoxic substances (such as radioisotopes, drugs, and toxins). The resulting immunoconjugates have shown promising results for cancer therapy. At least in the case of radioimmunoconjugates (antibodies labeled with radioisotopes), both preclinical and clinical studies have demonstrated that they are more effective than immunotherapy with unconjugated antibodies.10,17−19 Radioimmunotherapy (RIT) offers the opportunity to selectively kill tumor cells while sparing normal tissues, and not every tumor cell must be targeted by antibodies. Instead, tumor cells can be destroyed by the “cross-fire” effect. In addition, the tumor therapeutic efficacy of RIT relies on the radiation dosimetry delivered to the tumor, and higher tumor uptake of a RIT agent is expected to cause higher antitumor effect. Therefore, the therapeutic efficacy of RIT can be predicted by determining the tumor uptake of the RIT agent via positron emission tomography (PET) or single-photon-emission computed tomography (SPECT) imaging. In our recent study, we found that RIT with 90Y-labeled panitumumab exhibited a significant antitumor effect in a mouse model that did not respond to panitumumab-based immunotherapy.10 90Y is a pure β− emitter and is normally not used for PET and SPECT imaging purposes. The corresponding 111In- or 86Y-labeled compound is thus usually used as the imaging surrogate for biodistribution and dosimetry determination.20,21 By contrast, 177Lu is a low-energy β− emitter with both β and γ emissions. The low β− energy can be used for radionuclide therapy, while the presence of γ emission allows SPECT imaging for quantification of biodistribution and determination of radiation dosimetry. In this study, we compared the in vitro and in vivo EGFR-targeting properties as well as the RIT efficacy of two 177Lu-labeled anti-EGFR antibodies (panitumumab and cetuximab) side-by-side in a UM-SCC-22B human head and neck squamous carcinoma mouse model, which was resistant to immunotherapy with either panitumumab or cetuximab. Our aim was to investigate which antibody would be more suitable for serving as a delivery vehicle for EGFR-targeted therapy of tumors that do not respond to immunotherapy.

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EXPERIMENTAL SECTION Chemicals and Reagents. The bifunctional chelator 1,4,7,10-tetraazadodecane-N,N′,N″,N‴-tetraacetic acid (DOTA) was purchased from Macrocyclics, Inc. 1-Ethyl-3-[3(dimethylamino)propyl]carbodiimide (EDC), N-hydroxysulfonosuccinimide (SNHS), and Chelex 100 resin were purchased from Sigma-Aldrich. Water and all buffers were passed through a Chelex 100 column (1 × 15 cm) before use in DOTA conjugation and radiolabeling procedures to ensure that aqueous buffers were metal free. PD-10 desalting columns were purchased from GE Healthcare. Female BALB/c normal 801

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Figure 1. (A) In vitro cytotoxic effect of panitumumab or cetuximab on UM-SCC-22B tumor cells. The cells were treated with various concentrations of panitumumab or cetuximab. After 144 h of incubation, the cell proliferation was determined by MTT assay (n = 7, mean ± SD). (B) Inhibition of 125I-Pan binding to EGFR on UM-SCC-22B cells by panitumumab and cetuximab (n = 4, mean ± SD). (C) Inhibition of 125I-Cet binding to EGFR on UM-SCC-22B cells by panitumumab and cetuximab (n = 4, mean ± SD).

Figure 2. Representative whole-body posterior NanoSPECT/CT images of UM-SCC-22B-bearing nude mice at 72 h after injection of 37 MBq of 177 Lu-Pan or 177Lu-Cet. For the blocking experiment, the tumor mice were coinjected with 50 mg/kg mouse body weight of cetuximab and 37 MBq of 177Lu-Cet (n = 4 per group). Tumors are indicated by white arrows.

or 177Lu-Cet (n = 4 per group). For the blocking experiment, the tumor mice were coinjected with 50 mg/kg body weight of cetuximab and 37 MBq of 177Lu-Cet. At 72 h postinjection (p.i.), the mice were anesthetized by inhalation of 2% isoflurane in oxygen and imaged using a NanoSPECT/CT camera. A total of 24 projections were acquired in a 256 × 256 acquisition matrix with a minimum of 50,000 counts per projection. Images were reconstructed using an ordered-subset expectation maximization (OSEM) algorithm. Prior to each SPECT session, cone beam CT (180 projections, 1 s/projection, 45 kVp) images were acquired using the NanoSPECT/CT system. The SPECT and CT fusion images were obtained using the automatic fusion feature of the InVivoScope software (Bioscan Inc., Washington, DC, USA). Biodistribution. UM-SCC-22B tumor-bearing mice were randomly divided into six groups (n = 5 per group). Each mouse was injected with 370 kBq of 177Lu-Pan or 177Lu-Cet via tail vein. At 24 h, 72 h, and 120 h p.i., mice were sacrificed, and tissues of interest (blood, heart, lung, liver, spleen, kidney, stomach, intestine, bone, muscle, and tumor) were harvested,

weighed, and counted. Organ uptake was calculated as percentage of injected dose per gram of tissue (% ID/g). Therapy Studies. UM-SCC-22B tumor-bearing nude mice with a uniform tumor size of ∼200 mm3 were divided into five groups. Each group was treated with PBS, panitumumab (10 mg/kg body weight every 3 days for four doses), cetuximab (10 mg/kg body weight every 3 days for four doses), 177Lu-Pan (14.8 MBq in a single dose), or 177Lu-Cet (14.8 MBq in a single dose) (n = 6 per group). Tumor size and body weight were measured every other day. The tumor volume was calculated using the following formula: volume = length × width2/2. Intratumor Microdistribution. Panitumumab or cetuximab was conjugated with NHS-Rhodamine ester using a previously described method.23 The Rhodamine−panitumumab (Rhodamine-Pan) and Rhodamine−cetuximab (Rhodamine-Cet) conjugates were purified by PD-10 desalting column. Nude mice bearing UM-SCC-22B tumor xenografts were injected with 300 μg of Rhodamine-Pan or RhodamineCet via tail vein. At 0, 10, or 48 h p.i., each mouse was sacrificed and perfused through the heart with 25 mL of PBS. The tumors 802

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were collected, frozen, and cut into 5 μm thick slices. The tumor slices were fixed with ice-cold acetone for 10 min and allowed to dry in the air for 30 min. After washing with PBS and blocking in 10% FBS for 1 h, the tumor slices were incubated with rat anti-mouse CD31 antibody (1:100; BD Biosciences) and then visualized with FITC-conjugated donkey anti-rat secondary antibody (1:200; Jackson Immuno-Research Laboratories, Inc.) under a microscope. Images were taken under the same conditions and displayed at the same scale. Statistical Analysis. Quantitative data are expressed as means ± SD. Means were compared using one-way analysis of variance (ANOVA) and Student’s t test. P values 80% compared with untreated cells. Cell Binding Assays. The EGFR-binding affinity of panitumumab was compared with cetuximab by competition binding assay using 125I-Pan or 125I-Cet as a radioligand. As shown in Figure 1B,C, both cetuximab and panitumumab inhibited the binding of 125I-Pan or 125I-Cet to UM-SCC-22B cells in a concentration-dependent manner. The IC50 values for panitumumab and cetuximab were (4.41 ± 0.14) × 10−10 M and (1.49 ± 0.15) × 10−10 M, respectively, using 125Ipanitumumab as the radioligand, and (3.60 ± 0.06) × 10−10 M and (1.03 ± 0.04) × 10−10 M, respectively, using 125Icetuximab as the radioligand. These two sets of cell-binding assays both demonstrated that cetuximab possessed a slightly higher in vitro UM-SCC-22B binding affinity than panitumumab. Small-Animal SPECT/CT Imaging. Small-animal SPECT/ CT imaging studies were performed to investigate the in vivo behaviors of 177Lu-Pan and 177Lu-Cet. Representative wholebody SPECT/CT images of UM-SCC-22B tumor-bearing mice at 72 h postinjection of 177Lu-Pan or 177Lu-Cet are shown in Figure 2. The SPECT/CT images of 177Lu-Pan and 177Lu-Cet exhibited a similar radiotracer distribution pattern to that observed in the planar gamma images (Figure S1 in the Supporting Information), but with much higher spatial resolution. The UM-SCC-22B tumors were clearly visualized with high tumor-to-background contrast for both 177Lu-labeled antibodies. Prominent uptake was also observed in the liver for both 177Lu-labeled antibodies, suggesting that they were mainly excreted through the hepatobiliary route. 177Lu-Pan exhibited much higher tumor uptake than did 177Lu-Cet (Figure 2A,B). Figure 2C shows the representative SPECT/CT of 177Lu-Cet at 72 h p.i. in the presence of excess cetuximab. Coinjection of an excess dose of cetuximab resulted in >90% blockage of tumor uptake for 177Lu-Cet, indicating the EGFR targeting specificity of 177Lu-Cet. Biodistribution Studies. Biodistribution studies of 177LuPan and 177Lu-Cet were performed in nude mice bearing UMSCC-22B xenografts. As shown in Figure 3A, the uptake of 177 Lu-Pan in UM-SCC-22B tumors was 20.92 ± 4.45, 26.10 ± 5.18, and 13.27 ± 1.94% ID/g at 24, 72, and 120 h p.i., respectively. Radioactivity level of 177Lu-Pan in blood was 5.43

Figure 3. (A) Biodistribution of 177Lu-Pan (370 kBq) in UM-SCC22B tumor-bearing mice at 24, 72, and 120 h after injection. (B) Biodistribution of 177Lu-Cet (370 kBq) in UM-SCC-22B tumorbearing mice at 24, 72, and 120 h after injection. Data were expressed as percentage of injected dose per gram (% ID/g) (n = 5, mean ± SD).

± 1.55% ID/g at 24 h p.i, followed by a rather rapid clearance by the end of 120 h (0.51 ± 0.34% ID/g). 177Lu-Pan was cleared predominantly through the hepatobiliary route as evidenced by higher liver uptake at all time points examined (27.41 ± 2.55, 23.54 ± 5.45, and 20.35 ± 0.60% ID/g at 24, 72, and 120 h p.i., respectively). The other organs such as heart, lung, kidney, stomach, bone, and muscle showed very low uptake. 177Lu-Cet also showed predominant liver uptake, while the uptake in heart, lung, kidney, stomach, intestine, bone, and muscle was relatively low. The liver uptake of 177Lu-Cet was significantly higher than that of 177Lu-Pan at 72 h (37.65 ± 6.77 vs 23.54 ± 5.45% ID/g, P < 0.05) and 120 h (25.69 ± 2.88 vs 20.35 ± 0.60% ID/g, P < 0.05) p.i. (Figure 3 and Figure S2A in the Supporting Information). The uptake of 177Lu-Cet in UMSCC-22B tumors was 15.67 ± 3.84, 15.72 ± 3.49, and 7.82 ± 2.36% ID/g at 24, 72, and 120 h p.i., respectively. The tumor uptake of 177Lu-Cet was lower than that of 177Lu-Pan at all time points examined, and the differences were statistically significant at 72 and 120 h p.i. (P < 0.05, Figure 3 and Figure S2B in the Supporting Information). The area under the curve (AUC) of 177Lu-Pan uptake in tumors was greater than that of 177 Lu-Cet (Figure S2B in the Supporting Information), indicating a greater radiation exposure of the tumor to 177LuPan compared with 177Lu-Cet. Tumor Therapy Studies. We investigated the in vivo tumor treatment efficacy of panitumumab- and cetuximabbased immunotherapy, as well as 177Lu-Pan- and 177Lu-Cetbased radioimmunotherapy. As shown in Figure 4A, a timedependent increase in tumor volume was observed in the PBS and immunotherapy groups, in which the tumors demonstrated average volumes over 1500 mm3 on day 12. These results indicated that treatment with panitumumab or cetuximab was well tolerated in UM-SCC-22B tumor mice. By contrast, a single-dose injection (14.8 MBq) of 177Lu-Pan or 177Lu-Cet 803

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homogeneously in UM-SCC-22B tumors compared with that of cetuximab. These results suggest that the relatively slow penetration of cetuximab through perivascular tissues was at least partially responsible for the lower UM-SCC-22B tumor uptake of cetuximab compared with panitumumab.

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DISCUSSION The promising antitumor activity of EGFR inhibitors (e.g., mAbs and TKIs) in the clinic has led to EGFR being one of the most investigated targets for tumor therapy.4 However, the intrinsic and acquired resistance of tumors to EGFR inhibitors is also well recognized. One approach that may revolutionize the treatment of nonresponsive tumors involves modifying EGFR-targeting mAbs to deliver cytotoxic substances (toxins, radioisotopes, or cytokines) to tumors. In the current study, we compared the in vitro and in vivo EGFR targeting properties of two anti-EGFR mAbs (panitumumab and cetuximab) and evaluated whether they can function as effective carriers for tumor-targeted delivery of therapeutics (e.g., radioisotopes). Increasing clinical evidence has indicated that EGFR-positive tumors are not certain to benefit from treatment with antiEGFR mAbs, although the detailed mechanism of tumor resistance still requires further investigation.24,25 We found that EGFR-positive UM-SCC-22B tumor did not respond to treatment with either panitumumab or cetuximab. Our in vitro therapy study showed that mAb treatment did not have a significant antitumor effect on UM-SCC-22B tumor cells (Figure 1A). Immunotherapy studies in the nude mouse model using several doses of panitumumab or cetuximab also failed to demonstrate any therapeutic effect (Figure 4A). These results are consistent with previous reports.10,17 Although both unconjugated mAbs had no significant effect on UM-SCC-22B tumor growth inhibition, our small-animal SPECT/CT imaging and biodistribution studies demonstrated that both mAbs labeled with 177Lu located to the tumor with high contrast compared to other organs except for the liver due to the hepatic clearance of antibodies (Figures 2 and 3). EGFR specificity was also confirmed by a blocking study, which is consistent with previous studies.7,26 Considering the high tumor accumulation of the antibodies, we speculated that 177Lu-labeled panitumumab and cetuximab could be used for EGFR-targeted RIT. We used a single dose of 14.8 MBq of 177Lu-labeled mAbs for RIT of immunotherapy-nonresponsive UM-SCC-22B tumors in this study. No evident weight loss was observed after 177Lu-Pan or 177 Lu-Cet treatment (Figure 4B), indicating that 14.8 MBq of 177 Lu-labeled mAbs was below the maximum tolerated dose of the animals. RIT significantly delayed tumor growth in both the 177 Lu-Pan and 177Lu-Cet treatment groups (Figure 4A). A single dose of around 10 μg of antibody was used for RIT, which is much lower than that used in immunotherapy (approximately 200 μg/dose for four doses) with unconjugated mAbs. Thus, we believe that the therapeutic effect of 177Lu-Pan and 177Lu-Cet resulted from the radiation dose delivered by panitumumab and cetuximab, and not by the immunotherapeutic effect of mAbs. The therapeutic efficacy of RIT depends on the tumor uptake value and the radiation dosimetry that is delivered to tumors. Therefore, the much higher antitumor effect of 177LuPan compared with 177Lu-Cet (Figure 4A) was most likely due to the higher tumor accumulation of 177Lu-Pan (Figure 3, Figure S2B in the Supporting Information). The efficient tumor targeting of an antibody relies on many factors, including

Figure 4. (A) Growth curves of UM-SCC-22B tumors in five groups of mice after injection of PBS, four doses of cold panitumumab (10 mg/kg, every 3 days), four doses of cold cetuximab (10 mg/kg, every 3 days), 14.8 MBq of 177Lu-Pan, and 14.8 MBq of 177Lu-Cet, respectively (mean ± SD, n = 6 per group). (B) Body weight change over time of the UM-SCC-22B tumor-bearing nude mice administrated with PBS, 14.8 MBq of 177Lu-Pan, or 14.8 MBq of 177Lu-Cet (mean ± SD, n = 6 per group).

produced dramatically enhanced antitumor activity compared with PBS. The tumors in the 14.8 MBq 177Lu-Cet group demonstrated significant growth inhibition compared with the control group from day 6 (P < 0.05). On day 30, a tumor relapse was observed in this group, and the average tumor volume exceeded 1500 mm3 on day 36. In the 14.8 MBq 177LuPan treatment group, tumor growth was almost completely inhibited up to day 36 posttreatment. Although the radioimmunotherapy groups exhibited significantly enhanced antitumor effect, we observed neither mortality nor noticeable body weight loss among the mice treated with 177Lu-Pan or 177 Lu-Cet (Figure 4B). These results suggest that the 14.8 MBq dose used in this study had no observable toxicity on mice. Microdistribution of Antibodies in Tumors. To investigate the location of antibodies in tumor tissues, Rhodamine-Pan or Rhodamine-Cet was injected into nude mice bearing UM-SCC-22B tumor xenografts. After circulation for 0−48 h, the tumor tissues were harvested, cut into slices, and costained with CD31. As shown in Figure 5, both antibodies demonstrated patterns of increased perfusion in the tumor tissues from 0 to 48 h. Immediately after injection (0 h), both antibodies were concentrated near the tumor vasculature, whereas at 48 h, the antibodies were homogenously distributed within the tumors. Compared with the location of cetuximab, panitumumab exhibited a longer diffusive distance from the blood vessels at 0 h (Figure 5B). At 10 and 48 h, the fluorescence signal of panitumumab diffused slightly more 804

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Figure 5. Localization of panitumumab and cetuximab in UM-SCC-22B tumors. (A) Immunofluorescence staining of mouse CD31 was performed with tumor tissues harvested at 0, 10, and 48 h after injection of Rhodamine-Pan or Rhodamine-Cet. Red color from Rhodamine for panitumumab or cetuximab, green color from FITC for CD31, blue color from DAPI for visualization of nuclei. (B) Magnified images of the boxed areas of A.

Antibody pharmacokinetics also affect in vivo tumor targeting efficacy. Essentially, a prolonged circulation time in the blood pool leads to an increased chance of accumulation in the tumor tissue. We found that panitumumab was cleared from the circulation more slowly than cetuximab (Figure S4 in the Supporting Information), which is consistent with the results of Nayak et al.26 Panitumumab is a mAb of IgG2 subclass, whereas cetuximab belongs to the IgG1 subclass. Since IgG2 has a much lower binding affinity to Fc receptors than IgG1, we used mIgG blocking to avoid the effect of Fc receptors in the mouse blood pool on the pharmacokinetics of panitumumab and cetuximab. After blocking with mIgG, panitumumab still demonstrated slower blood clearance than cetuximab (Figure S4 in the Supporting Information). Another interesting finding is that the liver uptake of 177Lu-Pan was lower than that of 177Lu-Cet at all time points examined. Although the underlying mechanism is unclear, this observation emphasizes that 177Lu-Pan possessed superior properties (higher tumor uptake and lower liver uptake) to 177Lu-Cet for tumor-targeted RIT in the UM-SCC22B tumor mouse model. Note that panitumumab is a fully human IgG2 antibody, while cetuximab is a chimeric human mouse IgG1. Antibodies of different origins and subclasses may have significantly different in vivo pharmacokinetics in different animal models. Future studies investigating the RIT effects of 177 Lu-Pan and 177Lu-Cet in non-mouse models may be needed to further verify the findings of this study.

affinity, molecular weight, penetration, internalization, and catabolism properties of the antibody, as well as in vivo pharmacokinetics and nonspecific accumulation. Both panitumumab and cetuximab bind domain III of EGFR.27 Although they recognize different epitopes, panitumumab and cetuximab competed against each other for EGFR binding (Figure 1B,C), presumably due to the steric hindrance of the bulky IgG molecules. Two sets of cell competitive binding assays validated that cetuximab possessed higher EGFR-binding affinity than panitumumab (Figure 1B,C). This higher affinity could result in more rapid antigen recognition, and would subsequently lead to the higher binding and internalization of cetuximab than panitumumab in the UM-SCC-22B tumor cells at the early time points (Figure S3 in the Supporting Information). Under in vivo conditions, tumor-targeting efficacy requires sufficient binding affinity of the antibody to mediate tumor localization. However, it has been well recognized that antibodies with high affinity are certain to lead to high tumor accumulation. The proposed “binding site barrier” effect28,29 predicts that as the affinity of antibodies increases, the amount of antibodies available to diffuse into the tumor decreases, leading to a reduction in tumor penetration.30 Our tumor microdistribution study validated that cetuximab demonstrated impaired tumor penetration compared with panitumumab, which, at least in part, led to the lower tumor uptake and subsequently the lower antitumor effect of 177Lu-Cet compared with 177Lu-Pan. 805

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CONCLUSION In this study, we described the in vitro and in vivo characterization of two 177Lu-labeled anti-EGFR mAbs, namely, panitumumab and cetuximab, in a preclinical mouse model. Both 177Lu-Pan and 177Lu-Cet exhibited favorable tumor targeting efficacy, and significant antitumor effects were achieved after a single-dose injection of 177Lu-Pan or 177LuCet. 177Lu-Pan induced more effective tumor growth inhibition due to its higher tumor accumulation. Our results suggest that panitumumab and cetuximab can serve as effective carriers for tumor-targeted delivery of radiation, and that radioimmunotherapy is a promising strategy for targeted therapy of EGFRpositive tumors, especially for immunotherapy-nonresponsive tumors. Their high tumor-targeting efficacy would also allow panitumumab and cetuximab to be modified with other therapeutic moieties, such as chemical drugs and toxins, for EGFR-targeted cancer therapy.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information and methods; representative planar gamma images of UM-SCC-22B tumor-bearing mice after injection of 177Lu-Pan or 177Lu-Cet (with or without blocking); radioactivity uptake (% ID/g) in liver and tumor of UM-SCC22B tumor-bearing mice at 24, 72, and 120 h after injection of 177 Lu-Pan or 177Lu-Cet; cell uptake and internalization of 125IPan or 125 I-Cet in UM-SCC-22B tumor cells; blood pharmacokinetics of 125I-Pan or 125I-Cet in female normal BALB/c mice. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*Zhaofei Liu: Medical Isotopes Research Center, University, 38 Xueyuan Road, Beijing 100191, China; fax, 86-10-82802871; e-mail, [email protected]. *Fan Wang: Medical Isotopes Research Center, University, 38 Xueyuan Road, Beijing 100191, China; fax, 86-10-82801145; e-mail, [email protected].

Peking tel and Peking tel and

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Chao Liu and Dr. Xiaodan Shi for excellent technical assistance. This work was supported by “973” projects (2013CB733802 and 2011CB707703), National Natural Science Foundation of China (NSFC) projects (81125011, 81222019, 30930030, 81000625, and 81201127), grants from the Ministry of Science and Technology of China (2011YQ030114, 2012ZX09102301-018, and 2012BAK25B03-16), grants from the Ministry of Education of China (31300 and BMU20110263), grants from Beijing Natural Science Foundation (7132131 and 7132123), and a grant from Beijing Nova Program (Z121107002512010).

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