Abstract
Vascular endothelial growth factor receptor 2 (VEGFR2) is traditionally regarded as an important therapeutic target in a wide variety of malignancies, such as hepatocellular carcinoma (HCC). We previously generated a murine-human anti-VEGFR2 chimeric Fab (cFab), named FA8H1, which has the potential to treat VEGFR2-overexpressing solid tumors. Here, we investigated whether FA8H1 can be used as a carrier in molecularly targeted therapy in HCC xenograft models. FA8H1 was labeled with 131I and two HCC xenograft models were generated using BEL-7402 (high VEGFR2-expressing) and SMMC-7721 (low VEGFR2-expressing) cells, which were selected from five HCC cell lines. The biodistribution of 131I-FA8H1 was determined in both models by Single-Photon Emission Computed Tomography and therapeutic effects were monitored in nude mice bearing BEL-7402 xenografts. Finally, we determined the involvement of necrosis and apoptotic pathways in treated mice using immunohistochemistry. 131I-FA8H1 levels were dramatically reduced in blood and other viscera. The therapeutic effect of 131I-labeled FA8H1 in the BEL-7402 model was significantly better than that by 131I and FA8H1 alone. We observed extensive necrosis in the treated tumors and both FasL and caspase 3 were up-regulated. Thus, 131I-anti-VEGFR2 cFab has the potential to be used for molecularly targeted treatment of HCC overexpressing VEGFR2.
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Introduction
Human liver cancer, particularly hepatocellular carcinoma (HCC), is the sixth most common neoplasm worldwide and the third highest cause of cancer deaths worldwide1. Most HCC patients are diagnosed at an advanced stage, when traditional treatments are not effective2,3. Despite the advances in surgery, liver transplantation and systemic chemotherapy, the survival rate of HCC patients has not improved much over recent decades2,3. Monoclonal antibodies (mAbs) can be used for molecular imaging as well as cancer-specific vehicles to deliver therapy to the tumor site4,5. However, the use of murine mAbs is limited in the clinic because of their high immunogenicity, large molecular weight and the risk of human anti-mouse antibody (HAMA) responses6,7. Murine-human chimeric Fab (cFab) was prepared from a murine antibody. cFab offers several advantages over whole murine IgG: first, molecular weight of murine-human chimeric Fab (cFab) is approximately 50 kDa and only one-third of the size of full-length IgG. Secondly, cFab reduces the HAMAs responses as it is prepared by recombining whole murine variable regions with human constant regions8,9,10,11,12.
Many cFabs have been studied under pre-clinical or clinical development and have become one of the most prolific drug classes in oncology13,14,15. We previously produced a high-affinity murine anti-vascular endothelial growth factor receptor 2 (VEGFR2) mAb (A8H1) using mouse hybridoma technology16 and constructed anti-VEGFR2-cFab (FA8H1) containing the variable region from A8H1 and the constant region from human IgG. The chimeric Fab maintained the specificity for the VEGFR2 antigen17.
VEGFR2 plays an important role in angiogenesis in a wide variety of malignancies18,19,20, such as HCC21. Our previous study confirmed the prognostic significance of VEGFR2 overexpression in HCC22. VEGFR2 has also been investigated as an anticancer target23,24,25. In fact, one VEGFR2 mAb, ramucirumab (IMC-1121B) is currently being tested in the treatment of human cancer26,27.
Radioimmunotherapy (RAIT) involves the use of mAbs in combination with therapeutic radionuclides, which have been increasingly used in the clinical setting28,29. For example, both yttrium-90-ibritumomab tiuxetan (Zevalin) and 131I-labeled Tositumomab (Bexxar) are FDA-approved to treat non-Hodgkin’s lymphoma (NHL)30,31,32.
In this study, we investigated the therapeutic efficacy of radioiodinated anti-VEGFR2-cFab (FA8H1) on human HCC xenografts. We determined the biodistribution of 131I-labeled FA8H1 and its therapeutic effects in vivo and the hematological toxicities from the iodinated antibody were assessed. Finally, we determined the involvement of necrosis and apoptotic pathways in the therapeutic effects of 131I-labeled FA8H1.
Materials and methods
Cell Lines and Culture
Five HCC cell lines, BEL-7402, QCY-7701, SMMC-7721, HepG-2 and SK-HEP-1, were purchased from the cell bank of the Chinese Academy of Science in Shanghai, China. They were cultured in DMEM medium (Gibco, Invitrogen, USA) containing 10% fetal bovine serum (FBS) at 37 °C and 5% CO2.
Fluorescence-Activated Cell Sorting (FACS)
HCC cell lines were treated with FA8H1 at 20 μg/ml overnight at 4 °C. After washing with phosphate-buffered saline (PBS), they were incubated with 0.01% rhodamine-conjugated anti-human Fab antibody (Rockland, USA). Cells were then centrifuged and washed and subsequently analyzed on a BD FACS Calibur using the Cell Quest program. Background staining was obtained by replacement of the primary antibody with the sonicated supernatant of E. coli TOP10F´17. And the experiment was repeated by displacement of the primary antibody with PBS as a negative control which was consistent with the control using the sonicated bacterial supernatant.
Radiolabeling of Anti-VEGFR2-cFab
Murine-human chimeric anti-VEGFR2-Fab (FA8H1) was previously generated in our laboratory17. The chloramine-T method33 was used to label the antibody with 131I. Briefly, 2.0 mCi (74 MBq) of 131I (Gaotong, Chengdu, China), 100 μg of FA8H1 and 200 μL of 0.2 M phosphate buffer (pH 8.0) were added to vials coated with 50 μg Iodogen (Sigma-Aldrich, St. Louis, MO) and incubated for 10 minutes at room temperature. Then the mixture was separated from free iodide by passing over an equilibrated PD-10 desalting column (GE, Niskayuna, NY, USA). The labeling efficiency was determined in a Perkin Elmer 1470 Automatic Gamma counter (Fremont, CA, USA). The radiochemical purity (RCP) of 131I-FA8H1 was assessed by a trichloroacetic acid (TCA) assay, as described elsewhere33,34 and the stability of 131I -FA8H1 in vitro was determined by incubating of the 131I-FA8H1 in murine blood with heparin at 37 °C for 24 h.
Immunoreactivity of Radiolabeled Anti-VEGFR2-cFab
HCC cell lines were harvested by scraping using TrypLE Express (Invitrogen, USA) and washed with PBS (pH 7.4). A total of 2 × 106 cells were re-suspended in 1 ml PBS (pH 7.4) containing 0.2% BSA and incubated with 10 μg/ml 131I-FA8H1 in a 37 °C water bath for 1 h. Cells were washed twice and spun at 2,000 rpm for 10 min. The radioactivity of the pellets was then read by a gamma reader. Same number of cells were incubated with the presence of a 200-fold molar excess of 131I-labelled unrelated cFab (human-murine chimeric antibody against protective antigen of Bacillus anthracis, which was prepared in our lab)35 to obtain the nonspecific binding as the blank (NSB, nonspecific binding). The assay was repeated three times.
Human HCC Xenograft Model
Two HCC cell lines with the highest and lowest VEGFR2 expression among the five cell lines were used to develop two human liver cancer xenografts. Six-week-old female athymic nude (nu/nu) mice (National Rodent Laboratory Animal Resources, Shanghai Branch) were injected with 2 × 106 HCC cells in the left hind limb per 100 μl DMEM medium without FBS by subcutaneous implantation. For all studies, mice had access to laboratory food and water ad libitum. Drinking water contained 0.1% potassium iodide to block thyroid uptake of iodine. All experimental protocols were approved by the Research Ethics Committee of the Nanjing Medical University and Jiangsu Institute of Nuclear Medicine, China. All experimental procedures involving animals and their care were carried out in accordance with the Nanjing Medical University and Jiangsu Institute of Nuclear Medicine for Animal Experimentation and the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Biodistribution Studies
Total 36 tumor-bearing nude mice with high VEGFR2 expression were randomly divided into six groups (n = 6/group) when tumors had reached 0.5–0.8 cm in diameter as measured by external caliper measurement. Animals were injected with 10 μCi 131I-FA8H1 through the tail vein under light inhalation anesthesia. Then whole-body images of each mouse were acquired at 6, 12, 24 and 48 h post-injection by Single-Photon Emission Computed Tomography (SPECT, Skylight; Philip, Amsterdam, the Netherlands). The mice were sacrificed at 1, 6, 12, 24, 48 and 72 h and tumors and tissues (blood, brain, heart, liver, spleen, kidney and lung) were removed for measurement of 131I-FA8H1 accumulation using a gamma counter. The results were calculated as a percentage of the injected dose per gram of tissue (%ID/g) [34].
Imaging studies
Both (n = 6/group) HCC xenograft models, were used on imaging study when the tumor’s dimension achieved 1.0 cm in size. Prior to the imaging session, mice were anaesthetised with 13 mg/kg xylazine and 87 mg/kg ketamine. In brief, each mouse was injected with 200 μCi 131I-FA8H1 through the mouse tail vein. Whole-body images of each mouse were acquired at 6, 12, 24 and 48 h post-injection by SPECT.
Radioimmunotherapy with 131I-FA8H1
A total of 48 mice injected with BEL-7402 cells were divided into six groups when the average diameter of the tumors reached 0.3 cm. Three groups of mice (8 mice per a group) were administered varying amounts of 131I-FA8H1 (100, 200 and 300 μCi) via the tail vein at 2-week intervals under light inhalation anesthesia. The other three groups of mice (8 mice per a group) served as controls, receiving 300 μCi 131I along with 300 μg unlabeled FA8H1 and PBS in the same volume. Tumor volumes were measured once a week for 42 days and calculated according to the following equation: tumor volume (TV) = π/6 × a × b2, where a is the longest diameter and b is the shortest diameter, as we described elsewhere36. These animals were sequentially killed by CO2 asphyxiation. Tumors were removed, weighed and stored for further use.
Hematoxylin-Eosin (H&E) Staining
Tumor were formalin-fixed and paraffin-embedded. All sections were cut to 4 μm on a manual rotary microtome (Leica RM2235, Germany). The sections for H&E were stained by an Automated Slide Stainer (Leica ST5020, Germany) and cover slipped with a Robotic Coverslipper (Leica CV5030, Germany). The extent of tumor necrosis was divided into four grades, including without or including illegibility necrosis (-), spotty necrosis (+), small schistic necrosis (++) and big schistic necrosis (+++).
Immunohistochemistry (IHC) and Scoring
Deparaffinized sections (4 μm-thick) from paraffin blocks were separately stained as we described previously37,38 using the following primary antibodies: rabbit polyclonal to Fas Ligand (1:1000, ab15285, Abcam, USA) and rabbit polyclonal to Caspase 3 (1:1000, ab4051, Abcam, USA). Secondary antibody was Envision goat anti-rabbit HRP (DAKO, USA). The evaluation of the immunostaining of these proteins was blinded to the pathologists (Huang J and Feng Z) by an independent observation simultaneous design. The sum of the percentages and intensity scores was used as the final staining score, as described previously22.
Statistical Analysis
Using the STATA V.9.0 Statistics package(version 9.0; Stata Corp, College Station, TX, USA), the analysis of variance (ANOVA) and the least significant difference (LSD) method were used to assess changes in tumor weight and tumor volume. Mean ± SD was calculated for each group. The degree of tumor necrosis and the disparate expression of FasL or caspase 3 in tumor tissues were examined using the Kruskal-Willis test. For all analyses, data were analyzed using STATA and a P value <0.05 was considered to be statistically significant.
Results
Native VEGFR2 Antigen Expression in HCC Cell Lines
To compare the expression of native VEGFR2 in HCC cell lines, FACS was performed to analyze the five human HCC cell lines. All lines showed various degrees of binding with anti-VEGFR2 antibody (A8H1) in the following order, from highest to lowest based on the geometric mean fluorescence: BEL-7402, QCY-7701, HepG2, SK-HEP-1 and SMMC-7721 (Fig. 1a).
Binding and Immunoreactivity of Radioiodinated Antibody
The radiochemical purity of the labeled antibody was found to be >95.5% and the specific activity of 131I-FA8H1 ranged from approximately 1.0 to 1.5 mCi/mg. Radiopharmaceutical quality control was confirmed by layer chromatography. The amount of free nuclide in blood fell from 98 to 97.5% in 24 h at 37 °C. Specific binding between 131I-FA8H1 and native VEGFR2 in HCC cells was confirmed with a gamma counter by measuring binding between the cells and 131I-FA8H1, representing the total binding, versus a radiolabeled unrelated cFab, representing non-specific binding (Fig. 1b). The specific binding of the FA8H1 antibody was substantial after comparing total binding and non-specific binding, especially when the fact that a 200-fold molar excess of radiolabeled non-specific antibody was used is taken into account. These results were consistent with the data obtained by flow cytometry analysis and previous western blotting data for VEGFR2 in HCC cell lines22. BEL-7402 and SMMC-7721 had the highest and lowest native VEGFR2 expression of the five HCC cell lines, respectively and were thus chosen as the suitable cell lines for subsequent experiments.
Biodistribution Studies in Vivo
As shown in Fig. 1c, radioactivity levels were greatly reduced in blood and other viscera and elevated to the highest levels (18.31% (±1.31)) in tumors at 24 h. With time, blood activity and levels (9.57% (±0.46)) in other normal tissues presented discontinuous and weaker expression, while good retention of activity in tumors could be detected at 72 h (F = 5.33, P < 0.0001). The biological half-life of the 131I-FA8H1 in the BEL-7402 xenograft was estimated to be 120 h from this portion of the decay curve.
Effect of 131I-FA8H1 in vivo imaging on HCC model
SPECT images were acquired for individual mice bearing BEL-7402 HCC model and SMMC-7721 HCC model, which separately had high and low VEGFR2 expression levels. Since injected with the 131I-FA8H1, the tumor in the left hindlimb of these mice was visible within 6 hours after tracer injection in the two kinds of tumor models. As the metabolism of these animals, the image of brain and other viscera get more and more down-expression along with time passing. At 24 h post-injection, the tumor in the higher VEGFR2 expression of BEL-7402 HCC xenograft was easily seen and the representative patterns were listed in Fig. 2a, which supported the consequence of biodistribution in vivo study in some degree.
Changes in Tumor Volume and Weight in the HCC Xenografts
To reduce the number of animals used, we focused this experiment on mice bearing BEL-7402 tumors which showed the highest expression level of VEGFR2 among the five HCC cell lines tested. As shown in Fig. 2b,c, no anti-tumor activity was observed in the PBS control group. Prominent inhibition of tumor volume and weight with high dose of 300 μCi 131I-FA8H1 was observed and remarkable growth-inhibitory effects could be seen in the 300 μg FA8H1 alone group or the 300 μCi 131I alone group as compared with the PBS control group. No significant difference was found in tumor growth between the 300 μg FA8H1 alone group and the 300 μCi 131I group. Furthermore, the anti-tumor result was significantly enhanced with the elevated dose of 131I-FA8H1 (100, 200 and 300 μCi) during the process of tumor growth (F = 9.88, P < 0.001, tumor volume; F = 701.45, P < 0.001, tumor weight), the detail could be seen in the Fig. 2b,c.
Histopathologic Tumor Necrosis in HCC Tissue
The morphological changes characteristic of tumor necrosis could be easily observed using H-E staining. As shown in Table 1 and Fig. 3a, the category and the extent of necrosis were disparate with distinct therapeutics. Although necrosis was not significant in the PBS group, fibrinoid necrosis was identified in the FA8H1 alone group. Moreover, coagulative necrosis presented to a minor extent in the 131I group and in large areas in the three 131I-FA8H1 groups. Moreover, we observed different levels of radionecrosis resulting from the various doses of 131I-FA8H1 treated in the three groups with obvious statistical difference (χ2 = 33.253, P < 0.0001).
Effects of Treatment through Apoptotic Pathways
The expression of two apoptosis indicator proteins, FasL and caspase 3, in the tumor tissues was evaluated by immunohistochemical staining (Fig. 3a,b). Compared with the HCC tissue of the PBS group, FasL and caspase 3 expressions in the other five groups were both increased, especially in the three 131I-FA8H1 groups (P < 0.001; Table 2).
Discussion
In targeted therapy to human malignant tumors, the ideal therapeutic drug only affects tumor cells, having no influence on normal cell proliferation and does not cause toxicity in vivo39. VEGFR2 was initially considered to be one of the most important targets in anticancer treatment and its targeting could meet the above requirements19,40. Our previous work demonstrated that the VEGFR2 expression in liver cancer was much higher than in normal liver tissues and this high VEGFR2 expression was associated with the poor outcome of these patients22. Moreover, we established an active murine-human chimeric Fab antibody (FA8H1), which is added to the list of potential therapeutic agents against solid tumors overexpressing VEGFR217.
In the current research, the chimeric Fab antibody was radioiodinated with 131I to investigate its therapeutic efficacy. A minimal biodistribution study was performed for this experiment prior to the therapy studies to confirm selective tumor localization and retention. The biodistribution study showed that tumors with high VEGFR2 expression could be clearly detected at appropriate time points and that the retention of the 131I-labeled chimeric antibody was limited in other normal organs. This result was in agreement with previous reports of anti-VEGFR2 antibody as an imaging agent in an in vivo tumor model41.
The therapeutic effect of the 131I-FA8H1 was confirmed in this study showed that its administration led to a reduction in tumor weight and volume; thus, 131I-FA8H1 may be a promising tool to treat cancer in vivo. Our findings provided evidence that the function of 131I-labeled chimeric anti-VEGFR2 Fab in vivo relies heavily on the recognition of VEGFR2 protein.
We also investigated the degree of necrosis under the targeted therapy using the 131I-labeled antibody. Apoptosis was confirmed by monitoring FasL and caspase 3 levels. These data revealed the necrosis and apoptosis pathways were activated by 131I or 131I-FA8H1. Then, the effect of treatment with the 131I-labeled chimeric anti-VEGFR2 Fab was determined. To our knowledge, these observations were seldom made in previous studies19,42,43 that reported the anti-tumor role of radionuclide labeled mAbs.
There were some limitations of this study. First, our results lack clues regarding the detailed molecular mechanism involved in the binding of the anti-VEGFR2 antibody and VEGFR2 antigen on the tumor cell surface. Moreover, the side-effects and toxicity issues were ignored in our present investigation. Furthermore, following observations of neutralizing effectiveness of FA8H1 in the HCC animal model, we did not perform further experiments to verify and expand the neutralizing antibody. In addition, the biodistribution and RAIT were studied in mice, but it remains to be determined whether those conclusions will also apply to human patients with HCC. Also in this investigation, we mainly focused on the cancer cells in the tumors; since VEGFR2 plays an important role in the formation of tumor angiogenesis, we did not examine the changes in new blood vessels formation in tumors resulting from the targeted treatment. Finally, it remains to be determined if FA8H1 is specific to HCC or whether it plays a similar functional role in other human malignancies.
Since we have established that VEGFR2 expression is low in adult human liver22,44 and that HCC can be treated in a mice model via the caudal vein, we believe 131I- FA8H1 has clinical translational potential in the targeted treatment of human HCC with high expression of VEGFR2. On this basis, this study clearly indicates that 131I-labeled chimeric anti-VEGFR2 Fab could be used as a therapeutic drug for RAIT through targeting VEGFR2 antigen with anti-apoptotic and necrosis effects in HCC xenograft model in mice. Thus further studies are needed to determine the mechanisms involved in the neutralizing effect of FA8H1 on HCC tumorigenesis.
Additional Information
How to cite this article: Huang, J. et al. Molecularly Targeted Therapy of Human Hepatocellular Carcinoma Xenografts with Radio-iodinated Anti-VEGFR2 Murine-Human Chimeric Fab. Sci. Rep. 5, 10660; doi: 10.1038/srep10660 (2015).
References
Zhang, A., Sun, H. & Wang, X. Power of metabolomics in diagnosis and biomarker discovery of hepatocellular carcinoma. Hepatology 57, 2072–2077 (2012).
El-Serag, H. B. Hepatocellular carcinoma. N. Engl. J. Med. 365, 1118–1127 (2011).
Forner, A., Llovet, J. M. & Bruix, J. Hepatocellular carcinoma. Lancet 379, 1245–1255 (2012).
Deckert, P. M. Current constructs and targets in clinical development for antibody-based cancer therapy. Curr. Drug. Targets 10, 158–175 (2009).
Leitha, T. Nuclear medicine: proof of principle for targeted drugs in diagnosis and therapy. Curr. Pharm. Des. 15, 173–187 (2009).
Arlen, M., Arlen, P., Tsang, A., Wang, X. & Gupta, R. The therapeutic value of monoclonal antibodies directed against immunogenic tumor glycoproteins. J Cancer 1, 209–222 (2010).
Lazar, G. A., Desjarlais, J. R., Jacinto, J., Karki, S. & Hammond, P. W. A molecular immunology approach to antibody humanization and functional optimization. Mol. Immunol. 44, 1986–1998 (2007).
Schlapschy, M. et al. Functional humanization of an anti-CD16 Fab fragment: obstacles of switching from murine {lambda} to human {lambda} or {kappa} light chains. Protein Eng. Des. Sel. 22, 175–188 (2009).
Yang, X. M. et al. High efficiency reconstitution of a human-mouse chimeric Fab of CAb-1 antibody specific to human colon cancer. Scand J. Immunol. 68, 12–21 (2008).
Burtet, R. T. et al. Production of a recombinant Fab in Pichia pastoris from a Monocistronic expression vector. J. Biochem. 142, 665–669 (2007).
Wu, J. et al. Identification and confirmation of differentially expressed fucosylated glycoproteins in the serum of ovarian cancer patients using a lectin array and LC-MS/MS. J. Proteome Res. 11, 4541–4552 (2012).
Ding, G. et al. A human/murine chimeric fab antibody neutralizes anthrax lethal toxin in vitro. Clin. Dev. Immunol. 2013, 475809 (2013).
Beck, A. & Reichert, J. M. Therapeutic Fc-fusion proteins and peptides as successful alternatives to antibodies. MAbs 3, 415–416 (2011).
McIntyre, J. A. & Faulk, A. W. Autoantibody potential of cancer therapeutic monoclonal antibodies. Int. J. Cancer 127, 491–496 (2010).
Congy-Jolivet, N., Probst, A., Watier, H. & Thibault, G. Recombinant therapeutic monoclonal antibodies: mechanisms of action in relation to structural and functional duality. Crit. Rev. Oncol. Hematol. 64, 226–233 (2007).
Huang, J. et al. A high-affinity human/mouse cross-reactive monoclonal antibody, specific for VEGFR-2 linear and conformational epitopes. Cytotechnology 62, 61–71 (2010).
Huang, J. et al. An active murine-human chimeric Fab antibody derived from Escherichia coli, potential therapy against over-expressing VEGFR2 solid tumors. Appl. Microbiol. Biotechnol. 91, 1341–1351 (2011).
Tugues, S., Koch, S., Gualandi, L., Li, X. & Claesson-Welsh, L. Vascular endothelial growth factors and receptors: anti-angiogenic therapy in the treatment of cancer. Mol. Aspects Med. 32, 88–111 (2011).
Wicki, A. et al. Targeting tumor-associated endothelial cells: anti-VEGFR2 immunoliposomes mediate tumor vessel disruption and inhibit tumor growth. Clin. Cancer Res. 18, 454–464 (2012).
Hamerlik, P. et al. Autocrine VEGF-VEGFR2-Neuropilin-1 signaling promotes glioma stem-like cell viability and tumor growth. J. Exp. Med. 209, 507–520 (2012).
Kampen, K. R. The mechanisms that regulate the localization and overexpression of VEGF receptor-2 are promising therapeutic targets in cancer biology. Anticancer Drugs 23, 347–354 (2012).
Huang, J. et al. Prognostic significance and potential therapeutic target of VEGFR2 in hepatocellular carcinoma. J. Clin. Pathol. 64, 343–348 (2011).
Gule, M. K. et al. Targeted therapy of VEGFR2 and EGFR significantly inhibits growth of anaplastic thyroid cancer in an orthotopic murine model. Clin. Cancer Res. 17, 2281–2291 (2011).
Patten, S. G. et al. VEGFR2 heterogeneity and response to anti-angiogenic low dose metronomic cyclophosphamide treatment. BMC Cancer 10, 683 (2010).
Chae, S. S. et al. Angiopoietin-2 interferes with anti-VEGFR2-induced vessel normalization and survival benefit in mice bearing gliomas. Clin. Cancer Res. 16, 3618–3627 (2010).
Spratlin, J. Ramucirumab (IMC-1121B): Monoclonal antibody inhibition of vascular endothelial growth factor receptor-2. Curr. Oncol. Rep. 13, 97–102 (2011).
Reichert, J. M. Antibody-based therapeutics to watch in 2011. MAbs 3, 76–99 (2011).
DeNardo, G. & DeNardo, S. Dose intensified molecular targeted radiotherapy for cancer-lymphoma as a paradigm. Semin. Nucl. Med. 40, 136–144 (2010).
Steiner, M. & Neri, D. Antibody-radionuclide conjugates for cancer therapy: historical considerations and new trends. Clin. Cancer Res. 17, 6406–6416 (2011).
Liu, Z. et al. Radioimmunotherapy of human colon cancer xenografts with 131I-labeled anti-CEA monoclonal antibody. Bioconjug Chem. 21, 314–318 (2010).
Turner, J. H. Defining pharmacokinetics for individual patient dosimetry in routine radiopeptide and radioimmunotherapy of cancer: Australian experience. Curr. Pharm Des. 15, 966–982 (2009).
Illidge, T. M. et al. Phase 1/2 study of fractionated (131)I-rituximab in low-grade B-cell lymphoma: the effect of prior rituximab dosing and tumor burden on subsequent radioimmunotherapy. Blood 113, 1412–1421 (2009).
Xu, N. et al. Molecular imaging application of radioiodinated anti-EGFR human Fab to EGFR-overexpressing tumor xenografts. Anticancer Res. 29, 4005–4011 (2009).
Chekhonina, V. P. et al. [Biodegradation of 125I-labeled monoclonal antibodies after introvenous administration to rats with experimental C6 glioma]. Biomed. Khim 53, 532–540 (2007).
Hao, L. et al. Preparation and evaluation of human-murine chimeric antibody against protective antigen of Bacillus anthracis. Int. J. Mol. Sci. 15, 18496–18507 (2014).
Feng, J. et al. Involvement of FoxQ1 in NSCLC through regulating EMT and increasing chemosensitivity. Oncotarget 5, 9689–9702 (2014).
Sun, R. et al. Prognostic value of LAMP3 and TP53 overexpression in benign and malignant gastrointestinal tissues. Oncotarget 5, 12398–12409 (2014).
Zhang, H. et al. ROR1 expression correlated with poor clinical outcome in human ovarian cancer. Sci. Rep. 4, 5811 (2014).
Rosa, D. D., Ismael, G., Lago, L. D. & Awada, A. Molecular-targeted therapies: lessons from years of clinical development. Cancer Treat. Rev. 34, 61–80 (2008).
Cheong, S. J. et al. Evaluation of the therapeutic efficacy of a VEGFR2-blocking antibody using sodium-iodide symporter molecular imaging in a tumor xenograft model. Nucl. Med. Biol. 38, 93–101 (2011).
Chopra, A. 18F-Labeled N-succinimidyl-4-fluorobenzoate-conjugated rat anti-mouse vascular endothelial growth factor receptor 2 monoclonal antibody linked to microbubbles. Molecular Imaging and Contrast Agent Database (MICAD) [Internet]. Bethesda (MD): National Center for Biotechnology Information (US) ; 2004-2013, 1–4 (2004).
Stantz, K. M., Cao, M., Cao, N., Liang, Y. & Miller, K. D. Monitoring the longitudinal intra-tumor physiological impulse response to VEGFR2 blockade in breast tumors using DCE-CT. Mol. Imaging Biol. 13, 1183–1195 (2011).
Otsuji, E. et al. Application of 99mTc labeled chimeric Fab fragments of monoclonal antibody A7 for immunoscintigraphy of pancreatic carcinoma. J. Surg. Oncol. 84, 160–165 (2003).
Huang, J. et al. The patterns and expression of KDR in normal tissues of human internal organs. J. Mol. Histol. 42, 597–603 (2011).
Acknowledgements
This study was supported by the State Major Basic Research Development Program of China (973 Project, No. 2010CB933902), the postdoctoral study grant (2013M541706) from China, the postdoctoral study (1301069B) from the Human Resources and Social Security Department of Jiangsu, China.
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Z. F. and Z. J. designed the study; Q. T. and B. L. collected the tissue samples; H. Y. carried out the experiments with 131I ; Q. T. performed the FACS and IHC analysis; J. H. and Z. F. collected data and participated in the evaluation of the IHC; J. H. drafted the manuscript; Z. F. supervised the study. All authors read and approved the final manuscript.
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Huang, J., Tang, Q., Wang, C. et al. Molecularly Targeted Therapy of Human Hepatocellular Carcinoma Xenografts with Radio-iodinated Anti-VEGFR2 Murine-Human Chimeric Fab. Sci Rep 5, 10660 (2015). https://doi.org/10.1038/srep10660
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DOI: https://doi.org/10.1038/srep10660
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