A monoclonal antibody (E-cadherin delta 9–1) directed against a characteristic E-cadherin mutation (in-frame deletion of exon 9), found in diffuse-type gastric cancer but not in any normal tissue, was conjugated with the high linear energy transfer α-emitter 213Bi and tested for its binding specificity in s.c. and i.p. nude mice tumor models. After intratumoral application in s.c. tumors expressing mutant E-cadherin, the 213Bi-labeled antibody was specifically retained at the injection site as shown by autoradiography. After injection into the peritoneal cavity, uptake in small i.p. tumor nodules expressing mutant E-cadherin was 17-fold higher than in tumor nodules expressing wild-type E-cadherin (62% injected dose/g versus 3.7% injected dose/g). 78% of the total activity in the ascites fluid was bound to free tumor cells expressing mutant E-cadherin, whereas in control cells, binding was only 18%. The selective binding of the 213Bi-labeled, mutation-specific monoclonal antibody E-cadherin δ 9–1 suggests that it will be successful for α-radioimmunotherapy of disseminated tumors after locoregional application.

The use of radiolabeled MAbs2 in radioimmunotherapy is limited because of the lack of tumor-specific antigens. In most cases reported thus far, tumor antigens that serve as targets are not tumor-specific, being overexpressed by tumor cells and also at a lower level by normal cells. Thus far, only one tumor-specific MAb has been reported that recognizes a mutant form of the epidermal growth factor receptor (EGFR vIII) that is found on different tumor types but not on normal human tissue (1). This antibody has been labeled with 125I, 131I, and the α-emitter 211At, and it appears to be a promising candidate for radioimmunotherapy (2, 3).

In-frame deletions of exons 8 or 9 in the mRNA coding for the cell adhesion molecule E-cadherin are characteristic of diffuse-type gastric carcinomas. These mutations alter cell adhesion functions and contribute to the diffuse spread of this cancer type (4, 5). A rat MAb, designated d9MAb, was generated that specifically reacts with mutant E-cadherin lacking exon 9 but not with the wild-type protein. d9MAb was found to react with 13% (22 of 172) of E-cadherin-expressing diffuse-type gastric cancers (6). Because of this specific tumor cell targeting, d9MAb coupled with the α-emitting radionuclide 213Bi could have a significant potential for the locoregional radioimmunotherapy of disseminated, diffuse-type gastric carcinoma, which is often associated with i.p. spread of single malignant cells leading to peritoneal carcinomatosis. The advantages of α-particles are their high LET and their short range of a few cell diameters. These features result in a high localized energy deposition, even in single target cells, and minimal irradiation of surrounding normal tissue.

We have established an i.p. tumor model using cells expressing E-cadherin with an exon 9 deletion that mimics the clinical situation in human gastric cancer with i.p. tumor spread that is known to be a crucial process in diffuse-type gastric cancer. The free i.p. application of a tumor-specific MAb labeled with an appropriate radionuclide seems to be an effective treatment of peritoneal tumor spread. Here we report that the d9MAb coupled with the α-emitter 213Bi specifically binds to mutant E-cadherin after locoregional application.

Antibody.

The rat MAb recognizing mutant E-cadherin lacking exon 9 was generated as described previously (6). Briefly, a 13-amino acid peptide spanning the fusion junction between exons 8 and 10 of mutant E-cadherin with an exon 9 deletion was injected into Lou/C rats i.p. and s.c. for immunization. After fusion of the immune rat spleen cells with a myeloma cell line, hybridoma supernatants were tested by a solid phase immunoassay using the mutation-specific peptide coupled to BSA. A tumor cell-specific, MAb against the delta 9 peptide, referred to as d9MAb (clone 6H8) was selected for the studies described below.

Conjugation of Chelate to d9MAb and Radiolabeling.

d9MAb was conjugated to SCN-CHX-A″-DTPA as described previously (7, 8). The number of chelates per antibody ranged from 5 to 10 as determined by a standard 111In-assay (9). For comparative binding studies, both the MAb chelate construct and the MAb without chelate were labeled with 125I according to the Iodogene method. 213Bi (t1/2 = 46 min) was eluted from a 225Ac/213Bi generator provided by the Institute for Transuranium Elements, Karlsruhe, Germany (10), with 0.1 m HCl/0.1 m NaI as the BiI4/BiI52− anion. The eluant was adjusted to pH 5.3 with 2 m ammonium acetate, and ∼100 μg of the chelated antibody were added and allowed to react for 5 min. The 213Bi-immunoconjugate was purified by size exclusion chromatography (Pharmacia PD-10) with 2 ml of PBS.

The coupling of 111In (InCl3, Mallinckrodt) to the chelated d9MAb was carried out using the 213Bi-protocol omitting NaI. The 111In-immunoconjugate was applied for scintigraphic imaging of i.p. retention and biodistribution. The labeling efficiency was assayed via TLC with instant thin-layer chromatography paper (Gelman Sciences).

Cell Lines.

The human MDA-MB-435S mammary carcinoma cell line (American Type Culture Collection, Manassas, VA) was transfected with exon 9-deleted E-cadherin cDNA and wild-type E-cadherin cDNA, respectively (5). The cells were grown at 37°C in a humidified atmosphere with 5% CO2 in DMEM containing 4.5 g/l glucose supplemented with 10% FCS. For selection of the transfected MDA-MB-435S cells Geneticin was added to the cell medium. The cells were harvested by rinsing the monolayer with 1 mm EDTA and counted in a hemocytometer.

Determination of Antigen Density of Mutant E-Cadherin Transfected Cells and Binding Characteristics of the Labeled MAb.

The antigen density and binding characteristics of the radiolabeled MAb were analyzed by Scatchard analysis. Mutant E-cadherin transfected cells (106) were incubated with increasing concentrations of d9MAbs labeled with 125I or 213Bi. Specific binding was confirmed by the failure of radiolabeled d9MAb to bind to cells expressing wild-type E-cadherin. Antigen density was also determined for unlabeled and chelate-coupled MAbs by indirect immunofluorescence with flow cytometry. The fluorescence signal was quantified by a calibration curve established with fluorescence quantitation beads (Quantibrite PE; Becton Dickinson).

Animal Models.

To investigate the specific binding properties of 213Bi-labeled d9Mab, two different tumor models, a s.c. solid tumor model as well as an i.p. tumor model, were established. For that purpose, 4–5 week old female athymic mice were inoculated s.c. or i.p. with 1 × 107 cells expressing either mutant E-cadherin or wild-type E-cadherin as a negative control. After i.p. injection, two mice were sacrificed every 2 days until day 12 and thereafter at weekly intervals for histological examination of i.p. tumor progression. All experiments with mice were performed in accordance with the guidelines for the use of living animals in scientific studies and the “German Law for the Protection of Animals.”

Biodistribution Studies.

Mice, bearing s.c. tumors of 100–200 mg derived from both mutant and wild-type E-cadherin transfected cells (4–5 weeks after tumor cell inoculation), were injected i.v. with 3.7 MBq (100 μCi) of 213Bi-d9MAb to study the accumulation in tumors and organs at 45 min and at 3 h postinjection. In addition, 37 kBq (1 μCi) of the radioimmunoconjugate were injected directly into the tumor at three different sites. After injection, the tumors were removed, immediately frozen, cut into 8-μm sections, and analyzed for intratumoral distribution of the radioimmunoconjugate via exposure (15 min) to a high-sensitive Micro Imager system, which is capable of a spatial resolution of 10–15 μm (Biospace Measures, Paris, France).

Animals bearing small i.p. tumor nodules (0.1–2.0 mm in diameter) with or without ascites (∼3–4 weeks after i.p. tumor cell inoculation) received an injection of 3 MBq (80 μCi) of 213Bi-d9MAb into the peritoneal cavity and were sacrificed at 45 min and 3 h postinjection. Tumor nodules and various normal tissues were removed, washed, and weighed, and the activity was determined by gamma counting using the 440-keV γ-emission of 213Bi. The results were expressed as a percentage of the injected dose/g of tissue (%ID/g). Each reported value represents the mean and the SD of eight animals. Ascites volume was measured, and, after centrifugation, the activity in the pellet and the supernatant was determined.

To obtain scintigraphic images with optimal resolution (Eγ111In, 172 and 249 keV; Eγ213Bi, 440 keV) and to gain information about the long-term retention (t1/2111In, 2.6 d; t1/2213Bi, 46 min) of the immunoconjugate in mice bearing i.p. tumor nodules expressing either mutant or wild-type E-cadherin and in mice without tumor, scintigraphic images were taken at 3, 24, and 48 h after i.p. injection of 111In-labeled d9MAb (740 kBq). Immediately after the 48 h scintigram, the animals were sacrificed and the distribution of 111In immunoconjugate in representative organs was determined and expressed as %ID/g.

Statistical Analysis.

Unpaired Student’s t tests were performed to compare the mean values. Ps ≤ 0.05 were considered statistically significant.

Antibody Specificity.

Specific binding of the rat d9MAb reacting with mutant, but not with wild-type, E-cadherin was demonstrated by Western blot and immunohistochemistry. In human tissue, d9MAb reacted only with tumor cells of diffuse-type gastric cancer with an exon 9 deletion and did not show any cross-reaction to any normal human tissue similar to the clone 7E6, as described previously (6).

Radiolabeling and Tumor Cell Binding of d9MAb.

The labeling efficiency of CHX-A"-DTPA-d9MAb with 213Bi was >90% at a specific activity of 1.48 GBq/mg (40 mCi/mg). The binding characteristics of 213Bi- and 125I-labeled d9MAb to MDA-MB-435S cells transfected with mutant E-cadherin were evaluated by Scatchard analysis. With both 125I-labeled and 213Bi-conjugated MAbs, 5.5 × 104 binding sites/cell and a dissociation constant of 1.9 nmol/l were determined. These results were confirmed by flow cytometry using unconjugated and CHX-A"-DTPA-conjugated MAbs. The results demonstrated that conjugation and 213Bi-labeling of the MAb do not influence the binding characteristics. The binding of both radiolabeled MAbs to cells expressing wild-type E-cadherin was <4% compared with mutant E-cadherin-expressing cells.

Development of the Tumor Models.

s.c. tumors of 100–200 mg in weight developed 4–5 weeks after s.c. inoculation of 1 × 107 tumor cells.

Up to day 6 after i.p. inoculation of mutant and wild-type E-cadherin-transfected cells, single tumor cells and small tumor-cell clusters consisting of ∼100 cells could be detected histologically in the peritoneal cavity. After day 10, tumor attachment to the visceral organs and the peritoneum could be demonstrated microscopically. Beginning from day 20 after tumor cell inoculation, macroscopic tumor nodules in the mesenterium ranging from 0.1 to 2 mm in diameter could be observed (Fig. 1,a). Histological sections from these tumor nodules showed tumor cells on the serosa and also invasive tumor cells with a desmoplastic reaction (Fig. 1,b). The tumor cells on the serosa often lost their cell-to-cell contact, and isolated tumor cells or cell clusters could be detected in the ascites. Forty percent of the animals developed ascites that contained up to 1 × 108 tumor cells/ml as single cells and as cell clusters (Fig. 1 c). At this stage of tumor development, the animals were used for biodistribution studies after i.p. injection of the 213Bi immunoconjugate.

Biodistribution Studies of 213Bi- and 111In-d9MAb.

In the s.c. tumor model, the activity concentration of 213Bi-d9MAb 3 h after i.v. injection was lower than in the blood; this was as expected for intact MAbs, which slowly diffuse from the circulation into solid tumor tissue. However, binding in tumors expressing mutant E-cadherin was 3-fold higher than in tumors expressing wild-type E-cadherin.

After intratumoral injection in s.c. tumors, the specific binding of 213Bi-d9MAb to mutant E-cadherin could be demonstrated by autoradiographic images. Fig. 2,a shows local retention of the 213Bi immunoconjugate at the three injection sites in the center of the tumor that expressed E-cadherin with an exon 9 deletion. Retention is also seen at the three puncture sites on the tumor periphery. In contrast, tumors expressing wild-type E-cadherin did not show any specific retention of the 213Bi coupled MAb (Fig. 2 b).

In the i.p. tumor model, 213Bi-d9MAb was injected into the peritoneal cavity 3–4 weeks after the inoculation of tumor cells expressing mutant E-cadherin or wild-type E-cadherin, and the biodistribution was quantified at 45 min and 3 h postinjection. The results are summarized in Table 1. In animals that had not developed ascites, a high specific uptake of up to 62 ± 14% ID/g at 45 min and 58 ± 19% ID/g at 3 h was observed in small tumor nodules expressing mutant E-cadherin. In the wild-type E-cadherin model, uptake was only 3.7 ± 1.0% ID/g at 45 min and 3.4 ± 0.9 at 3 h. In all other tissues, the uptake of 213Bi immunoconjugate was low. The low 213Bi accumulation in the kidneys, which are known to accumulate free bismuth, indicates the stability of the immunoconjugate. In animals bearing tumors expressing wild-type E-cadherin that does not bind the MAb, however, uptake in normal tissue was statistically significantly higher than in animals expressing mutant E-cadherin. In mice with ascites (up to 5 ml), the concentration of 213Bi-d9MAb in tumor nodules and the other organs was statistically significantly reduced compared with animals without ascites, depending on the volume of ascites and the number of tumor cells in the fluid. This result suggests that the antibody was rapidly and firmly bound to free accessible mutant E-cadherin on tumor cells in the ascites. After centrifugation of ascites with cells expressing mutant E-cadherin, 78% of the 213Bi activity was recovered in the cell pellet in contrast with 18% bound in the pellet of ascites from cells expressing wild-type E-cadherin.

Scintigraphic images of mice bearing small tumor nodules, expressing mutant E-cadherin or wild-type E-cadherin, and of mice without tumor obtained 48 h after i.p. injection of 111In-d9Mab, are shown in Fig. 3. In mice without tumor, the activity is mainly distributed in the blood pool of heart, lungs, and liver (Fig. 3,a). A similar activity distribution was found for mice bearing tumors that expressed wild-type E-cadherin (Fig. 3,c). This indicates that in both cases most of the activity was reabsorbed from the peritoneal cavity. Conversely, in the mouse with multiple i.p. tumor nodules expressing mutant E-cadherin, a considerable amount of activity was retained in the peritoneal cavity, resulting in a clearly visible lower background activity compared with the two controls (Fig. 3 b). Tissue distribution data of 111In were in accordance with the results of the scintigraphic images. Activity accumulation in tumor nodules expressing mutant E-cadherin, was 56% ID/g compared with 4.8% ID/g in controls. Activity concentration of 111In in the blood of animals inoculated with wild-type E-cadherin-expressing cells and of animals without tumor cell inoculation was 11% ID/g compared with 5% ID/g in animals with tumor nodules expressing mutant E-cadherin.

Early i.p. dissemination of tumor cells is a crucial event in the course of gastric carcinoma, resulting in peritoneal carcinomatosis and rapid deterioration of the patient’s clinical status. Apart from a few experimental therapeutic strategies, there is currently no specific treatment for peritoneal cancer spread.

Effective treatment of the i.p. compartment would require locoregional administration of a cytotoxic substance into the peritoneal cavity that could specifically bind to diffusely spread tumor cells and tumor cell clusters. Monoclonal antibodies that specifically recognize tumor cell antigens coupled with a radionuclide with high LET are promising candidates.

Because the d9MAb used in our experiments specifically binds to mutant E-cadherin expressed by diffuse-type gastric carcinoma, it is an ideal vehicle to attach radionuclides to gastric carcinoma cells that have spread diffusely into the peritoneal cavity.

By choosing the appropriate radionuclide, the range of the cytotoxic effect can be matched to the size of the tumor. For the radioimmunotherapy of malignancies with large tumor masses, β-emitting radionuclides such as 131I, 188Re, or 90Y, with mean tissue ranges of 0.9 to 3.9 mm, have been coupled to MAbs. For selective irradiation of single tumor cells or small tumor cell clusters, the new approach of labeling tumor-specific MAbs with α-emitting nuclides seems to be very promising. The α-particles emitted by 212Bi, 211At, and 213Bi have short ranges of only 50–100 μm and a high LET of ∼100 keV/μm that deposit a large amount of energy within a few cell diameters. α-Emitter immunoconjugates have proven to be powerful therapeutic agents in animal experiments (11, 12, 13), especially for malignancies that spread on the surface of the body cavities, such as ovarian cancer and malignant meningitis (14, 15, 16).

We developed a nude mouse model for i.p. tumor spread similar to that which occurs in patients with diffuse-type gastric cancer. In this model, d9MAb demonstrated high and specific binding to small tumor nodules established from tumor cells expressing mutant E-cadherin, whereas binding of d9MAb to tumors expressing wild-type E-cadherin was comparatively low. In addition, the 213Bi-labeled d9MAb bound to the tumor cells in the i.p. cavity within less than one half-life of 213Bi (46 min). The binding remained stable at least 3 h after injection, when 94% of the injected 213Bi activity had decayed at the tumor site.

The number of antigen molecules on the E-cadherin-transfected tumor cells was calculated to be 5.5 × 104/cell by Scatchard analysis. At a specific activity of 1.48 GBq/mg, 213Bi-labeled d9MAb can attach 40 α-particles to a tumor cell. It has been reported in a number of cell lines that 3–9 α-particles bound/cell can reduce clonogenic cell survival to as low as 10% (17, 18, 19). Because the antigen density on human diffuse gastric carcinoma cells as shown by immunohistochemistry may exceed that of our tumor model, binding of 213Bi immunoconjugates should be sufficient to guarantee destruction of almost all of the tumor cells. By increasing the specific activity without the loss of immunoreactivity caused by radiolysis, the specificity of binding and the therapeutic efficiency could probably be improved further.

The beneficial therapeutic effects of α-emitter-immunoconjugates are currently being evaluated in two clinical trials. Patients suffering from acute myeloic leukemia are being treated with 213Bi-labeled HuM195 MAb recognizing CD33, a differentiation antigen expressed in leukemic cells. More than one-half of the 17 patients treated thus far have shown a reduction of leukemic cells in the peripheral blood, and a few also have shown decreased numbers of bone marrow blast cells (20). The MAb 81C6 specifically binds to the matrix glycoprotein tenascin that is expressed by glioma cells but not by normal brain tissue. The 211At labeled antibody has been applied locally to the surgical cavity created by the glioma resection with promising results (16).

The results obtained in our experimental model with the d9MAb labeled with 213Bi suggest that this radioimmunoconjugate and similar ones targeting other E-cadherin mutations (e.g., exon 8 deletion) should be tested in clinical therapeutic trials for a subgroup of diffuse-type gastric carcinoma patients. This would be the first application of such a method in disseminated gastro-intestinal tumors.

Fig. 1.

An example of the development of i.p. carcinomatosis with ascites 5 weeks after injection of 1 × 107 cells expressing mutant E-cadherin. a, macroscopic view showing development of small tumor nodules in the peritoneum (arrow). b, histological section of an i.p. nodule with tumor cells on the serosa (arrow head) and invasive tumor cells in the mesenteric adipose tissue (arrow; ×200; H&E staining). c, single tumor cells (arrow heads) and small cell clusters (arrow) in the ascites fluid with a nonspecific inflammatory reaction (×400; modified May-Grünwald-Giemsa staining).

Fig. 1.

An example of the development of i.p. carcinomatosis with ascites 5 weeks after injection of 1 × 107 cells expressing mutant E-cadherin. a, macroscopic view showing development of small tumor nodules in the peritoneum (arrow). b, histological section of an i.p. nodule with tumor cells on the serosa (arrow head) and invasive tumor cells in the mesenteric adipose tissue (arrow; ×200; H&E staining). c, single tumor cells (arrow heads) and small cell clusters (arrow) in the ascites fluid with a nonspecific inflammatory reaction (×400; modified May-Grünwald-Giemsa staining).

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Fig. 2.

a, autoradiography of an 8-μm tumor section 1 h after intratumoral injection of 213Bi-d9MAb in a tumor expressing mutant E-cadherin (activity retention at the three injection sites in the center of the tumor [arrow heads] and at the puncture sites at the tumor periphery [arrows]). b, autoradiography of a 8 μm tumor section, 1 h after intratumoral injection of a 213Bi-d9MAb in wild type E-cadherin tumors (no specific activity retention).

Fig. 2.

a, autoradiography of an 8-μm tumor section 1 h after intratumoral injection of 213Bi-d9MAb in a tumor expressing mutant E-cadherin (activity retention at the three injection sites in the center of the tumor [arrow heads] and at the puncture sites at the tumor periphery [arrows]). b, autoradiography of a 8 μm tumor section, 1 h after intratumoral injection of a 213Bi-d9MAb in wild type E-cadherin tumors (no specific activity retention).

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Fig. 3.

Scintigrams of mice 48 h after i.p. injection of 740 kBq (20 μCi) 111In-d9MAb. a, mouse without tumor cell injection showing blood pool mainly in the heart, lungs, and liver; no visible retention of activity in the peritoneal cavity. b, mouse with tumors expressing mutant E-cadherin; besides some blood pool activity, there is a clearly visible activity accumulation in peritoneal tumor nodules (arrows). c, mouse with i.p. tumors expressing wild-type E-cadherin showing blood pool mainly as in a.

Fig. 3.

Scintigrams of mice 48 h after i.p. injection of 740 kBq (20 μCi) 111In-d9MAb. a, mouse without tumor cell injection showing blood pool mainly in the heart, lungs, and liver; no visible retention of activity in the peritoneal cavity. b, mouse with tumors expressing mutant E-cadherin; besides some blood pool activity, there is a clearly visible activity accumulation in peritoneal tumor nodules (arrows). c, mouse with i.p. tumors expressing wild-type E-cadherin showing blood pool mainly as in a.

Close modal

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2

The abbreviations used are: MAb, monoclonal antibody; d9MAb, E-cadherin delta 9–1 MAb; LET, linear energy transfer.

Table 1

Biodistribution of 213Bi d9MAb in animals with i.p. tumors expressing mutant or wild-type E-cadherin, 45 min and 3 h after i.p. injectiona

OrganMutant E-cadherin without ascitesMutant E-caderin with ascitesWild-type E-cadherin without ascites
45 min3 h45 min3 h45 min3 h
Blood 1.1 ± 0.4 2.6 ± 0.9 0.4 ± 0.1 1.4 ± 0.4 2.8 ± 0.6 5.9 ± 1.4 
Tumor 62 ± 14 58 ± 19 7.1 ± 3.4 9.3 ± 4.2 3.7 ± 1.0 3.4 ± 0.9 
Heart 0.5 ± 0.1 1.1 ± 0.3 0.1 ± 0.03 0.5 ± 0.1 1.5 ± 0.4 2.1 ± 0.3 
Lung 0.7 ± 0.2 1.4 ± 0.4 0.2 ± 0.08 0.6 ± 0.2 1.1 ± 0.3 2.0 ± 0.6 
Spleen 1.0 ± 0.4 1.2 ± 0.5 0.3 ± 0.07 0.9 ± 0.2 1.6 ± 0.6 1.9 ± 0.8 
Stomach 2.1 ± 1.0 1.7 ± 0.4 0.6 ± 0.1 0.8 ± 0.3 3.3 ± 0.9 4.1 ± 1.3 
Bowel 1.4 ± 0.3 1.2 ± 0.3 0.8 ± 0.3 0.9 ± 0.3 2.8 ± 0.6 2.9 ± 0.7 
Peritoneum 3.9 ± 1.8 3.2 ± 1.4 2.5 ± 0.9 3.1 ± 1.2 2.3 ± 0.1 1.8 ± 0.3 
Kidney 3.6 ± 1.4 5.1 ± 1.9 2.8 ± 1.2 4.0 ± 0.8 4.1 ± 1.3 6.8 ± 2.3 
Liver 1.2 ± 0.4 2.1 ± 0.8 0.5 ± 0.1 1.2 ± 0.4 2.5 ± 0.3 3.8 ± 0.9 
Muscle 0.1 ± 0.08 0.4 ± 0.1 0.09 ± 0.03 0.2 ± 0.06 0.4 ± 0.1 0.6 ± 0.1 
Ascites   14.4 ± 6.3 17.3 ± 5.6   
OrganMutant E-cadherin without ascitesMutant E-caderin with ascitesWild-type E-cadherin without ascites
45 min3 h45 min3 h45 min3 h
Blood 1.1 ± 0.4 2.6 ± 0.9 0.4 ± 0.1 1.4 ± 0.4 2.8 ± 0.6 5.9 ± 1.4 
Tumor 62 ± 14 58 ± 19 7.1 ± 3.4 9.3 ± 4.2 3.7 ± 1.0 3.4 ± 0.9 
Heart 0.5 ± 0.1 1.1 ± 0.3 0.1 ± 0.03 0.5 ± 0.1 1.5 ± 0.4 2.1 ± 0.3 
Lung 0.7 ± 0.2 1.4 ± 0.4 0.2 ± 0.08 0.6 ± 0.2 1.1 ± 0.3 2.0 ± 0.6 
Spleen 1.0 ± 0.4 1.2 ± 0.5 0.3 ± 0.07 0.9 ± 0.2 1.6 ± 0.6 1.9 ± 0.8 
Stomach 2.1 ± 1.0 1.7 ± 0.4 0.6 ± 0.1 0.8 ± 0.3 3.3 ± 0.9 4.1 ± 1.3 
Bowel 1.4 ± 0.3 1.2 ± 0.3 0.8 ± 0.3 0.9 ± 0.3 2.8 ± 0.6 2.9 ± 0.7 
Peritoneum 3.9 ± 1.8 3.2 ± 1.4 2.5 ± 0.9 3.1 ± 1.2 2.3 ± 0.1 1.8 ± 0.3 
Kidney 3.6 ± 1.4 5.1 ± 1.9 2.8 ± 1.2 4.0 ± 0.8 4.1 ± 1.3 6.8 ± 2.3 
Liver 1.2 ± 0.4 2.1 ± 0.8 0.5 ± 0.1 1.2 ± 0.4 2.5 ± 0.3 3.8 ± 0.9 
Muscle 0.1 ± 0.08 0.4 ± 0.1 0.09 ± 0.03 0.2 ± 0.06 0.4 ± 0.1 0.6 ± 0.1 
Ascites   14.4 ± 6.3 17.3 ± 5.6   
a

%ID/g, mean ± SD; n = 8.

We thank Dr. Martin W. Brechbiel at the NIH for providing SCN-CHX-A″-DTPA chelate. We acknowledge the kind assistance of Roger Molinet and Ramon Carlos-Marquez in the separation of 225Ac and in the preparation of 213Bi generators. We thank Susanne Daum and Stephanie Alam for technical assistance and James Mueller for carefully reading the manuscript.

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