Re-188 Enhances the Inhibitory Effect of Bevacizumab in Non-Small-Cell Lung Cancer

The malignant behaviors of solid tumors such as growth, infiltration and metastasis are mainly nourished by tumor neovascularization. Thus, anti-angiogenic therapy is key to controlling tumor progression. Bevacizumab, a humanized anti-vascular endothelial growth factor (VEGF) antibody, plus chemotherapy or biological therapy can prolong survival for cancer patients, but treatment-related mortality is a concern. To improve inhibitory effect and decrease side-effects on non-small-cell lung cancer (NSCLC), we used Re-188, which is a β emitting radionuclide, directly labeled with bevacizumab for radioimmunotherapy in a human A549 tumor model. Cytotoxic assay data showed that, after 188ReO4− or 188Re-bevacizumab at different concentration for 4 and 24 h, a time- and radioactivity does-dependent reduction in cell viability occurred. Also, an apoptosis assay conformed great apoptosis in the 188Re-bevacizumab group compared with controls and other treatment groups. In vivo, tumor volumes in the 188Re-bevacizumab (11.1 MBq/mice) group were not reduced but growth was delayed compared with other groups. Thus, 188Re-bevacizumab enhanced the therapeutic effect of bevacizumab, suggesting a potential therapeutic strategy for NSCLC treatment.


Introduction
Lung cancer is the leading cause of cancer death worldwide and represents a significant financial and social burden [1]. Non-small-cell lung cancer (NSCLC), the mostly common cancer subtype, accounts for 85% of all new lung cancer diagnoses [2]. Due to late onset of clinical manifestations, patients are commonly diagnosed at advanced or inoperable-stages. Recently, chemotherapy with platinum plus one other anti-tumor agents is recommended for first-line chemotherapy, but side-effects are a concern [3]. Although maintenance therapy followed by second-line cytotoxic chemotherapy is common, advanced stage patients did not live one year beyond median survival [4]. Thus, effective, low side-effect therapeutic methods for NSCLC treatment are needed.
Tumor progression (growth, infiltration and metastasis) are reportedly supported by tumor neovascularization. Thus, tumor angiogenesis is thought to be an approach for treating cancers [5,6]. Bevacizumab, a humanized monoclonal antibody (MAb), that specifically binds to vascular endothelial growth factor (VEGF) and can block neovascularization, was approved by the FDA [7]. In randomized clinical trials, survival benefits were improved when bevacizumab was added to standard chemotherapy regimens to treat metastatic colorectal cancer and advanced NSCLC [7,8]. Radioimmunotherapy (RIT) is an alternative for patients with advanced cancers. Using selective targeting to cancer-associated antigens on the tumor-cell surface, monoclonal antibody-mediated radionuclide can deliver high-dose therapeutic radiation to cancer cells with minimal exposure of normal cells [9][10][11]. The first radiolabeled antibody for anti-tumor therapy, 90 Y-murine anti-CD20 antibody, ibritumomab, offered a satisfactory therapeutic response, and was approved for clinical practice in 2002 [12]. Coupled to 131 I, tositumomab has been used to treat chronic lymphocytic leukemia or small lymphocytic lymphoma in first remission. Also, patients with leukemias or lymphomas enjoyed more survival benefits from RIT than without RIT treatment [13][14][15]. However, for solid tumor, which are radio-resistant and less accessible to MAbs, a concern remains about limited clinical efficacy [16].
According to Lucas, 90 Y and 188 Re were regarded as the best candidates for solid tumor treatment such as for NSCLC [17]. In addition, considering the appropriate half-life of 16.9 h (0.7 d), therapeutic beta radiation (Eβ = 2.118 Mev) and easy used in-home, 188 Re was selected as the radioisotope for RIT in this study. The radiotherapeutic agent 188 Re-bevacizumab was designed, and its therapeutic efficacy was evaluated in tumor models of NSCLC.

Radiolabeling
The synthesis of 188 Re-bevacizumab was achieved by a two-step reaction ( Figure 1A). For paper chromatography studies, the Rf values of radiolabeled bevacizumab and 188 Re-colloid were 0.0-0.1, and the Rf value of free or unbound 188 ReO 4 − was 0.8-0.9 in normal saline. In contrast, for the mixed solvent system v ethanol :v ammonia :v water = 2:5:1 the Rf value of radiolabeled bevacizumab and free 188 ReO 4 − were 0.8-0.9, and the Rf value of radiolabeled bevacizumab and 188 Re-colloid were 0.0-0. in acetic acid buffer solution (pH = 4.5) for 30 min at room temperature (RT). Purification was performed with disposable G25 PD-10 desalting columns (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA) and a radiochemical purity of more than 98% was achieved. After purification, radiolabeled products were used for studies. In this present study, the influence on radiolabeling efficiency by pH was also investigated. The optimal pH value was 4.5, as the results in Figure 1A. According to the published literature [18][19][20], the direct radiolabeling of 188 Re to antibodies occurs within a pH range from 4 and 6, and it is proposed that a radiolabeling pH value lower than 4 could compromise the immunoreactivity of the antibodies, while a higher pH value increases the percentage of unbound 188 ReO 4 − .

Stability in Vitro
After purification, 188 Re-bevacizumab was diluted and incubated in mouse serum and normal saline (1:5 and 1:1, respectively). Radiolabeling was measured at 4, 12 and 24 h at 37 • C ( Figure 1C). After 12 h of incubation, over than 95% of labelled products remained intact in both normal saline and serum. For 24 h, less than 10% decomposition was observed in mouse serum, suggesting a favorable stability in vitro.

Cytotoxicity Assay (CCK-8)
Cell viability of all treated groups at different times points is shown in Figure 2, where the data show that the viability was time-and dose-or radioactive dose-dependent. Cells incubated with fresh medium were the most viable, whereas 188 Re-bevacizumab (1.85 MBq) treatment reduced viability the most.      Increasing the radioactivity dose from 37 kBq to 1.85 MBq proportionally decreased cell viability at 4 h and 24 h in all groups. Thus, A549 cells are more sensitive to 188 Re-bevacizumab compared to bevacizumab alone at equal concentration. How bevacizumab inhibits cell growth is unknown, but Wang suggested that cytotoxicity in tumor cells after bevacizumab administration may be due to endoplasmic reticulum stress, which accelerates apoptosis, or autocrine VEGF [21].
Molecules 2016, 21, 1308 4 of 9 Increasing the radioactivity dose from 37 kBq to 1.85 MBq proportionally decreased cell viability at 4 h and 24 h in all groups. Thus, A549 cells are more sensitive to 188 Re-bevacizumab compared to bevacizumab alone at equal concentration. How bevacizumab inhibits cell growth is unknown, but Wang suggested that cytotoxicity in tumor cells after bevacizumab administration may be due to endoplasmic reticulum stress, which accelerates apoptosis, or autocrine VEGF [21].

Micro SPECT/CT Imaging
To eliminate the influence on therapeutic efficiency by 188 ReO4 − before and after treatment, 99m Tc-MAG3-bevacizumab was used to monitor angiogenic changes during treatment. Whole body images and relevant axial slices of pre-and post-therapy A549 xenograft mouse treated with 188 Re-bevacizumab are depicted in Figure 4. Images acquired at 4 h after injection showed intense uptake of drug by lung, liver, spleen, kidney and bladder. Tumor uptake before treatment was more intensive than post-treatment, suggesting effective anti-angiogenic efficacy of 188 Re-bevacizumab, and treatment was ceased. Bevacizumab has a long half-life and binds to VEGF-A, a key mediator of angiogenesis [22]. Thus, the accumulation of 99m Tc-MAG3-bevacizumab in organs may be explained by organ mesenchyme secretion of VEGF [23].

Micro SPECT/CT Imaging
To eliminate the influence on therapeutic efficiency by 188 ReO 4 − before and after treatment, 99m Tc-MAG 3 -bevacizumab was used to monitor angiogenic changes during treatment. Whole body images and relevant axial slices of pre-and post-therapy A549 xenograft mouse treated with 188 Re-bevacizumab are depicted in Figure 4. Images acquired at 4 h after injection showed intense uptake of drug by lung, liver, spleen, kidney and bladder. Tumor uptake before treatment was more intensive than post-treatment, suggesting effective anti-angiogenic efficacy of 188 Re-bevacizumab, and treatment was ceased. Bevacizumab has a long half-life and binds to VEGF-A, a key mediator of angiogenesis [22]. Thus, the accumulation of 99m Tc-MAG 3 -bevacizumab in organs may be explained by organ mesenchyme secretion of VEGF [23].

Assessment of Treatment Effect in Vivo
Bevacizumab (1.5 mg/kg or 30 μg per mice) and 188 Re-bevacizumab (11.1 MBq/30 μg per mice) were separately injected into the tail vein treatment groups weekly and tumor sizes and body weight were recorded ( Figure 5). Compared with controls, tumor growth in the 188 Re-bevacizumab and bevacizumab treatment groups were obviously delayed with no significant regression (p value = 0.018 < 0.05). Although there was no statistical difference (p value = 0.0561 > 0.05) between the 188 Re-bevacizumab treatment group and the bevacizumab treatment group, more pronounced inhibition was observed in the 188 Re-bevacizumab treatment group. According to a previous report [24], dosimetry analyses of 177 Lu-DOTA-RS7 showed that complete remissions were obtained in the maximal tolerated dose group in tumor-bearing nude mice. Thus, in order to gain more benefit from 188 Re-bevacizumab

Assessment of Treatment Effect in Vivo
Bevacizumab (1.5 mg/kg or 30 µg per mice) and 188 Re-bevacizumab (11.1 MBq/30 µg per mice) were separately injected into the tail vein treatment groups weekly and tumor sizes and body weight were recorded ( Figure 5).

Assessment of Treatment Effect in Vivo
Bevacizumab (1.5 mg/kg or 30 μg per mice) and 188 Re-bevacizumab (11.1 MBq/30 μg per mice) were separately injected into the tail vein treatment groups weekly and tumor sizes and body weight were recorded ( Figure 5). Compared with controls, tumor growth in the 188 Re-bevacizumab and bevacizumab treatment groups were obviously delayed with no significant regression (p value = 0.018 < 0.05). Although there was no statistical difference (p value = 0.0561 > 0.05) between the 188 Re-bevacizumab treatment group and the bevacizumab treatment group, more pronounced inhibition was observed in the 188 Re-bevacizumab treatment group. According to a previous report [24], dosimetry analyses of 177 Lu-DOTA-RS7 showed that complete remissions were obtained in the maximal tolerated dose group in tumor-bearing nude mice. Thus, in order to gain more benefit from 188 Re-bevacizumab Compared with controls, tumor growth in the 188 Re-bevacizumab and bevacizumab treatment groups were obviously delayed with no significant regression (p value = 0.018 < 0.05). Although there was no statistical difference (p value = 0.0561 > 0.05) between the 188 Re-bevacizumab treatment group and the bevacizumab treatment group, more pronounced inhibition was observed in the 188 Re-bevacizumab treatment group. According to a previous report [24], dosimetry analyses of 177 Lu-DOTA-RS7 showed that complete remissions were obtained in the maximal tolerated dose group in tumor-bearing nude mice. Thus, in order to gain more benefit from 188 Re-bevacizumab treatment, dosimetry analyses will be performed in our future work. Animal weight gain one week after treatment in the treatment groups was slowed and food intake was less, but these differences were not statistically different (p value = 0.7792 > 0.05).
Effective radiotherapy requires maximum therapeutic radioactivity accumulation in tumors and long-time retention. Micro SPECT/CT images show that 99m Tc-bevacizumab was taken up by tumors and cleared slowly. Similarly characteristics of bevacizumab permit the delivery of a high-dose 188 Re to tumor tissues and minimizes exposure of normal tissue.

Materials and Reagents
Solvents and regents used in radiolabeling reactions were of analytical grade, used without any further purification and purchased from Sigma-Aldrich (St. Louis, MO, USA), BD Biosciences (New York, NJ, USA). Bevacizumab (Avastin) was from Roche Pharma Ltd. (Basel, Switzerland). 188 Re was obtained as its perrhenate by elution from an alumina-column-based 188 W/ 188 Re generator with normal saline (LaiTai Biotechnology, Jiangsu, China). Na 99m TcO 4 was supplied by Shanghai GMS Pharmaceutical Co., Ltd. (Shanghai, China). Flexible silica gel 60 F254 plates was purchased from Merck (Darmstadt, Germany) and used for thin-layer chromatography (TLC) studies. Micro SPECT/CT imaging was performed with a Nano SPECT/CT scanner (Mesido, Budapest, Hungary) at the Cancer Center of Shanghai. Human lung A549 adenocarcinoma cells were purchased from the Chinese Type Culture Collection (Chinese Academy of Sciences, Shanghai, China). Male nude mice (4-6 weeks-old, 20-25 g) obtained from Slac Biotechnology (Shanghai, China) which were bred in a SPF laboratory animal facility at Fudan University were used as xenograft models. All animal experiments were conducted in accordance with relevant guidelines and regulations, and were approved by the Institutional Animal Care and Committee of Fudan University.

Stability Study in Vitro
To estimate the stability of 188 Re-bevacizumab in vitro, a sample (10 µL) from the final preparation was incubated in normal saline (10 µL) and diluted in mice serum (50 µL) at 4 h,12 h and 24 h at 37 • C. The analysis was repeated three times.
Labeling efficiency and stability was measured by spotting samples on BSA blocked instant thin-layer chromatography silica gel (ITLC-SG). When the eluent of normal saline reached the front, the strip was removed, dried and cut into 1-cm sections and counted. All radioactive measurements were conducted on a gamma radioimmunoassay counter (GC-1200, Zhongjia Co., Ltd., Hefei, China). Radiolabeling of 188 Re-bevacizumab = 100% − colloidal (%) − unbound 188 ReO 4 − (%).
Cytotoxicity of 188 Re-bevacizumab was evaluated with a cell count kit-8 (CCK-8) according to the manufacturer's instruction. A549 cells were trypsinized and suspended at a concentration of 5 × 10 4 /mL and when they reached 80% confluence, 5 × 10 3 cell were seeded and cultured with 100 µL complete medium with 10% FBS in 96-well plates coated with 0.05% rat tail collagen. After culturing for 24 h, medium was removed and replaced with 100 µL fresh medium containing bevacizumab at different concentrations, 188 Re (1, 10, 50 µCi/mL) and equal amounts of 188 Re-bevacizumab. A 549 cells were incubated in each desired medium as stated above for 4 h and 24 h. To evaluate cell viability, 10 µL of CCK-8 solution was added to each well, and 96-well plates were continuously incubated at 37 • C for 2 h. The OD values were read at 450 nm to measure cell viability as following: cell viability (%) = [OD (treated group) − OD (blank)] / [OD (control group) − OD (blank)] × 100%.

Tumor Xenografts and Treatment Design
Tumor xenografts were established in nu/nu male mice via injection of approximately 6 × 10 6 A549 cells in the left shoulder area. When tumor volumes reached 150 mm 3 , mice were randomly divided into three groups of fifteen animals as follows: controls, bevacizumab treated, and 188 Re-bevacizumab treated. Furthermore, A549 tumor models were treated weekly for three weeks as follows: (1) normal saline every week (control group, 100 µL i.v.); (2) bevacizumab (1.5 mg/kg/week, 100 µL, i.v.); (3) 188 Re-bevacizumab (11.1 MBq/week/mouse, 100 µL, i.v.); Tumor volumes and animal weights were recorded weekly. Tumor volumes were estimated using the equation of 0.5 × length × (width) 2 . All animals were observed daily to assess general health.

Micro SPECT/CT Imaging
Micro-SPECT/CT imaging was following the described method [29] in three mice before the initial treatment with 188 Re-bevacizumab and after final treatment at 3 weeks. 99m Tc-MAG 3 -bevacizumab (500 µCi/50 µg/mice) was injected, and then CT and SPECT imaging was performed 4 h later.

Conclusions
188 Re-bevacizumab offered high yield with direct labeling and had favorable stability in vitro. 188 Re-bevacizumab enhanced tumor inhibition for NSCLC in both in vivo and in vitro studies.