This study shows the feasibility of using arsenic radioisotopes to label a monoclonal antibody directed against anionic phospholipids on the surface of tumor vascular endothelium. Tumor selective targeting was observed in vivo and confirmed by biodistribution analysis and histology.
The two isotopes we selected for the present studies were 74
As, a potential clinical PET imaging isotope, and 77
As, a potential therapeutic isotope. 74
17.8 days) has a long half life that allows imaging several days after administration of labeled antibody. Optimal tumor imaging in humans is often achieved 3 days or more after administration of a labeled antibody, when the levels of free antibody have declined relative to those specifically bound or retained by the tumor (30
38.8) has a high energy β-
emission suitable for antitumor therapy. Both isotopes, like other isotopes of arsenic, can be attached through stable covalent linkages to antibodies. In addition, arsenic does not accumulate in the thyroid or undergo transchelation to metal-binding blood and tissue proteins.
Jennewein and Rösch have developed efficient methods for isolating arsenic from irradiated germanium oxide targets to provide arsenic in a form that is useful for labeling sensitive biomolecules (7
). They have also developed novel methods for linking arsenic to biomolecules. Here, we show that monoclonal antibodies can be labeled efficiently with 74
As or 77
As to produce radioimmunoconjugates having full antigen-binding activity and high in vitro
and in vivo
stability. [*As]bavituximab was stable for several days when incubated in serum. Very little nonspecific uptake of radioactivity by the liver was seen in rats injected with [*As]bavituximab or [*As]rituximab, indicating that the labeled antibodies have high in vivo
stability and that transfer of *As to serum proteins and uptake by the liver is minimal. This contrasts with the use of radioiodine for antibody labeling, where dehalogenation and high thyroid uptake are considerable. Instability is less of a problem for antibodies labeled with metal ions (e.g., 64
Cu) since the advent of improved chelating agents.
Biodistribution studies showed high selectivity of bavituximab toward tumor tissue. Within 48 h, the tumor to muscle ratio approached 10 and reached almost 500 by 72 h (). The tumor to liver ratio exceeded 20 by 72 h. [74As]bavituximab showed 30-fold to 50-fold higher absolute uptake in tumor than did the control antibody [74As]rituximab. The ex vivo biodistribution matches the tumor uptake observed by imaging, with higher localization of bavituximab being seen in the tumor than in any normal tissues. Both *As-labeled bavituximab and rituximab accumulated in the spleen, possibly due to nonspecific capture of immunoglobulin or metabolites by the reticuloendothelial system. We did not observe preferential accumulation of [*As]bavituximab in the liver or spleen, which would be expected if bavituximab bound to phosphatidylserine-expressing blood cells being cleared by these organs.
The PET and planar scintigraphy studies showed pronounced localization of bavituximab to solid Dunning prostate R3227-AT1 tumors. Localization was seen in the periphery of the tumor and in various central regions, in agreement with prior PET studies with FDG or perfusion MRI (32
). We have previously observed that phosphatidylserine-positive vessels are present in both the periphery and the core of tumors. It is likely that the peripheral location of the radioactivity seen with [*As]bavituximab in the present study is because this is the region of tumors that typically has the most abundant and functional blood supply. Some of the bavituximab was probably free in the blood of peripheral vessels or had diffused into peripheral tumor regions because a similar peripheral distribution was seen with the nonbinding rituximab control antibody. Heterogeneous localization of bavituximab was also observed throughout the central regions of the tumor. This central localization was antigen-specific because relatively little localization was seen in central tumor regions with the rituximab control antibody. Immunohistochemical examination confirmed that the bavituximab was bound to the endothelium of the central tumor regions with little staining of necrotic regions being visible. The heterogeneous staining with bavituximab is probably because some tumor regions have more hypoxia, acidity, or inflammatory cytokines than others, leading to variable levels of phosphatidylserine exposure on the tumor endothelium. We have previously examined multiple different types of mouse and human tumors growing in mice, and all have phosphatidylserine-expressing tumor vascular endothelium (10
). The percentage of phosphatidylserine-positive vessels ranged from 16% to 41%. Thus, we anticipate that vascular imaging observed with bavituximab in the present studies will extend to other tumor types. The Dunning prostate R3227-AT1 tumor has small areas of focal necrosis scattered throughout the tumor (37
). The lack of strong localization of bavituximab to these necrotic regions could be related to difficulties of access associated with high interstitial pressure and inadequate lymphatic drainage. However, in a previous study using a different anti-phosphatidylserine antibody (9D2) and different tumors, staining of necrotic tumor tissue was observed in addition to the endothelium at later time points (10
). The apparent difference in the ability of the two antibodies to localize to necrotic regions may relate to idiosyncrasies of the tumor models or to differences in the ability of the two antibodies to resist proteolysis after binding. It is also possible that the cofactor protein β2-glycoprotein I, which is needed for phosphatidylserine binding by bavituximab but not 9D2, does not efficiently penetrate into extravascular tissues or is degraded rapidly by proteolytic enzymes within the tumor interstitium.
The present labeling chemistry can also be applied to other radioarsenic isotopes. 72
As has a half life of 26 h, suitable for imaging with antibody Fab′ and F(ab′)2
fragments and other biomolecules having intermediate half lives. The abundance of positrons for 72As is 88%, which is higher than in other commonly used positron emitters, such as 64
Cu (18.0% β+
12.7 h) or 124
I (23.0% β+
4.2 days). Arsenic provides two potentially therapeutic isotopes: 77
226 keV), as used in the present study, and 76
1.068 keV; see Supplementary Table S1). The multiple isotopes of arsenic potentially offer additional applications, such as combined imaging/dosimetry and radioimmunotherapy. Another advantage of arsenic is that, unlike iodine, it does not subject the thyroid to high irradiation. The doses of arsenic used for imaging with [74
As]bavituximab are also several orders of magnitude below toxic levels, so that even if 74
As were released from the antibody, no toxicity would be expected. However, the arsenic isotopes do not include a emitters, which, because of their short path length, could be advantageous for vascular targeted therapies.
In conclusion, we have exploited the unique properties of arsenic radioisotopes to achieve clear imaging of tumors with an antibody, bavituximab, directed against a tumor vessel marker. Radioarsenic-labeled bavituximab shows promise as a vascular imaging agent for tumor detection and dosimetry in man.