CN103505727A - Application of novel function of CD146 targeted as co-receptor of vascular endothelial growth factor receptor-2 (VEGFR-2) in anti-tumor angiogenesis treatment - Google Patents

Abstract

The invention discloses application of a novel function of a CD146 targeted as a co-receptor of a vascular endothelial growth factor receptor-2 (VEGFR-2) in anti-tumor angiogenesis treatment. In the invention, the condition that a cell adhesion molecule CD146 is the co-receptor of the VEGFR-2 is proposed for the first time, and combined application of an anti-CD146 antibody and an anti-VEGF antibody for inhibition of tumor angiogenesis can become a new strategy for clinical treatment of cancers. Compared with the conventional single molecule-targeted antibody drug for tumor treatment, tumor growth can be more effectively inhibited by combination of two different molecule-targeted antibodies; the CD146 can assist the co-receptor to more effectively transmit a VEGF-mediated downstream signal as the co-receptor of the VEGFR-2, so as to achieve an angiogenesis function; an action mechanism of combination of the anti-CD146 antibody and the anti-VEGF antibody for tumor treatment manly comprises the functions, such as common inhibition of activation of a VEGF-VEGFR-2 signal channel, migration of endothelial cells, angiogenesis and the like by combination of the two antibodies, so as to more effectively inhibit humor growth (such as a pancreatic cancer and the like). Combined application of the two antibodies or cooperation with other clinical treatment means (for example, clinical chemoradiotherapy) is bound to become a new strategy for inhibition of tumor angiogenesis; meanwhile, the CD146 can be applied to basic researches of a tumor angiogenesis mechanism and the like.

Description

Targeting the novel function of CD146 as a VEGFR-2 co-receptor in anti-tumor angiogenesis therapy

technical field

The present invention relates to compositions useful in the treatment of tumors. Specifically, the present invention relates to a combination of anti-CD146 antibody and anti-VEGF antibody, which can be used for treating tumors. More specifically, the composition of the anti-CD146 monoclonal antibody AA98 or its functional fragments and the anti-VEGF monoclonal antibody bevacizumab or its functional fragments involved in the present invention can jointly inhibit the activation and activation of the VEGF-VEGFR-2 signaling pathway. The endothelial cell migration, angiogenesis and other functions caused by it can more effectively inhibit tumor growth, and more specifically inhibit pancreatic tumors. the

Background technique

Cancer is currently the number one killer that endangers human life and health. The annual incidence of malignant tumors in my country is about 2.6 million. People who died of cancer accounted for more than 20% of the total number of deaths, ranking first in the cause of death of urban residents (according to 2005 statistics). the

At present, drug therapy is an important aspect in the three conventional treatments of tumors <radiotherapy, chemotherapy and surgery>. At present, there are many factors that affect the therapeutic effect of tumor drugs, including drug specificity, transport mode, permeability and induction of tumor drug resistance. Among them, the specificity and drug resistance of drugs are the key problems that need to be solved urgently in tumor therapeutics. Anti-tumor vascular therapy has become an important means of tumor treatment due to its advantages of tumor specificity, broad-spectrum, no or low drug resistance. Its theoretical basis is mainly the concept of angiogenesis proposed by Folkman in the 1970s, that is, new blood vessels provide nutrients and oxygen supply to tumors, thereby promoting tumor growth and metastasis. the

A large number of studies have found that there are more than 30 factors related to tumor angiogenesis, such as vascular endothelial growth factor (vascular endothelial growth factor, VEGF), transforming growth factor (transforming growth factor-, TNF), fibroblast growth factor (fibroblast growth factor, FGF), tumor necrosis factor (tumor necrosis factor, TNF), angiopoietins (angiopoietins) and so on. Among them, VEGF has been proved to be the most important highly specific growth factor directly acting on angiogenesis. Tumor cells can synthesize and periodically secrete VEGF. The secreted VEGF binds to VEGF receptors (VEGFRs) on vascular endothelial cells, thereby promoting tumor angiogenesis. The signal transduction of VEGF to promote angiogenesis is mainly realized through its combination with VEGFR-2. Most of the currently marketed anti-angiogenic drugs are designed with key signaling molecules in these signal transduction pathways as drug targets, such as anti-VEGF monoclonal antibody Bevacizumab (Bevacizumab, Roche Company), anti-VEGFR- 2 monoclonal antibody DC101 and so on. At the same time, studies have found that VEGFR-2 has co-receptors, such as CD44, which can interact with VEGFR-2 and act as co-receptors to regulate VEGFR-2-mediated angiogenesis. Therefore, targeting such co-receptors can also effectively inhibit tumor angiogenesis and thus tumor growth. the

At the same time, there are still some problems and bottlenecks in antibody immunotherapy targeting tumor blood vessels. For example, antibody therapy often cannot completely kill all tumor cells, and the remaining cells are prone to relapse after drug withdrawal. Moreover, single-target antibody therapy has limitations and cannot achieve effective therapeutic effects. Therefore, finding new therapeutic targets and discovering more effective therapeutic strategies is an urgent problem to be solved. the

Contents of the invention

The present invention proposes for the first time that CD146 is a co-receptor of VEGFR-2, and the combination of anti-CD146 monoclonal antibody AA98 and anti-VEGF monoclonal antibody Bevacizumab (Bevacizumab, Roche Company) can be used as a new targeted drug for treating tumors . In animal tumor models (such as human pancreatic cancer), compared with a single antibody drug treatment of tumors, the combination of AA98 and bevacizumab can more effectively inhibit tumor angiogenesis and thereby inhibit tumor growth. the

The application of anti-CD146 antibody AA98 combined with bevacizumab antibody in anti-tumor therapy is based on the following important scientific discoveries: (1) CD146 interacts with VEGFR-2 and is a co-receptor of VEGFR-2; CD146 monoclonal antibody AA98 can block the interaction between CD146 and VEGFR-2, thereby blocking the activation of VEGFR-2 and the activation of downstream signals (Akt and p38/NF-κB) induced by VEGF stimulation; (3) Anti- CD146 monoclonal antibody AA98 inhibits the migration and angiogenesis of endothelial cells induced by VEGF; (4) In the nude mouse tumor-bearing model (human pancreatic cancer cell line SW1990), the combination of AA98 and bevacizumab can inhibit tumor blood vessels more effectively produced to inhibit tumor growth. the

In summary, as a co-receptor of VEGFR-2, CD146 can regulate and assist the transmission of VEGF-VEGFR-2 signals, thereby affecting the migration and angiogenesis of endothelial cells. Compared with AA98 or bevacizumab alone, our self-developed anti-CD146 functional antibody AA98 combined with bevacizumab can more significantly inhibit tumor angiogenesis and thereby inhibit tumor growth in nude mouse tumor-bearing models. Therefore, we propose that targeting CD146 as a co-receptor of VEGFR-2 can more effectively inhibit tumor growth, and the pharmaceutical composition of anti-CD146 monoclonal antibody AA98 and bevacizumab is a new targeted drug for treating tumors. AA98 can inhibit the activation of signaling pathways caused by VEGF by inhibiting the interaction between VEGFR-2 and CD146, thereby blocking angiogenesis and cutting off the nutrient supply of tumors, thereby inhibiting tumor growth. The combination of AA98 and bevacizumab can more effectively cut off the activation of signaling pathways that determine tumor growth, and more significantly inhibit tumor growth. the

Specifically, the present invention provides the following:

1. A composition comprising an anti-CD146 antibody or a functional fragment of the anti-CD146 antibody and an anti-VEGF antibody or a functional fragment of the anti-VEGF antibody, the composition is used to inhibit tumor angiogenesis, thereby inhibiting tumor growth. the

2. The composition according to item 1, wherein the tumor is a pancreatic tumor. the

3. The composition according to item 1 or 2, wherein the anti-CD146 antibody is anti-CD146 monoclonal antibody AA98, and the anti-VEGF antibody is bevacizumab. the

4. Use of the composition according to item 1 in the preparation of a medicament for inhibiting tumor growth. the

5. The use according to item 4, wherein the tumor is a pancreatic tumor. the

6. The use according to item 4 or 5, wherein the anti-CD146 antibody is anti-CD146 monoclonal antibody AA98, and the anti-VEGF antibody is bevacizumab. the

The present invention will be further described below in conjunction with specific examples. the

Description of drawings

From the following detailed description in conjunction with the accompanying drawings, the above-mentioned features and advantages of the present invention will be more apparent, wherein:

Figure 1. CD146 interacts with VEGFR-2 at the molecular level. A, in human umbilical vein endothelial cells (HUVECs), the co-immunoprecipitation method found the interaction between CD146 and VEGFR-2; B, in HEK293T, the co-immunoprecipitation method verified the interaction between CD146 and VEGFR-2; C, in vitro pull -down experiments found that there is a direct interaction between the extracellular region of CD146 and the extracellular region of VEGFR-2;

Figure 2. Anti-CD146 monoclonal antibody AA98 blocks VEGF-induced activation of VEGFR-2 signaling pathway (B-F) by blocking the interaction between CD146 and VEGFR-2 (A);

Figure 3. In vitro, AA98 inhibits VEGF-induced endothelial cell migration (A) and angiogenesis (B);

Figure 4. In the tumor-bearing mouse model, compared with AA98 or bevacizumab administered alone, the combination of AA98 and bevacizumab can inhibit tumor growth more effectively. (A) Schematic diagram of tumor size in different administration groups; (B) Compared with AA98 or bevacizumab administered alone, the combined administration of AA98 and bevacizumab can inhibit the tumor size more effectively; (C) comparison Compared with AA98 or bevacizumab alone, AA98 combined with bevacizumab can inhibit the tumor weight more effectively; (D) Compared with AA98 or bevacizumab alone, AA98 and bevacizumab Combined anti-tumor administration can more effectively inhibit tumor angiogenesis. the

Detailed ways

Antibody AA98 (Yan, X et al. A novel anti-CD146 monoclonal antibody, AA98, inhibits angiogenesis and tumor growth. Blood. 2003; 102 (1): 184-191.), AA1 (produced independently by this laboratory, Deposit institution: China General Microbiology Collection Center; deposit number: CGMCC No.2310; deposit date: 2007.12.28) etc. can be obtained according to the descriptions of Chinese patent application number 99107586.2 (CN1234405) and Chinese patent application number 200810057260.7 (CN101245101). Bevacizumab was purchased from Roche Company. the

Example 1: Interaction between CD146 and VEGFR-2 at the cellular level;

Both CD146 and VEGFR-2 are markers of endothelial cells and are key molecules in the process of angiogenesis, but their connection is not clear. In order to study the relationship between the two, we confirmed the interaction between CD146 and VEGFR-2 at the cellular level by co-immunoprecipitation and other methods. Anti-CD146 monoclonal antibody AA98 can block the interaction between CD146 and VEGFR-2. the

Main materials: human umbilical vein endothelial cell line (HUVECs) (ATCC, CRL-1730), HEK293T cell line stably transfected with CD146cDNA (ATCC, CRL-11268) (HEK293T/CD146), etc. the

Main reagents: cell lysate, PBS, mouse anti-CD146 monoclonal antibody AA1 (produced by our laboratory independently. Preservation agency: China General Microbiology Collection Center; preservation number: CGMCC No.2310; preservation date: 2007.12.28), rabbit Anti-VEGFR-2 antibody (purchased from Cell Signaling Technology Company, catalog number #2479), His-sCD146 protein (purchased from Sino Biological Inc., catalog number 50794-M08H), Fc-VEGFR-2 protein (purchased from Sino Biological Inc., Product number 10012-H02H), Fc protein (purchased from Sino Biological Inc., product number 10702-HNAH), VEGF protein (purchased from Upstate Biotechnology company, product number B500014). the

Main method: co-immunoprecipitation and in vitro pull-down experiment, the specific method is as follows:

Co-immunoprecipitation:

1) Human umbilical vein endothelial cells (1×10 ⁷ /ml) or transiently transfected with VEGFR-2 plasmid (10 μg) (Lena Claesson-Welsh et al. EMBO J. 2005 July 6; 24(13): 2342-2353 .) HEK293T/CD146 was inoculated in a 100mm culture dish. When the cell density reached 90%, the cells were gently scraped off, centrifuged at 4°C for 5 minutes and collected in an Ep tube.

2) Add 600μl lysate RIPA Buffer (50mM Tris-HCl, pH 7.4, 1% NP-40, 0.25% Na-deoxycholate, 150mM NaCl, 1mM EDTA, 1mM Na3VO4, 1mM NaF, 1mM PMSF and 1mM proteinase inhibitors cocktails (protease Inhibitors were purchased from Roche, Cat. No. 04693116001)), lysed on ice for 30 minutes, and centrifuged (12,000 g) at 4° C. for 15 minutes. the

3) Aspirate the supernatant as a cell lysate, and after measuring the protein concentration by the Bradford method, dilute the total protein concentration to 1 mg/ml. the

4) Add 20 μl protein G-Agarose to the lysate and incubate at 4°C for 1 hour to remove proteins non-specifically bound to 20 μl protein G-Agarose. the

5) Take the supernatant by centrifugation, add 2 μg of CD146 monoclonal antibody AA1 or rabbit anti-VEGFR-2 antibody, and incubate at 4°C for 2 hours. the

6) Add 50 μl protein G-Agarose again and incubate at 4°C for 1 hour. the

7) Discard the supernatant by centrifugation, wash the precipitated agarose beads 3 times with PBS containing protease inhibitors, add 100 μl of loading buffer solution for 5 minutes each time, vortex for 2 minutes, cook at 100°C for 10 minutes, and centrifuge to take the supernatant. the

8) The processed protein sample and the reserved sample of the whole cell lysate are used for Western blot detection together. the

In vitro pull-down experiment:

1) Melt 200ng of Fc-VEGFR-2 protein or 200ng of Fc protein and 200ng of His-CD146 protein in 500μl PBS EP tube, incubate at 4°C for 1 hour. the

2) Add 20 μl protein G beads and incubate at 4°C for 1 hour. the

3) Centrifuge at 4°C for 5 minutes, discard the supernatant, wash the precipitated agarose beads 3 times with PBS containing protease inhibitors, after 5 minutes each time, add 50 μl of loading buffer, vortex for 2 minutes, cook at 100°C for 10 minutes. the

4) The processed protein samples were used for Western blot detection. the

The results are shown in Figure 1. In HUVECs (A), CD146 can bind to VEGFR-2 while being captured by anti-CD146 antibody AA1, and vice versa, indicating that there is an interaction between CD146 and VEGFR-2. In HEK293T/CD146 (B), in the cells transfected with VEGFR-2, the rabbit anti-VEGFR-2 antibody can simultaneously capture VEGFR-2 and CD146, but in the cells expressing only CD146, it cannot be detected in the results of Western blot To CD146, this result verified the conclusion of A that there is an interaction between CD146 and VEGFR-2. In in vitro pull-down experiments, as shown in (C), there is a direct interaction between CD146 and VEGFR-2. In addition, HUVECs were incubated with anti-CD146 monoclonal antibody AA98 or AA1 (100 μg/ml), and after 1 hour at 37°C, they were washed three times with cell culture medium, and the above-treated cells were used for co-immunoprecipitation experiments, such as (D ) showed that in HUVECs, anti-CD146 monoclonal antibody AA98 could block the interaction between endogenous CD146 and VEGFR-2. The above results indicate that CD146 interacts directly with VEGFR-2 in endothelial cells, and the anti-CD146 monoclonal antibody AA98 can block the interaction between the two. the

Example 2: The anti-CD146 antibody blocks the activation of the VEGFR-2 signaling pathway caused by VEGF stimulation by blocking the interaction between CD146 and VEGFR-2;

The adhesion molecule CD146 has a key role in angiogenesis. As a co-receptor of VEGFR-2, CD146 regulates the VEGFR-2 signaling pathway. Anti-CD146 monoclonal antibody AA98 can block the interaction between CD146 and VEGFR-2, thereby blocking the activation of VEGFR-2 signaling pathway induced by VEGF stimulation. the

Incubate HUVECs with anti-CD146 monoclonal antibody AA98 or AA1 (100μg/ml), and after 1 hour at 37°C, wash with cell culture medium three times, then stimulate with VEGF (50ng/ml) and lyse the cells for biochemical analysis path. As shown in Figure 2(A), AA98 was able to block the interaction between endogenous CD146 and VEGFR-2 in HUVECs. As shown in (B-F), VEGF stimulation (5 minutes) can cause the phosphorylation of VEGFR-2, as well as the activation of downstream signaling molecules such as Akt (15 minutes), p38 (15 minutes), and NF-κB (10 hours). AA98 can block the activation of VEGFR-2 and downstream molecules Akt, p38, NF-kB induced by VEGF stimulation. It shows that CD146 can participate in and regulate the activation of VEGF-VEGFR-2 signaling pathway. the

Example 3: Anti-CD146 antibody inhibits endothelial cell migration and angiogenesis induced by VEGF stimulation;

Angiogenesis mainly provides nutrients for tumors and provides pathways for tumor cell metastasis, so it plays a vital role in tumor growth. Inhibition of angiogenesis can inhibit tumor growth to a great extent. VEGF is the main cytokine secreted by tumor cells, and the signaling pathway mediated by it ultimately determines tumor angiogenesis. CD146 participates in and regulates VEGF-VEGFR-2 signaling pathway, and anti-CD146 antibody inhibits the migration and angiogenesis of endothelial cells stimulated by VEGF. the

Main materials: human umbilical vein endothelial cell line, 96-well Transwell plate (Corning HTS Transwell-96 Cell Migration Products), etc. the

Main reagents: VEGF (purchased from Upstate Biotechnology, Cat. No. B500014), anti-CD146 antibodies AA98, AA1, mouse IgG (mIgG, purchased from Sigma-Aldrich, Cat. No. I5381), Matrigel (without growth factors, BD Biosciences, Cat. No. 354234 ). the

Main methods: cell migration experiment, endothelial cell angiogenesis experiment. The specific method is as follows:

Cell Migration Assay:

1) HUVECs cells were resuspended in complete medium to make a single cell suspension (1×10 ⁵ /ml).

2) Add RPMI-1640 medium (purchased from Gibco, Cat. No. 31800-022) (200 μl/well) containing 10% fetal bovine serum to the lower chamber of the Transwell, and add cell suspension (100 μl/well) to the upper chamber, and set three times for each treatment. parallel holes),

3) Add antibody AA98 or AA1 (100 μg/ml) and stimulator VEGF (100 ng/ml) to the cells in the upper chamber. Incubate overnight in a 37°C carbon dioxide incubator. the

4) Wipe off the cells on the upper layer of the membrane with a cotton swab, peel off the membrane of the 96-well transwell plate with tweezers, and place the lower layer of the membrane on a glass slide. the

5) After the cells in the lower layer were fixed with 4% paraformaldehyde at room temperature for 15 minutes, they were stained with 1% crystal violet for 15 minutes, and the number of cells in each field of view was recorded under a microscope. the

The endothelial cell angiogenesis experiment is improved on the basis of the experimental method established by Nagata et al. The specific operation is as follows: 

1) HUVECs cells were resuspended in complete medium to make a single cell suspension (1×10 ⁵ /ml).

2) Coat ice-bathed Matrigel (50 μl/well) in a 96-well plate, and solidify at 37° C. for 30 minutes. the

3) Add 100 μl of cell suspension to each well, and simultaneously add the stimulating agent VEGF (100 ng/ml) and antibody AA98 or AA1 (100 μg/ml) accordingly. the

4) Incubate overnight in a 37°C incubator, then observe and take pictures under an inverted microscope. the

The experimental results are shown in Figure 3(A), VEGF can increase the migration ability of endothelial cells and, compared with the AA1 control group, the anti-CD146 antibody AA98 can significantly reduce the migration ability of endothelial cells induced by VEGF stimulation, and the inhibition rate is about 53%. Similarly, stimulating HUVECs with VEGF can promote HUVECs to form blood vessel-like structures. However, AA98 significantly inhibited the angiogenic ability of endothelial cells induced by VEGF stimulation relative to the AA1-treated group. The above results confirmed that CD146 is necessary for the angiogenesis process mediated by VEGFR-2. the

Example 4: In a tumor-bearing nude mouse model, compared with AA98 or bevacizumab alone, the combination of AA98 and bevacizumab can more significantly inhibit human pancreatic cancer tumor angiogenesis and thus tumor growth. the

During the occurrence and development of tumors, angiogenesis provides nutrients for tumors and provides pathways for tumor cell metastasis. At present, targeting tumor angiogenesis has become one of the main strategies for clinical treatment of tumors. Animal experiments have confirmed that the combination of AA98 and bevacizumab can inhibit tumor growth more effectively. the

Experimental method: 60 4-week-old female BalB/C nude mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were selected and randomly divided into 6 groups to be treated with different antibodies, 10 in each group. 1×10 ⁷ human pancreatic cancer cell line SW1990 cells (ATCC, CRL-2172) were subcutaneously injected on the back (resuspended in 100 μl PBS) respectively. When the tumor volume reached 0.06cm ³ , intraperitoneal injection of antibodies was started, namely mIgG (purchased from Sigma-Aldrich, Cat. No. I5381; 200 μg/monkey), hIgG (purchased from Zhongshan Jinqiao, Cat. No. ZDR-5001; 200 μg/monkey), AA1 (200 μg/body), AA98 (200 μg/piece), bevacizumab (purchased from Roche; 200 μg/piece), AA98+bevacizumab (AA98 and bevacizumab were 200 μg/piece, a total of 400 μg/piece) , 2 times a week, measure the length and width of the tumor in order to calculate the tumor volume. After 4 weeks, all the mice were sacrificed by neck dislocation, and the tumors were peeled off. After the tumors were weighed, they were fixed with 4% PFA for 24 hours, embedded in paraffin, sectioned, and analyzed by immunohistochemistry. The following is the immunohistochemical process of paraffin section:

1) Take out the slice, dewax in xylene solution at 37°C twice, 30 minutes each time;

2) Hydrate in absolute ethanol × 2-95%-80%-70%-50%-30% and distilled water, room temperature for 5 minutes each time;

3) 0.3% hydrogen peroxide/methanol solution was treated at 37°C in the dark for 30 minutes to eliminate the activity of endogenous peroxidase, and washed three times with PBS;

4) pH 6.0 citric acid repair solution, 100°C water bath for 30 minutes for antigen heat repair, and natural cooling;

5) 5% normal sheep serum (purchased from Zhongshan Jinqiao Company, product number ZLI-9021) was blocked at 37°C for 1 hour;

6) Add primary antibody diluted in PBS (rabbit anti-CD31 polyclonal antibody, purchased from Abcam, Cat. No. ab28364; 1:50 dilution), and incubate overnight at 4°C;

7) Wash three times with PBS; goat anti-rabbit-biotin (purchased from Zhongshan Jinqiao Company, Cat. No. ZB-2010; 1:1000 dilution) was incubated at 37°C for 1 hour, and washed three times with PBS;

8) Avidin-HRP (purchased from Hyclone-pierce, Cat. No. N100; 1:1000) was incubated at 37°C for 45 minutes;

9) The prepared DAB (purchased from Zhongshan Jinqiao Company, Cat. No. ZLI-9032; diluted 1:1000) was developed in the dark for 2-7 minutes, and counterstained with hematoxylin. the

10) Gradual dehydration: 50-70-80-90-100-100% ethanol-xylene, dry in air, and seal with neutral resin.

11) Filming in the microscopic imaging system. the

The results showed that, as shown in FIG. 4 , in the experimental group of the combination of AA98 and bevacizumab, the inhibitory effect of the composition on tumor growth was more obvious. Compared with the control group (mIgG or hIgG group), the tumor volume inhibition percentage of the composition was 70%, while the inhibition rate of AA98 or bevacizumab alone was only 46% or 48%. Tumors treated with the combination of AA98 and bevacizumab had less vessel density, 40% or 30% of the vessel density of the AA98 or bevacizumab alone groups, respectively. The above results indicate that the combination of AA98 and bevacizumab can more effectively inhibit tumor angiogenesis and thus inhibit tumor growth. the

Claims (6)

1. A composition comprising an anti-CD146 antibody or a functional fragment of the anti-CD146 antibody and an anti-VEGF antibody or a functional fragment of the anti-VEGF antibody, the composition is used to inhibit tumor angiogenesis, thereby inhibiting tumor growth. 2. The composition of claim 1, wherein the tumor is a pancreatic tumor. 3. The composition according to claim 1 or 2, wherein the anti-CD146 antibody is anti-CD146 monoclonal antibody AA98 and the anti-VEGF antibody is bevacizumab. 4. Use of the composition according to claim 1 in the preparation of a medicament for inhibiting tumor growth. 5. The use according to claim 4, wherein the tumor is a pancreatic tumor. 6. The use according to claim 4 or claim 5, wherein the anti-CD146 antibody is anti-CD146 monoclonal antibody AA98, and the anti-VEGF antibody is bevacizumab.

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