Epidermal Growth Factor Receptor–Targeted Radioimmunotherapy of Human Head and Neck Cancer Xenografts Using 90Y-Labeled Fully Human Antibody Panitumumab

Published OnlineFirst August 3, 2010; DOI: 10.1158/1535-7163.MCT-10-0444

Preclinical Development Molecular Cancer Therapeutics Epidermal Growth Factor Receptor–Targeted Radioimmunotherapy of Human Head and Neck Cancer Xenografts Using 90Y-Labeled Fully Human Antibody Panitumumab

Zhaofei Liu1, Yan Liu1, Bing Jia1, Huiyun Zhao1, Xiaona Jin2, Fang Li2, Xiaoyuan Chen3, and Fan Wang1

Abstract Panitumumab (ABX-EGF or Vectibix), the first fully human monoclonal antibody targeting epidermal growth factor receptor (EGFR), was approved by the Food and Drug Administration for treatment of patients with metastatic colorectal cancer. Here, we report for the first time the radioimmunotherapy − (RIT) of EGFR-positive human head and neck cancer in a nude mouse model using pure β emitter 90Y-labeled panitumumab. Biodistribution and planar γ-imaging studies were carried out with 111In- DOTA-panitumumab. The RIT efficacy of 90Y-DOTA-panitumumab was evaluated in UM-SCC-22B tumor model. CD31, Ki67, terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling, and H&E staining were done on UM-SCC-22B tumor sections after treatment. The tumor uptake of 111In-DOTA- panitumumab in UM-SCC-22B tumor-bearing nude mice was 26.10 ± 4.93, 59.11 ± 7.22, 44.57 ± 9.80, 40.38 ± 7.76, and 14.86 ± 7.23 % injected dose per gram of tissue at 4, 24, 72, 120, and 168 hours after injection, respectively. Immunotherapy with cold panitumumab (four doses of 10 mg/kg) did not cause significant antitumor effect. RIT with a single dose of 100 μCi 90Y-DOTA-panitumumab caused significant tumor growth delay and improved the survival in UM-SCC-22B tumor model. A single dose of 200 μCi 90Y-DOTA-panitumumab led to almost complete tumor regression (tumor volumes were 34.83 ± 11.11 mm3 and 56.02 ± 39.95 mm3 on days 0 and 46 after treatment, respectively). Histopathologic analysis of tumors and normal organs further validated the therapeutic efficacy and limited systemic toxicity of 90Y-DOTA-panitumumab. The high tumor uptake and prolonged tumor retention, as well as effective therapy, reveal that 90Y-DOTA-panitumumab may be a promising radioimmunotherapeutic agent to treat EGFR-positive solid tumors. Mol Cancer Ther; 9(8); 2297–308. ©2010 AACR.

Introduction or IV; ref. 3). In contrast to many other cancers in which metastasis is the primary cause of death, local or regional Head and neck squamous cell carcinoma (HNSCC) is an recurrences are the most common causes of treatment epithelial cancer arising in the mucosa of the upper aero- failure or death in patients with HNSCC (4). About 50% digestive tract. Each year, more than half a million new of patients who develop recurrent disease have a very cases of HNSCC are diagnosed in the world (1, 2). poor prognosis, with a median overall survival of only Although surgery or radiotherapy can generally lead to a 6 to 9 months (5). Therefore, an effective systemic treat- good prognosis for early-stage (stage I or II) HNSCC, there ment for the local or regional recurrences of HNSCC is is still a high failure rate for the advanced stage (stage III urgently needed to improve the survival rate of patients. Radioimmunotherapy (RIT) using radiolabeled monoclonal antibodies (mAb) directed against tumor-associated Authors' Affiliations: 1Medical Isotopes Research Center, Peking University; 2Department of Nuclear Medicine, Peking Union Medical antigens offers the opportunity to selectively irradiate College Hospital, Beijing, China and 3Laboratory for Molecular Imaging tumor cells while sparing normal tissues. An important and Nanomedicine, National Institute of Biomedical Imaging and Bioengineering, NIH, Bethesda, Maryland advantage of RIT compared with other treatment ap- Note: Supplementary material for this article is available at Molecular proaches is that radionuclides can kill adjacent tumor Cancer Therapeutics Online (http://mct.aacrjournals.org/). cells. As a consequence, not every tumor cell has to be Z. Liu and Y. Liu contributed equally to this work. targeted by antibodies but tumor cells still can be de- Corresponding Author: Fan Wang, Medical Isotopes Research Center, stroyed by the cross-fire effect. RIT has been implemen- Peking University, 38 Xueyuan Road, Beijing 100191, China. Phone: 86- ted effectively for the treatment of non–Hodgkin's 10-82801145; Fax: 86-10-82801145. E-mail: [email protected] lymphomas, and two Food and Drug Administration doi: 10.1158/1535-7163.MCT-10-0444 (FDA)–approved drugs, Zevalin and Bexxar (anti-CD20 ©2010 American Association for Cancer Research. mAbs labeled with 90Y and 131I, respectively), have been

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successfully used to treat relapsed or refractory non– use in DOTA conjugation and radiolabeling procedures Hodgkin's lymphomas (6). However, the success of RIT to ensure that aqueous buffers were metal-free. PD-10 in the therapy of bulky solid tumors has generally been desalting columns were purchased from GE Healthcare. limited possibly owing to the relative radioresistance of Female BALB/c nude mice (4–5 weeks of age) were ob- solid tumors and an inability to deliver sufficient dose tained from the Department of Experimental Animal, to bulky tumor without substantial bone marrow toxicity Peking University Health Science Center (Beijing, 90 (7). The intrinsic radiosensitivity of HNSCC (8–10) and China). YCl3 solution (in 0.05 mol/L HCl) was ob- the relative effectiveness of RIT for the treatment of small tained from Perkin-Elmer. Panitumumab was obtained lesions or minimal residual diseases justify the develop- from Amgen, Inc. ment of RIT for the treatment of minimal residua and recurrence of HNSCC. Cell culture and animal models Epidermal growth factor receptor (EGFR), a member of UM-SCC-22B human head and neck squamous carcinoma the ErbB family of receptor tyrosine kinases, is over- cell line was obtained from the University of Michigan expressed in a variety of tumors, including head and (14). MDA-MB-435 human melanoma (15, 16) cell line neck (80–100%), renal (50–90%), lung (40–80%), breast was purchased from the American Type Culture Collection. (14–90%), colorectal (25–77%), ovarian (25–70%), prostate UM-SCC-22B cells were grown in DMEM supplemented (39–47%), glioma (40–63%), pancreas (30–50%), and blad- with 10% fetal bovine serum (FBS) at 37°C in humidified der (31–48%; ref. 11). The expression of EGFR is associated atmosphere containing 5% CO2. MDA-MB-435 cells were with a more aggressive malignant phenotype and poor grown in L-15 medium supplemented with 10% FBS at prognosis. It has been reported that patients with high 37°C in humidified atmosphere containing 100% air. EGFR expression fared significantly worse for survival For UM-SCC-22B tumor and MDA-MB-435 tumor- 6 (12). For these reasons, EGFR has been chosen as an bearing animal models, the UM-SCC-22B cells (3 × 10 ) 6 μ attractive candidate for targeted therapies of tumors, or MDA-MB-435 cells (3 × 10 ) at a volume of 100 L including head and neck carcinoma. Recent studies with were inoculated s.c. into the right thigh (for RIT studies) γ cetuximab (Erbitux), a FDA-approved chimeric mAb or right upper flanks (for biodistribution and -imaging inhibitor of EGFR, have shown clinical benefits across studies) of female BALB/c nude mice. The animals were the range of treatment settings in advanced head and used for therapy studies when the tumor size reached ∼ 3 neck cancer (4). Panitumumab (also known as ABX- 50 mm , whereas the animals were used for biodis- EGF or Vectibix), another anti-EGFR mAb, is the first tribution and imaging studies when the tumor size ∼ 3 fully human mAb approved by the FDA. Panitumumab reached 200 mm . All animal experiments were done has been used to the treatment of EGFR-expressing metas- in accordance with the Guidelines of Peking University tatic colorectal cancer with disease progression (13). The Health Science Center Animal Care and Use Committee. clinical data indicate that panitumumab is well tolerated, does not require premedication, and is associated with a Immunofluorescence staining very low incidence of human anti-mouse antibodies. UM-SCC-22B cell staining study was done as previous- Currently, the radiometal isotope 90Y (maximum ly described with some modifications (17). Briefly, 70% to − β energy, 2.27 MeV; half-life, 2.67 days) attracts increas- 85% confluent tumor cells grown in 35-mm MatTek glass- ing interest in the field of RIT. 90Y is a pure high-energy bottomed culture dishes were fixed using 4% paraformal- − β emitter and is considered to be retained intracellularly dehyde for 10 minutes. After blocking with 10% FBS in after antibody internalization, thereby increasing the PBS for 30 minutes, the cells were incubated with panitu- μ retention time of the isotope within the tumor (6). In this mumab (5 g/mL) for 1 hour. The cells were then washed – study, we labeled the fully human anti-EGFR mAb pani- with PBS and stained with FITC-coupled mouse anti tumumab with 90Y and investigated the therapeutic human IgG (hIgG; 1:100; Sigma) for 30 minutes. After potential of 90Y-panitumumab in UM-SCC-22B human the final wash with PBS, the cells were visualized using head and neck cancer xenografted mice. a Leica TCS-NT confocal microscope (Wetzler). Five-micrometer-thick UM-SCC-22B tumor tissue sections were cut from the frozen blocks, allowed to dry in Materials and Methods the air, and stored at −80°C. For immunofluorescence staining, the frozen UM-SCC-22B tumor sections were All commercially available chemical reagents were of warmed to room temperature, fixed with ice-cold 4% analytic grade and used without further purification. paraformaldehyde, washed with PBS, permeabilized The bifunctional chelator 1,4,7,10-tetraazadodecane-N, with 0.2% Triton X-100 for 10 minutes, and blocked N′,N″,N′″-tetraacetic acid (DOTA) was purchased from with 10% FBS for 1 hour at room temperature. After Macrocyclics, Inc. 1-Ethyl-3-[3-(dimethylamino)-propyl] a brief wash with PBS, the sections were incubated carbodiimide (EDC), N-hydroxysulfonosuccinimide with mAb panitumumab (10 μg/mL) for 1 hour at (SNHS), and Chelex 100 resin (50–100 mesh) were pur- 37°C and then visualized with FITC-coupled mouse chased from Sigma-Aldrich. Water and all buffers were anti-hIgG (1:100; Sigma) under a Leica TCS-NT confocal passed through a Chelex 100 column (1 × 15 cm) before microscope.

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EGFR-binding affinity of panitumumab at 37°C with constant shaking. 90Y-DOTA-panitumumab The EGFR-binding affinity of panitumumab was deter- was purified by the PD-10 column using PBS as the mined by saturation binding assay using previously eluent. The radioactive fractions containing 90Y-DOTA- described method with some modifications (18). Briefly, panitumumab were collected and passed through a 125I-panitumumab was prepared using the Iodogen 0.2-μm syringe filter for further in vitro and in vivo ex- method (19) and used as the radioligand. UM-SCC-22B periments. A similar procedure was used for 111In- cells were seeded in a Millipore 96-well filter plate DOTA-panitumumab and 90Y-DOTA-hIgG preparations. (105 per well) and then incubated with increasing concen- The radioimmunoreactive fraction of 90Y-DOTA- trations (0–17 nmol/L) of 125I-panitumumab. The total panitumumab was determined by incubating with volume for each well was adjusted to 200 μL with 1% bo- EGFR-positive UM-SCC-22B cells in suspension culture vine serum albumin (in PBS). The plate was incubated at under the condition of antigen excess (n = 3). The radio- 4°C for 2 hours. At the end of the incubation period, the immunoreactive fraction was calculated as follows: cells were filtered by the Millipore Multiscreen vacuum radioimmunoreactive fraction = bound activity on manifold and washed five times with cold PBS (pH 7.4). cells/totally added activity × 100%. The hydrophilic polyvinylidene difluoride filters were then collected, and the radioactivity was determined Biodistribution of 111In-DOTA-panitumumab using a gamma counter (Wallac 1470-002, Perkin-Elmer). UM-SCC-22B tumor-bearing mice (20–25 g) were ran- Nonspecific binding was determined in the presence of an domly divided into five groups (n = 4 per group). Ani- excess of cold panitumumab (>100-fold). A saturation mals were anesthetized by i.p. injection of sodium binding curve and Scatchard transformation were ob- pentobarbital at a dose of 45.0 mg/kg. Each mouse was K 111 tained by nonlinear regression analysis, and the d and injected with 185 kBq of In-DOTA-panitumumab via B 125 max (maximum number of binding sites) values of I- tail vein. The mice were sacrificed at 4, 24, 72, 120, and panitumumab were calculated using GraphPad Prism 168 hours after injection. Blood, heart, liver, spleen, lung, 4.0 software (GraphPad Software, Inc.). kidney, stomach, intestine, bone, muscle, and tumor were harvested, weighed, and measured for radioactivity in a DOTA conjugation and radiolabeling gamma counter. Organ uptake was calculated as percent- DOTA was activated by EDC and SNHS for 30 minutes age of injected dose per gram of tissue (% ID/g). Values with a molar ratio of DOTA/EDC/SNHS = 10:5:4 as pre- were expressed as mean ± SD (n = 4 per group). viously described (20). The DOTA-OSSu (10 μmol, calculated on the basis of SNHS) was added to panitumumab Planar γ-imaging γ (50 nmol) in 0.1 N NaHCO3 solution (pH 9.0). After stir- -Imaging was done on two UM-SCC-22B tumor- ring at 4°C overnight, DOTA-panitumumab was purified bearing mice. Animals were anesthetized with an i.p. in- by the PD-10 column. The concentration of DOTA- jection of sodium pentobarbital at a dose of 45.0 mg/kg. panitumumab was determined based on UV absorbance Each animal was administered ∼13 MBq of 111In-DOTA- at 280 nm using unconjugated panitumumab with known panitumumab in 0.2 mL of saline. Animals were placed concentrations as the standards. hIgG (as a control) was prone on a two-head γ-camera (GE Infinia Hawkeye) also conjugated with DOTA using the same protocol. equipped with a parallel-hole, middle-energy, and high- The number of DOTA per panitumumab was deter- resolution collimator. Anterior images were acquired at mined using previously reported method (20), and it 24, 72, and 96 hours after injection and stored digitally was calculated to be ∼9. To evaluate the effect of in a 128 × 128 matrix. The acquisition count limits were DOTA conjugation on the bioactivity of panitumumab, set at 200 k. competitive cell-binding assay was done using 125I- panitumumab as the EGFR-specific radioligand. Experi- RIT and immunotherapy ments were done on UM-SCC-22B cells. Briefly, 200,000 To assess the therapeutic efficacy of 90Y-DOTA-pani- counts per minute (cpm) of 125I-panitumumab in tripli- tumumab and cold panitumumab, UM-SCC-22B tumor- cate were added to UM-SCC-22B cells in the presence of bearing nude mice with a uniform tumor size of ∼50 increasing concentrations of unmodified panitumumab mm3 (9 days after inoculation) were chosen for this or DOTA-panitumumab. After washing the unbound study. For the 90Y-DOTA-panitumumab–based RIT radiotracer, the specific binding of 125I-panitumumab to study, the mice were segregated into five groups and UM-SCC-22B cells was analyzed by measuring the injected via tail vein with a single dose of 7.4 MBq radioactivity associated with the cells using a gamma (containing ∼6 μg DOTA-panitumumab; n =6)or3.7 ∼ μ n counter. The best-fit IC50 values were calculated by fit- MBq (containing 6 gDOTA-panitumumab; =5) ting the data with nonlinear regression using GraphPad of 90Y-DOTA-panitumumab, as well as 3.7 MBq (con- Prism 4.0 software. taining ∼6 μg DOTA-hIgG) of 90Y-DOTA-hIgG (n = 5), 90 ∼ μ n n YCl3 in 0.05 N HCl was added to DOTA-panitumumab panitumumab ( 6 g; = 5), and saline ( = 5), respec- conjugate at a reaction ratio of 0.68 μgofDOTA- tively. For the cold panitumumab-based immunothe- panitumumab per MBq of 90Y. The reaction mixture was rapy study, one group of six mice was injected via tail incubated in 0.2 N sodium acetate buffer for 30 minutes vein with four doses of panitumumab (10 mg/kg)

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every 3 days. The tumor size and body weight were Results measured daily. The tumor volume was calculated using the following formula: volume = 4/3π (1/2 length × In vitro EGFR targeting of panitumumab 1/2 width × 1/2 height). Mice were euthanized when UM-SCC-22B cells and tumor tissues were incubated the tumor size exceeded the volume of 1,500 mm3 with panitumumab and then visualized with FITC- or the body weight lost >20% of its original weight. coupled secondary antibody. It was found that panitu- Tumors, livers, and kidneys were harvested after the mumab bound strongly to both UM-SCC-22B cells and therapeutic studies and subjected to histopathologic tumor tissues (Fig. 1A). The staining was mostly on the examinations. Survival curve plots and Kaplan-Meier cell membrane, as EGFR is mainly expressed on the cell analysis were carried out using Prism 4.0 software. surface. K To determine the binding affinity ( d) and the number B 125 EGFR specificity of RIT of binding sites per UM-SCC-22B cell ( max)for I- To evaluate the EGFR-specific therapy of 90Y-DOTA- panitumumab, saturation binding assay was done. As panitumumab, we did the RIT study in MDA-MB-435 shown in Fig. 1B, panitumumab bound to EGFR on cell K tumor-bearing nude mice, which express very low level surface with a high affinity ( d of 1.33 ± 0.29 nmol/L). of EGFR (21). Tumor-bearing animals were separated Scatchard analysis revealed an average of 1.83 ± 0.01 × 105 into two groups (n = 6 per group) and received either available binding sites per UM-SCC-22B cell. 3.7 MBq of 90Y-DOTA-panitumumab or saline. These animals were then subjected to tumor size and body weight DOTA conjugation and radiolabeling measurement as described above. The competitive cell-binding assay revealed that DOTA- panitumumab inhibited the binding of 125I-panitumumab Histologic analysis to UM-SCC-22B cells in a dose-dependent manner After RIT studies, the UM-SCC-22B tumors and major (Fig.1C).TheIC50 values for DOTA-panitumumab and organs (livers and kidneys) of mice were harvested. panitumumab were 0.11 ± 0.02 and 0.10 ± 0.01 nmol/L, The tumor samples were immediately frozen in OCT respectively, showing that DOTA-panitumumab pos- medium (Sakura Finetek USA, Inc.) and then cut into sessed similar EGFR-binding avidity with panitumumab. 5-μm-thick slices for immunofluorescence staining. 90Y-DOTA-panitumumab was generated at an aver- Terminal deoxynucleotidyl transferase–mediated dUTP age yield of ∼80%, and the radiochemical purity was nick end labeling (TUNEL), CD31, and Ki67 staining >99% after PD-10 column purification. Using UM- were carried out on UM-SCC-22B tumor sections to SCC-22B cells, the immunoreactive fraction of 90Y- investigate the status of tumor apoptosis, tumor vascu- DOTA-panitumumab was measured to be 74.4 ± 2.9%. lature, and tumor cell proliferation, respectively. Fluo- rescent TUNEL assay was conducted by following the Biodistribution of 111In-DOTA-panitumumab Instruction Manual of In Situ Cell Death Detection kit Biodistribution studies were done using female (Roche) as previously described (22). CD31 and Ki67 BALB/c nude mice bearing UM-SCC-22B human head staining were done as previously described (22, 23). and neck cancer xenografts. The biodistribution data Briefly, frozen UM-SCC-22B tumor sections were fixed for 111In-DOTA-panitumumab are summarized in with cold acetone, blocked with 10% donkey serum, Fig. 2A. 111In-DOTA-panitumumab was cleared rapidly and incubated with rat anti-mouse CD31 antibody from blood with 30.97 ± 1.76, 10.54 ± 0.66, 2.00 ± 1.49, (1:100; BD Biosciences) and rabbit anti-human Ki67 1.62 ± 1.24, and 0.20 ± 0.20 % ID/g at 4, 24, 72, 120, (1:100, NeoMarkers), respectively. After washing with and 168 hours after injection, respectively. At these time PBS, the tumor sections were then incubated with points, the tumor uptake of 111In-DOTA-panitumumab FITC-conjugated goat anti-rat or Cy3-conjugated don- was 26.10 ± 4.93, 59.11 ± 7.22, 44.57 ± 9.80, 40.38 ± key anti-rabbit secondary antibodies (1:200; Jackson 7.76, and 14.86 ± 7.23 % ID/g, respectively. The maxi- ImmunoResearch Laboratories), respectively. The slides mum tumor uptake of 111In-DOTA-panitumumab oc- were mounted with 4′,6-diamidino-2-phenylindole and curred at 24 hours after injection and then decreased visualized under a Leica TCS-NT confocal microscope. slowly. Compared with other normal organs, 111In- To investigate the toxicity of RIT, liver and kidney sam- DOTA-panitumumab showed rapid and high uptake ples were fixed in 10% formalin/PBS and embedded in in liver, with 15.71 ± 1.36 % ID/g at 4 hours after injec- paraffin. Histologic sections of 5- to 8-μm thickness were tion, and then decreased to 7.05 ± 3.37 % ID/g at 168 prepared and stained with H&E for routine histopatho- hours after injection. Spleen uptake of 111In-DOTA-pani- logic examination. tumumab was relatively constant, ranging from 6.01 ± 0.50 to 6.46 ± 3.26 % ID/g from 4 to 168 hours after Statistical analysis injection. The area under curve of 111In-DOTA-panitu- Quantitative data are expressed as means ± SD. mumab uptake in tumors was significantly more than Means were compared using one-way ANOVA and that in other normal organs (Fig. 2B), indicating a much Student's t test. P values of <0.05 were considered sta- greater radiation exposure to the tumor than to normal tistically significant. organs.

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Figure 1. A, immunofluorescence staining of UM-SCC-22B tumor cells and tissues for EGFR using panitumumab as the primary antibody. B, saturation binding of 125I-panitumumab on UM-SCC-22B cells. C, in vitro inhibition of 125I-panitumumab binding to EGFR on UM-SCC-22B human head and neck cancer cells by DOTA-panitumumab and panitumumab. Points, mean (n = 3); bars, SD.

γ-Imaging then grew more quickly and reached an average volume The imaging studies of 111In-DOTA-panitumumab of 1,496.4 ± 91.18 mm3 on day 15 after treatment. The were done in BALB/c nude mice bearing UM-SCC-22B tumors in the 3.7 MBq 90Y-DOTA-panitumumab group human head and neck cancer xenografts. Figure 3 illus- displayed significant growth inhibition compared with trates the representative γ-images of tumor-bearing the control groups from day 4 to day 17 (P < 0.01), but mice administered with ∼13 MBq of 111In-DOTA- after 17 days, an obvious tumor relapse was observed. panitumumab. The planar γ-images showed a similar In the 7.4 MBq 90Y-DOTA-panitumumab group, the trend with the biodistribution data. At 24 hours after tumor growth was almost completely inhibited up to injection, UM-SCC-22B tumors could be clearly visualized day 46 after treatment, and only one mouse in this and the activity accumulated in the liver was also obvious group showed tumor relapse on day 48. Almost no due to the hepatic clearance of 111In-DOTA-panitumumab. UM-SCC-22B tumor inhibition effect was observed With the clearance of radioactivity from normal organs, in the panitumumab-based immunotherapy group the much better tumor contrast was observed at 72 and (10 mg/kg × four doses). The findings suggest that 96 hours after injection. 90Y-DOTA-panitumumab exerted a strong therapeutic effect against UM-SCC-22B tumor xenografts compared RIT and immunotherapy with cold panitumumab-based immunotherapy. All The therapeutic efficacy of 90Y-DOTA-panitumumab mice in the therapy studies showed no significant body was investigated in EGFR-positive UM-SCC-22B tumor- weight loss (<20% of original weight) after treatment bearing nude mice. As shown in Fig. 4A, single injec- (Fig. 5A), showing no significant acute toxicity for tion of low-dose panitumumab (∼6 μg) did not induce 90Y-DOTA-panitumumab. significant tumor growth inhibition compared with Kaplan-Meier analysis was done and is shown in saline. The tumors in the 3.7 MBq 90Y-DOTA-IgG group Fig. 4B. The mice were sacrificed when the tumor size grew somewhat slower than those in saline and cold reached ≥1,500 mm3 or body weight lost ≥20% of their panitumumab groups during the first 10 days, and original values. From the Kaplan-Meier curves, it was

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Figure 2. A, biodistribution of 111In-DOTA-panitumumab in BALB/c nude mice bearing UM-SCC-22B tumors at 4 h, 1 d, 3 d, 5 d, and 7 d after injection. Columns, mean % ID/g (n = 4); bars, SD. B, the time–% ID/g uptake of 111In-DOTA- panitumumab in tumor, blood, liver, kidney, and muscle. Points, mean (n = 4); bars, SD. Data were obtained from A.

found that mice administrated with 7.4 MBq 90Y- (21). As shown in Fig. 5B, compared with saline group, DOTA-panitumumab (median survival time, >80 days) the tumors in the 3.7-MBq 90Y-DOTA-panitumumab or 3.7 MBq 90Y-DOTA-panitumumab (median survival group exhibited a slight growth delay between day time, 35 days) had a significantly longer survival time 12 and day 18 after treatment. Thereafter, however, than that administrated saline (median survival, 17 days), the tumors in the two groups both grew rapidly and single dose of panitumumab (∼6 μg; median survival, there was no significant difference on the tumor size 14 days), four doses of panitumumab (10 mg/kg; after day 22 after treatment. The slight MDA-MB-435 median survival, 14 days), or 3.7 MBq 90Y-DOTA-IgG tumor growth inhibition at early time points was most (median survival, 17 days; P < 0.01). The mice that likely due to the nonspecific tumor accumulation be- received 7.4 MBq 90Y-DOTA-panitumumab also had a cause of the enhanced permeability and retention ef- significantly prolonged survival time compared with fect, subsequently leading to the radiation-mediated those that received 3.7 MBq 90Y-DOTA-panitumumab cell killing of 90Y-DOTA-panitumumab. (P < 0.01). Histologic analysis EGFR specificity of RIT After RIT, UM-SCC-22B tumors were harvested, frozen, To substantiate the EGFR-specific therapy of 90Y- and cut into sections and then subjected to CD31, Ki67, DOTA-panitumumab, the RIT was also tested in the and TUNEL staining (Fig. 6). We carried out the CD31 MDA-MB-435 tumor-bearing nude mice, which served staining to study the effect of 90Y-DOTA-panitumumab as a tumor model with negligible EGFR expression (3.7 MBq) treatment on vascular damage. The tumor vessels

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in the 3.7 MBq 90Y-DOTA-panitumumab treatment group especially non–Hodgkin's lymphoma. The promising tend to have increased vascular fragmentation, dys- antitumor efficacy of intact antibody-based RIT agents morphic vessel structure, and reduced endothelial pre- is most likely due to their much higher and more reten- sence compared with the saline group. There was no tive tumor accumulation compared with other small- significantly decreased vasculature density in the 3.7 molecule–based agents. In this study, we successfully MBq 90Y-DOTA-IgG treatment group, but slightly in- prepared the 111In- and 90Y-labeled panitumumab, a fully creased vascular fragmentation can be observed. Treating human anti-EGFR mAb. 111In-panitumumab exhibited with unlabeled panitumumab can induce an obvious de- high and retentive tumor accumulation in UM-SCC- crease in the vasculature density, but no significant vascu- 22B human head and neck cancer xenografts, and 90Y- lar fragmentation is found. Ki67 analysis shows reduced panitumumab showed high tumor suppression efficacy cell proliferation in the 90Y-DOTA-panitumumab–treated in the mouse model. mice, whereas the mice in 90Y-DOTA-IgG, panitumumab, Given the apparent role of EGFR in tumor aggression and saline groups have significantly higher cell prolifera- and poor prognosis, as well as the high degree to which it tion index, respectively. To evaluate whether cell apoptosis seems to be overexpressed in almost all types of head and was involved in the 90Y-DOTA-panitumumab–enhanced neck cancers, blockade of EGFR pathway has been well regression on UM-SCC-22B tumors, the TUNEL assay investigated as a rational strategy for targeted therapy of was used to quantify cell apoptosis in tumor sections head and neck cancers. The chimeric anti-EGFR mAb from different groups. As shown in Fig. 6, 90Y-DOTA- Erbitux (cetuximab) has been reported to inhibit the panitumumab induced significantly higher percentage growth of EGFR-expressing carcinoma cells and inhibit of TUNEL-positive cells than 90Y-DOTA-IgG, panitumu- the invasion and metastasis of tumor cells in preclinical mab, and saline. studies (4, 28). In a preliminary clinical trial, the combi- We also did histopathologic examination to determine nation of cetuximab with radiotherapy has led to a par- the toxicity of therapeutic doses of 90Y-DOTA-panitumu- tial or complete response in patients with locoregional mab. We tested the H&E staining of the kidney and liver advanced HNSCC (29). With the successful applications, slices after RIT. No obvious histologic damage is ob- the FDA granted regulatory approval for the use of served in kidneys and livers from mice administrated cetuximab in combination with external radiation to treat with 3.7 or 7.4 MBq of 90Y-DOTA-panitumumab (Supple- locoregional advanced head and neck cancer in 2006. mentary Fig. S1), indicating the limited toxicity of the However, as cetuximab is a chimeric antibody, it is still activities used in these therapeutic studies. immunogenic and can produce allergic reaction in some patients (30, 31). Consequently, other new fully human Discussion anti-EGFR antibodies are currently undergoing preclinical evaluations. Because RIT can significantly reduce the Despite the initial introduction of RIT for tumor ther- mAb dose and destroy the tumor cells with low or with- apy some 30 years ago, this technology seems to reat- out antigen expression, herein, we tested the RIT efficacy tract an increasing interest most recently (22, 24–27). for head and neck cancer in xenografted animal model The renewed interest may be attributed, in part, to the using radiolabeled panitumumab, the first fully human successful application of 90Y- and 131I-labeled anti-CD20 mAb against EGFR. mAbs in the management of hematopoietic tumors, Panitumumab possessed high affinity for EGFR on K UM-SCC-22B cancer cells ( d of 1.33 ± 0.29 nmol/L; Fig. 1B). The in vitro analysis confirmed that DOTA conjugation of panitumumab did not impair the receptor affinity of panitumumab (Fig. 1C), and 90Y-DOTA- panitumumab possessed high radioimmunoreactivity (radioimmunoreactive fraction = 74.4 ± 2.9%) with EGFR-expressing UM-SCC-22B cells. We used 111In- DOTA-panitumumab as a biodistribution and imaging surrogate to predict the in vivo behaviors and radiation dosimetry of 90Y-DOTA-panitumumab because it has been reported that 111In- and 90Y-labeled antibodies, proteins, and peptides are biologically equivalent with respect to their uptake in tumors and other major organs (32). The results from the biodistribution studies showed that the uptake of 111In-DOTA-panitumumab in EGFR-positive UM-SCC-22B tumors was much higher than that in normal organs and blood. The tumor Figure 3. Representative γ-images of BALB/c nude mice bearing the UM-SCC-22B human head and neck xenografts administered ∼13 uptake of radiotracer reached a maximum of 59.11 ± MBq 111In-DOTA-panitumumab at 1, 3, and 4 d after injection. Arrows 7.22 % ID/g at 24 hours after injection and remained indicate the location of tumors (n = 2 per group). high with time (14.86 ± 7.23 % ID/g at 7 days after

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Figure 4. A, growth curves of UM-SCC-22B tumors in six groups of mice after i.v. injection of 7.4 MBq 90Y-DOTA-panitumumab, 3.7 MBq 90Y-DOTA-panitumumab, 3.7 MBq 90Y-hIgG, cold panitumumab (∼6 μg), four doses of cold panitumumab (10 mg/kg) every 3 d, and saline. Points, mean (n =5–6 per group); bars, SD. B, Kaplan-Meier survival curves of UM-SCC-22B tumor-bearing nude mice that received 7.4 MBq 90Y-DOTA-panitumumab, 3.7 MBq 90Y-DOTA-panitumumab, 3.7 MBq 90Y-hIgG, cold panitumumab (∼6 μg), four doses of cold panitumumab (10 mg/kg) every 3 d, and saline. The mice were sacrificed when the tumor size reached ≥1,500 mm3 (n =5–6 per group).

injection). Furthermore, the low radioactivity accumula- uptake of 111In-DOTA-panitumumab in UM-SCC-22B tion in the bone suggested high stability of the 111In- tumors still showed the excellent targeting property of DOTA chelate and confirmed that the chelator was 111In-DOTA-panitumumab to EGFRs of human origin. suitable for 90Y-based therapy. Compared with some 90Y is a useful radioisotope for the targeted RIT of solid other EGFR-targeted antibodies (33, 34), 111In-DOTA- tumors due to its favorable physical characteristics. panitumumab in this study showed much higher tumor Recently, we determined the maximum tolerated dose uptake and lower liver radioactivity accumulation, of nude mice for 90Y-labeled mAb Abegrin, and it was which was likely due to the different EGFR recognition found that BALB/c nude mice could tolerate up to 200 properties and varied in vivo pharmacokinetics of dif- μCi of 90Y-Abegrin (22). As panitumumab is also an in- ferent antibodies. It should be noted that the biodistri- tact mAb similar to Abegrin, we used a maximum radio- bution of 111In-DOTA-panitumumab in the present activity dose of 200 μCi 90Y-DOTA-panitumumab for the study may not be a true mimicry of the clinical situa- RIT in this study. No evident weight loss was found after tion because panitumumab may not bind murine 90Y-DOTA-panitumumab treatment (Fig. 5A), indicating EGFRs that expressed in normal organs of mice. This that 200 μCi 90Y-DOTA-panitumumab was under the may also be partially responsible for the faster clear- maximum tolerated dose of tumor-bearing nude mice. ance of 111In-DOTA-panitumumab. However, the high RIT with 90Y-DOTA-panitumumab caused a significant

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EGFR-Targeted RIT with 90Y-Panitumumab

tumor growth delay and improved the survival in EGFR- of 90Y-DOTA-IgG compared with the saline control positive UM-SCC-22B tumor model (Fig. 4), and impor- group confirmed the EGFR-specific RIT of 90Y-DOTA- tantly, there was no significant radiation damage in the panitumumab. To evaluate the influence of EGFR dose-limiting organs such as liver and kidneys (Supple- expression level on the therapy outcome, we selected mentary Fig. S1). Achieving tumor treatment efficacy by MDA-MB-435 tumor model as a control. As there was 90Y-DOTA-panitumumab with limited whole-body toxic- a limited EGFR expression in MDA-MB-435 tumors ity is due to the much higher tumor uptake of panitumu- (21), it was assumed that the tumor uptake of 90Y- mab as determined by biodistribution study (Fig. 2A). As DOTA-panitumumab would be very low, resulting in reported in the RIT studies, a clear activity-dependent no tumor therapeutic efficacy. In fact, the tumor growth relationship in the efficacy of a single administration of curves revealed no statistical significance between 90Y- 90Y-DOTA-panitumumab was apparent in the established DOTA-panitumumab and control groups (Fig. 5B), tumor model. Treatment of tumor-bearing nude mice indicating the limited therapeutic efficacy of 90Y-DOTA- with 3.7 MBq 90Y-DOTA-panitumumab caused ∼20 days panitumumab in EGFR-negative tumors, which validated of tumor growth delay, but at day 34 after treatment, again that the antitumor effect of 90Y-DOTA-panitumumab almost all tumor reached a larger size (∼1,500 mm3). When was EGFR specific. using 7.4 MBq 90Y-DOTA-panitumumab for single-dose Although panitumumab showed high accumulation in therapy, the growth of all tumors was inhibited up to UM-SCC-22B tumors (Fig. 2A), the immunotherapy ∼50 days, and only one tumor showed recurrence at study using high doses of panitumumab did not show day 48 after treatment. The lack of therapeutic efficacy any therapeutic effect (Fig. 4A). Our results are consistent

Figure 5. A, mean body weight over time of the BALB/c nude mice administrated with 7.4 MBq 90Y-DOTA-panitumumab, 3.7 MBq 90Y-DOTA-panitumumab, 3.7 MBq 90Y-hIgG, panitumumab, or saline. Points, mean (n =5–6 per group); bars, SD. B, growth curves of the low EGFR-expressing MDA-MB-435 tumors in BALB/c nude mice after i.v. injection of 3.7 MBq 90Y-DOTA- panitumumab or saline. Points, mean (n =6 per group); bars, SD.

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Figure 6. Immunofluorescence staining of CD31, Ki67, and TUNEL for UM-SCC-22B tumor tissues from groups treated with 90Y-DOTA-panitumumab (3.7 MBq), 90Y-DOTA-IgG (3.7 MBq), panitumumab, and saline (control). Scale bar, 100 μm.

with a recent report showing that another anti-EGFR tration ability of the bulky immunoglobulins would pos- antibody, cetuximab (Erbitux), did not inhibit the growth sibly influence the effective tumor delivery of anti-EGFR of the UM-SCC-22B tumors as well (35). Several previous mAb (14, 40). Differing from panitumumab itself as the studies have also shown that some EGFR-positive tumors immunotherapeutic agent, 90Y-DOTA-panitumumab did not benefit from the treatment of anti-EGFR anti- exerts its antitumor effect by localizing continuous irradi- bodies (36–38). Our biodistribution study has shown the ation to the area of malignancy, thereby destroying not high UM-SCC-22B tumor uptake of panitumumab only the antibody-binding EGFR-positive tumor cells (Fig. 2A), but the high tumor uptake may not directly lead but also the unbound tumor cells and the EGFR-negative to effective antitumor activity with cold panitumumab due tumor cells by the cross-fire effect. to the disparity between the tumor EGFR expression and The easy availability, high-receptor binding affinity, the antibody treatment outcome, although the detailed and excellent tumor therapeutic efficacy of 90Y-DOTA- mechanism of UM-SCC-22B tumor resistance to panitumu- panitumumab as determined in this study warrant its fur- mab still needs further investigation. In contrast to ther possible clinical translation. In the meantime, it is immunotherapy,RITrelies on the cell-killing effect of radio- also worth investigating the combination of 90Y-DOTA- isotopes (i.e., the higher tumor accumulation of the radio- panitumumab with other therapeutic approaches in the activities would certainly lead to the more significant future. It is well documented that the inhibition of EGFR therapeutic efficacy because the tumor cells are usually sen- signaling can enhance the antitumor effect of radiation sitive to radiation). The high UM-SCC-22B tumor uptake of therapy (41, 42). Therefore, it would be interesting to in- panitumumab as determined by biodistribution study vestigate the synergistic effect of combining 90Y-DOTA- guaranteed the high antitumor effect of 90Y-panitumumab. panitumumab–based RIT with other EGFR inhibition The antiangiogenic effect of 90Y-DOTA-panitumumab approaches such as tyrosine kinase inhibitor and other was observed by the ex vivo immunofluorescence stain- blocking antibodies. In addition, in comparison with the − − ing result of significantly reduced CD31+ vascular endo- high-energy β emitter 90Y, 177Lu is a low-energy β emit- β E thelial cells (Fig. 6). Ki67 and TUNEL histopathologic ter with three -emissions [ max = 0.176 MeV (12%), 0.384 analyses revealed the reduced proliferation and the in- MeV (9%), and 0.497 MeV (79%)] and two γ-emissions creased apoptosis index of tumors in treatment animals, [113 keV (6.4%) and 208 keV (11%)]. Because of its low which further confirmed the therapeutic efficacy of 90Y- β-energy, 177Lu-labeled compounds might be useful for DOTA-panitumumab (Fig. 6). treatment of small and metastatic tumors (43). The pres- Increasing clinical evidence has shown the disparity ence of these two γ-emissions will allow scintigraphic between the EGFR expression level and the treatment imaging for quantification of biodistribution and determi- outcome of anti-EGFR mAb-based immunotherapeutic nation of radiation dosimetry in clinical settings. There- agents (37–39). Besides the EGFR expression level, some fore, 177Lu-labeled panitumumab may be more suitable additional factors such as tumor vasculature density and for the image-guided RIT of the local recurrence or micro- permeability, tumor interstitial pressure and binding site metastasis of EGFR-positive tumors. Moreover, treatment barrier, as well as the pharmacokinetics and tumor pene- of EGFR-positive tumors with 177Lu-labeled panitumumab

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EGFR-Targeted RIT with 90Y-Panitumumab

after pretreatment with 90Y-panitumumab may lead to Disclosure of Potential Conflicts of Interest the effective therapy of both large tumors and minimal residual tumors as a whole. No potential conflicts of interest were disclosed.

Conclusion Grant Support National Natural Science Foundation of China grants 30870728, The in vivo studies showed the high tumor uptake and 20820102035, and 30930030 (F. Wang) and 30900373 (B. Jia); “863” project prolonged tumor retention of 111In-DOTA-panitumumab grant 2007AA02Z467 (F. Wang); Ministry of Science and Technology of China grants 2009ZX09103-733 and 2009ZX09301-010 (F. Wang) and in UM-SCC-22B tumor-bearing nude mice. The RIT 2009ZX09103-746 (B. Jia); and Intramural Research Program of the NIH, studies of 90Y-DOTA-panitumumab showed the effective including the National Institute of Biomedical Imaging and Bioengineer- growth inhibition of EGFR-positive tumors. From these ing (X. Chen). 90 The costs of publication of this article were defrayed in part by the results, we believe that Y-DOTA-panitumumab is a payment of page charges. This article must therefore be hereby marked promising RIT agent for EGFR-positive tumor therapy. advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. To the best of our knowledge, this is the first preclinical 90 investigation of Y-labeled panitumumab for EGFR- Received 05/14/2010; accepted 06/15/2010; published OnlineFirst targeted RIT of head and neck carcinoma. 08/03/2010.

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2308 Mol Cancer Ther; 9(8) August 2010 Molecular Cancer Therapeutics

Epidermal Growth Factor Receptor−Targeted Radioimmunotherapy of Human Head and Neck Cancer Xenografts Using 90Y-Labeled Fully Human Antibody Panitumumab

Zhaofei Liu, Yan Liu, Bing Jia, et al.

Mol Cancer Ther 2010;9:2297-2308. Published OnlineFirst August 3, 2010.

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