86Y-CHX-A''-Diethylenetriamine pentaacetic acid-bevacizumab


Leung K.

Publication Details



In vitro Rodents



The vascular endothelial growth factor (VEGF) family is composed of five VEGF glycoproteins (VEGF-A, VEGF-B, VEGF-C, VEGF-D, and VEGF-E) and consists of at least six isoforms of various numbers of amino acids (121, 145, 165, 183, 189, and 206 amino acids) produced through alternative splicing (1). VEGF-A is composed of VEGF121, VEGF165, and VEGF189 isoforms, which are secreted by most cell types and are active as homodimers linked by disulfide bonds. VEGF121 does not bind to heparin like other VEGF species do (2). VEGF is a potent angiogenic factor that induces proliferation, sprouting, migration, and tube formation of endothelial cells. There are three high-affinity tyrosine kinase VEGF receptors (VEGFR-1, Flt-1; VEGFR-2, KDR/Flt-1; and VEGFR-3, Flt-4) on endothelial cells. Several types of non-endothelial cells, such as hematopoietic stem cells, melanoma cells, monocytes, osteoblasts, and pancreatic β cells, also express VEGFRs (1).

VEGF is overexpressed in various tumor cells and tumor-associated endothelial cells (3). Inhibition of VEGFR function has been shown to inhibit pathological angiogenesis as well as tumor growth and metastasis (4, 5). Radiolabeled VEGF has been developed as a single-photon emission computed tomography tracer for imaging solid tumors and angiogenesis in humans (6-8). However, several studies have shown that cancer treatments (photodynamic therapy, radiotherapy, and chemotherapy) can lead to increased tumor VEGF expression and subsequently to more aggressive disease (9, 10). Therefore, it is important to measure VEGF levels in the tumors to design better anticancer treatment protocols. Bevacizumab is a humanized antibody against VEGF-A (11) that binds to all VEGF-A isoforms. Bevacizumab is approved for clinical use in metastatic colon carcinoma and non-small cell lung cancer (12). VEGFR-2 has been shown to mediate most of the VEGF-A activation in tumor endothelial cells (13, 14). Nayak et al. (15) prepared 86Y-CHX-A''-diethylenetriamine pentaacetic acid-bevacizumab (86Y-CHX-A''-DTPA-bevacizumab) for imaging VEGF expression in nude mice bearing SKOV-3 human ovarian tumor xenografts.



Bifunctional CHX-A''-DTPA was used to conjugate bevacizumab to form CHX-A''-DTPA-bevacizumab (~150 kDa), which was purified with column chromatography (15). The DTPA/antibody ratio was determined to be 1.9. Next, 86Y solution (140–170 MBq (3.8–4.6 mCi)) was added to CHX-A''-DTPA-bevacizumab (0.33 nmol) in ammonium acetate buffer (pH 5–6). The reaction mixture was incubated for 30 min at room temperature, and 86Y-CHX-A''-DTPA-bevacizumab was purified with column chromatography. The yield of 86Y-CHX-A''-DTPA-bevacizumab was 60%–75% with a specific activity of >220 MBq/nmol (6.1 mCi/nmol).

In Vitro Studies: Testing in Cells and Tissues


Binding of 86Y-CHX-A''-DTPA-bevacizumab under excess antigen (VEGF165) conditions indicated that the immunoreactivity was 58%–66% (15). The non-specific binding was <6% in the assay.

Animal Studies



Nayak et al. (15) performed ex vivo biodistribution studies of 0.5 MBq (0.014 mCi) 86Y-CHX-A''-DTPA-bevacizumab (0.02 nmol) in nude mice (n = 4/group) bearing human SKOV-3 ovarian tumor (VEGF-A–positive) and MSTO-211H mesothelioma (VEGF-A–negative) xenografts. SKOV-3 tumor uptake values were 12% injected dose per gram (ID/g) at 0.5 d, 17% ID/g at 3 d, and 15% ID/g at 4 d. On the other hand, MSTO-211H tumor uptake values were 5% ID/g at 0.5 d, 6% ID/g at 3 d, and 6% ID/g at 4 d. The radioactivity level in the blood was 11% ID/g at 0.5 d, 7% ID/g at 3 d, and 6% at 4 d. The initial uptake in the liver was 14% ID/g at 0.5 d and decreased to 8% ID/g at 3 d. All other organs showed lower radioactivity levels than the SKOV-3 tumors at all these time points. Coinjection of bevacizumab (0.33 nmol) with 86Y-CHX-A''-DTPA-bevacizumab reduced the SKOV-3 tumor accumulation to 9% ID/g at 3 d after injection, whereas no inhibition was observed in the MSTO-211H tumors and non-tumor tissues.

Nayak et al. (15) studied the whole-body distribution of 2 MBq (0.056 mCi) 86Y-CHX-A''-DTPA-bevacizumab (0.02 nmol) with PET imaging in nude mice (n = 3) bearing human SKOV-3 ovarian tumor and MSTO-211H xenografts with static scans at various time points (1–3 d). The SKOV-3 tumors were clearly visualized at 1–3 d after injection, but the MSTO-211H tumors were not. The mean tumor residence times were similar (2.6–2.7 d). Co-injection of 0.33 nmol bevacizumab inhibited accumulation of radioactivity in the SKOV-3 tumors by 50% at 3 d after injection, whereas no inhibition was observed in the MSTO-211H tumors. There was a good correlation (r2 = 0.87) between the tracer accumulation measured with PET and tracer accumulation measured with ex vivo biodistribution studies.

Other Non-Primate Mammals


No publication is currently available.

Non-Human Primates


No publication is currently available.

Human Studies


No publication is currently available.

NIH Support

Intramural Research Program


Ferrara N. Vascular endothelial growth factor: basic science and clinical progress. Endocr Rev. 2004;25(4):581–611. [PubMed: 15294883]
Cohen T., Gitay-Goren H., Sharon R., Shibuya M., Halaban R., Levi B.Z., Neufeld G. VEGF121, a vascular endothelial growth factor (VEGF) isoform lacking heparin binding ability, requires cell-surface heparan sulfates for efficient binding to the VEGF receptors of human melanoma cells. J Biol Chem. 1995;270(19):11322–6. [PubMed: 7744769]
Soria J.C., Fayette J., Armand J.P. Molecular targeting: targeting angiogenesis in solid tumors. Ann Oncol. 2004;15 Suppl 4:iv223–7. [PubMed: 15477311]
Ferrara N. Vascular endothelial growth factor as a target for anticancer therapy. Oncologist. 2004;9 Suppl 1:2–10. [PubMed: 15178810]
Hicklin D.J., Ellis L.M. Role of the vascular endothelial growth factor pathway in tumor growth and angiogenesis. J Clin Oncol. 2005;23(5):1011–27. [PubMed: 15585754]
Li S., Peck-Radosavljevic M., Koller E., Koller F., Kaserer K., Kreil A., Kapiotis S., Hamwi A., Weich H.A., Valent P., Angelberger P., Dudczak R., Virgolini I. Characterization of (123)I-vascular endothelial growth factor-binding sites expressed on human tumour cells: possible implication for tumour scintigraphy. Int J Cancer. 2001;91(6):789–96. [PubMed: 11275981]
Li S., Peck-Radosavljevic M., Kienast O., Preitfellner J., Hamilton G., Kurtaran A., Pirich C., Angelberger P., Dudczak R. Imaging gastrointestinal tumours using vascular endothelial growth factor-165 (VEGF165) receptor scintigraphy. Ann Oncol. 2003;14(8):1274–7. [PubMed: 12881392]
Li S., Peck-Radosavljevic M., Kienast O., Preitfellner J., Havlik E., Schima W., Traub-Weidinger T., Graf S., Beheshti M., Schmid M., Angelberger P., Dudczak R. Iodine-123-vascular endothelial growth factor-165 (123I-VEGF165). Biodistribution, safety and radiation dosimetry in patients with pancreatic carcinoma. Q J Nucl Med Mol Imaging. 2004;48(3):198–206. [PubMed: 15499293]
Gorski D.H., Beckett M.A., Jaskowiak N.T., Calvin D.P., Mauceri H.J., Salloum R.M., Seetharam S., Koons A., Hari D.M., Kufe D.W., Weichselbaum R.R. Blockage of the vascular endothelial growth factor stress response increases the antitumor effects of ionizing radiation. Cancer Res. 1999;59(14):3374–8. [PubMed: 10416597]
Solban N., Selbo P.K., Sinha A.K., Chang S.K., Hasan T. Mechanistic investigation and implications of photodynamic therapy induction of vascular endothelial growth factor in prostate cancer. Cancer Res. 2006;66(11):5633–40. [PubMed: 16740700]
Wang Y., Fei D., Vanderlaan M., Song A. Biological activity of bevacizumab, a humanized anti-VEGF antibody in vitro. Angiogenesis. 2004;7(4):335–45. [PubMed: 15886877]
Ferrara N., Hillan K.J., Gerber H.P., Novotny W. Discovery and development of bevacizumab, an anti-VEGF antibody for treating cancer. Nat Rev Drug Discov. 2004;3(5):391–400. [PubMed: 15136787]
Folkman J. Role of angiogenesis in tumor growth and metastasis. Semin Oncol. 2002;29(6) Suppl 16:15–8. [PubMed: 12516034]
Ferrara N., Gerber H.P., LeCouter J. The biology of VEGF and its receptors. Nat Med. 2003;9(6):669–76. [PubMed: 12778165]
Nayak, T.K., K. Garmestani, K.E. Baidoo, D.E. Milenic, and M.W. Brechbiel, PET imaging of tumor angiogenesis in mice with VEGF-A targeted (86)Y-CHX-A''-DTPA-bevacizumab. Int J Cancer, 2010. [PMC free article: PMC2939172] [PubMed: 20473899]