Purpose: The locoregional application of tumor-specific antibodies conjugated with highly cytotoxic α-emitters is a promising new strategy for therapy of i.p. tumor cell dissemination. Using this approach, an antibody specifically targeting diffuse-type gastric cancer cells was coupled to the high linear energy transfer α-emitter 213Bi for treatment of i.p. tumor cell spread in a nude mouse model.

Experimental Design: Nude mice were inoculated with HSC45-M2 human gastric cancer cells expressing mutant d9-E-cadherin. Twenty-four h after cell inoculation, mice received i.p. injections of either 213Bi-d9MAb specifically binding to mutant d9-E-cadherin of HSC45-M2 cells or unspecific 213Bi-d8MAb (7.4 or 22.2 MBq). Survival of treated animals was monitored compared with controls that had been injected with nonlabeled monoclonal antibody (MAb) or saline. Toxicity was evaluated by WBC counts after injection of 1.85, 7.4, or 22.2 MBq and analysis of chromosomal aberrations of bone marrow cells after injection of 7.4, 14.8, or 22.2 MBq.

Results: Survival rates of control mice and of mice treated with 213Bi-MAbs differed significantly: the mean survival of untreated controls and mice that were given the nonlabeled antibody was 23 and 26 days. After injection of 22.2 MBq of the specific 213Bi-d9MAb or the unspecific 213Bi-d8MAb, mean survival was at least 143 or 130 days, respectively. Treatment with 7.4 MBq of 213Bi-d9MAb increased mean survival to at least 232 days and with 213Bi-d8MAb to at least 172 days. WBC counts decreased within 2 days after 213Bi-therapy but reached pretreatment values between day 14 and 21 after activity injection. Chromosomal aberrations in bone marrow cells could only be detected at day 1 after 213Bi-therapy. The frequency of chromosomal damages increased depending on the applied 213Bi-activity.

Conclusions: The therapeutic efficacy of the 213Bi-d9MAb together with a low bone marrow toxicity support the locoregional therapy for that subgroup of diffuse-type gastric carcinoma patients expressing d9-E-cadherin.

Peritoneal dissemination is the most devastating step in the course of solid gastrointestinal cancer. After complete tumor resection (R0-resection), recurrent disease often manifests in the peritoneal cavity because of an occult spread of single tumor cells or small tumor cell clusters.

Up to now, no effective therapeutic strategy has been established for this type of tumor cell spread. Chemotherapy or external radiation therapy involve severe side effects because of their unspecific mode of action. Therefore, a new locoregional treatment involving a specific targeting of malignant cells by cytotoxic substances would be of great benefit. To kill single tumor cells in the peritoneal cavity, a new locoregional therapeutic strategy based on tumor-specific MAbs3 conjugated to high linear energy transfer α-emitters seems highly promising (1, 2, 3).

Our approach takes advantage of the fact that 50% of the patients with diffuse-type gastric cancer show mutations in the gene coding for the cell adhesion molecule and tumor suppressor E-cad (4). These mutations, which are only present in tumor cells but not seen on all nonmalignant cells analyzed, may contribute to the diffuse spread of this type of cancer (5). In ∼20% of these mutations exon 9 (d9) or exon 8 (d8) are deleted in-frame from the E-cad mRNA because of splicing defects (6, 7). The resultant mutated E-cad protein still expressed on the surface of tumor cells serves as an ideal cancer-specific target for RIT of disseminated disease.

A MAb that specifically binds to the mutated d9 E-cad (d9MAb) but not to wild-type E-cad has previously been generated and coupled to the α-emitter 213Bi without loss of immunoreactivity (8). This immunoconjugate should be highly effective to destroy tumor cells expressing mutated d9 E-cad in the peritoneal cavity after locoregional application. The purpose of this study was to evaluate the therapeutic efficacy of the 213Bi-d9MAb in a model for disseminated spread of single tumor cells in the peritoneal cavity after locoregional administration compared with its bone marrow toxicity.

Antibodies.

Rat MAbs reacting with mutant E-cad lacking exon 8 (d8-E-cad) or exon 9 (d9-E-cad) were generated as described previously (6). The d9MAb (IgG2a, clone 6H8) specifically binds to HSC45-M2 gastric cancer cells because of expression of mutant d9-E-cad. The d8MAb (IgG2a, clone 7H1) recognizing mutant d8-E-cad does not bind to HSC45-M2 cells and was therefore used as a control.

213Bi Labeling of the Antibodies.

The α-emitter 213Bi, eluted from a 225Ac/213Bi generator system, was coupled to the antibodies (d8MAb or d9MAb) via the chelate SCN-CHX-A”-DTPA as described previously (9, 10). Briefly, MAbs conjugated to the chelate were incubated with BiI4/BiI52− from the 225Ac/213Bi generator system (provided by the Institute for Transuranium Elements, Karlsruhe) for 5–10 min in 0.4 m ammonium acetate buffer at pH 5.3 (11). The 213Bi-immunoconjugates were purified by size exclusion chromatography.

Cell Line.

The human HSC45-M2 stomach cancer cell line established from ascites and pleural effusion of a patient was reported to express d9-E-cad (12, 13). The cell line was kindly provided by Kazuyoshi Yanagihara from the National Cancer Center Research Institute (Tokyo, Japan). The cells were grown in DMEM supplemented with 10% FCS at 37°C in a humidified atmosphere with 5% CO2. Harvesting of the cells was achieved after rinsing the monolayer with PBS/1 mm EDTA. d9MAb specifically targets HSC45-M2 cells because of expression of mutant d9-E-cad.

Calculation of 213Bi-Immunoconjugate Binding on HSC45-M2 Cells.

To calculate binding efficiency of 213Bi-labeled d8MAb and d9MAb, radioimmunoconjugates (37 kBq; 50–100 ng of MAb) were incubated with 3 × 106 HSC45-M2 cells for 30 min on ice. After centrifugation (3 min, 1200 rpm), 213Bi-activity of cellular pellets and supernatants indicated the ratio of bound to unbound 213Bi-immunoconjugates.

Animal Model for i.p. Tumor Cell Dissemination.

The model for i.p. tumor cell dissemination of human diffuse type gastric cancer cells expressing d9-E-cad after resection of the solid tumor is similar to the model described earlier (8). Briefly, 1 × 107 HSC45-M2 cells in 0.5 ml of DMEM were injected i.p. into 6-week-old female nude mice. Tumor cell migration was assessed histologically at day 1, 4, 6, and 8 after inoculation. At the time of RIT 24 h after cell inoculation, tumor cells were still in the peritoneal cavity as single cells or small cell clusters.

All animal studies 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 of 213Bi-d9MAb.

One day after i.p. inoculation of 1 × 107 HSC45-M2 cells, the animals were given injections of 11.1 MBq of 213Bi-d9MAb either i.p. or i.v. At 45 min and 3 h after injection, animals were sacrificed in groups of six, various tissues were removed, weighed, and the 213Bi-activity was determined by γ-counting using the 440-keV γ-emission of 213Bi. The results are reported as % ID/g tissue.

RIT Studies.

For evaluation of the selective therapeutic efficacy of specific 213Bi-d9MAb and unspecific 213Bi-d8MAb mice were treated with a single i.p. injection of 7.4 MBq (n = 15 or 8) or 22.2 MBq (n = 11 or 8) of 213Bi-immunoconjugates 24 h after i.p. tumor cell inoculation. Controls were treated with saline (n = 42) or nonlabeled d9MAb (n = 8). Mice were observed up to day 245 after tumor cell inoculation or sacrificed as soon as ascites had developed.

Statistical analysis of survival data were performed using log rank test with P < 0.01 considered to be significant.

Determination of WBC Counts.

Approximately 50 μl of blood were obtained from nude mice by puncture of the jugular vein with heparinized syringes 2, 7, 14, 21, 28, 35, and 42 days after injection of 213Bi-d9MAb (1.85, 7.4, or 22.2 MBq) or of nonlabeled d9MAb. WBC counts were determined using a blood analyzer (SE 9000; Sysmex, Norderstädt, Germany) after dilution of the blood with a shedding reagent (Cellpack; Sysmex, Norderstädt, Germany). Counts are expressed in percentage of pretreatment values.

Analysis of Chromosomal Aberrations in Bone Marrow Cells.

Controls and animals treated with 7.4, 14.8, or 22.2 MBq of 213Bi-d9MAb were given injections of ConA (200 μg/mouse) 24 h and with Demecolcine (20 μg/mouse) 3 h before sacrifice. ConA induces proliferation of bone marrow cells, whereas Demecolcine arrests chromosomes in metaphase. At day 1, 3, 5, and 28 after i.p. 213Bi-therapy, chromosomes of bone marrow cells were analyzed microscopically. For that purpose, bone marrow was rinsed from the femur by lavage with HBSS medium. The cells were fixed in ethanol/acetic acid (3:1). Red blood cells were removed by hypotonic treatment with 0.56% KCl and washing with the fixans. After fixation, the cells were dropped onto cleaned micro slides, dried by heating at 45°C, and stained with 5% Giemsa solution. One hundred bone marrow cells/animal were analyzed to determine chromosomal aberrations.

213Bi Labeling of Antibodies.

213Bi labeling of d9MAb and d8MAb via the chelate SCN-CHX-A“-DTPA produced maximal specific activities of 1.48 GBq/mg MAb. The radiochemical purity of the labeled antibody fractions after gel filtration was between 95 and 99%. The stability of the 213Bi-immunoconjugates in PBS exceeded four half-lives of 213Bi (3 h) at room temperature.

Tumor Cell Binding of 213Bi-Immunoconjugates and Antigen Density on HSC45-M2 Cells.

Binding efficiencies of specific 213Bi-d9MAb and unspecific 213Bi-d8MAb toward HSC45-M2 cells differed significantly. 25% of 213Bi-d9MAb immunoconjugates bound to HSC45-M2 cells, but only 2% of 213Bi-d8MAb could be detected in the cell pellets. Weak binding of d8MAb might be attributable to unspecific interactions of d8MAb with cell surface molecules of HSC45-M2 cells. After incubation of HSC45-M2 cells with 213Bi-BSA conjugates, 0.5% of 213Bi-activity remained in the cellular pellet.

As determined by FACS analysis, using a standard curve made from polystyrene calibration beads covered with different numbers of antibodies, the number of d9-E-cad molecules expressed by HSC45-M2 cells was ∼3.5 × 105.

Biodistribution of 213Bi-d9MAb.

The biodistribution data of 213Bi-d9MAb obtained after i.v. or i.p. injection are summarized in Table 1. Because of the short half-life of 213Bi (46 min), only the data for 45 min and 3 h postinjection (i.e., one and four physical half-lives) are shown. It appears that the 213Bi-activity concentrations in blood and the tissues investigated remained significantly lower after i.p. compared with i.v. injection. In blood and all tissues investigated the activity concentrations decreased with time after i.v. injection and increased after i.p. injection. An accumulation of 213Bi-activity after i.v. injection was only detected in the kidneys.

Survival of Mice after 213Bi-Immunotherapy.

The survival curves for untreated mice and mice treated with the different activities of 213Bi-d9MAb or unspecific d8MAb 24 h after i.p. injection of HSC45-M2 tumor cells are shown in Fig. 1.

Untreated mice showed a mean survival of 23 days after i.p. injection of tumor cells. Mice treated with the nonlabeled d9MAb displayed an almost identical mean survival of 26 days (data not shown). The difference in survival of control mice and 213Bi-MAb-treated mice is statistically significant for all treated groups (P < 0.0001). The data clearly demonstrate that upon reduction of the injected 213Bi-activity survival increased. After injection of 22.2 MBq 213Bi-d9MAb mean survival was at least 143 days, whereas the mean survival of mice treated with the unspecific conjugate was 130 days. Treatment with 7.4 MBq 213Bi-d9MAb increased mean survival to at least 232 days and with unspecific 213Bi-d8MAb to at least 172 days. At day 245 after 213Bi-therapy, all animals that were still alive were killed for immunohistological examination showing no signs of tumor manifestation.

Leukocyte Counts.

WBC counts at different time points after 213Bi-therapy are shown in Fig. 2. In untreated mice and mice that received injections of nonlabeled d9MAb, a slight increase in WBC counts to ∼140% of the pretreatment value was observed within 42 days. All animals treated with 213Bi-immunoconjugates showed a decrease in WBC within 2 days after therapy: to 87% of the pretreatment value after 1.85 MBq, to 55% after 7.4 MBq, and to 32% after 22.2 MBq. Pretreatment values were reached between day 14 and 21 after 213Bi-MAb injection. WBC counts increased to 182% after 7.4 MBq and to 151% after 22.2 MBq of 213Bi-MAb at approximately day 42 postinjection.

Chromosomal Aberrations in Bone Marrow Cells.

Radiotoxicity of 213Bi-MAb was evaluated by quality and quantity of the chromosomal aberrations. Injection of nonlabeled MAb or saline did not induce any chromosomal aberrations (Fig. 3 a).

After 213Bi-d9MAb injection three types of aberrations with different categories were distinguished. Cells with moderate aberrations designate singular fragments or exchanges. Cells with heavy aberrations designate both several fragments and/or several exchanges (Fig. 3,b). Cells with multiple damages are those with exchange patterns characteristic of more than two chromosomes in combination with severe fragmentation showing an appearance like premature chromosome condensation phenomenon (Fig. 3,c). At day 1 after application of the 213Bi-d9MAb, chromosomal aberrations were observed in 48% of bone marrow cells after 22.2 MBq, in 31% after 14.8 MBq, and in 17% after 7.4 MBq (Fig. 4). At days 3, 5, and 28 after 213Bi-therapy, chromosomal aberrations were no longer detectable, indicative of rapid elimination of damaged cells.

Intraperitoneal tumor cell dissemination as the precursor of a peritoneal carcinomatosis is a crucial step in the course of solid gastrointestinal malignancies. Because of peritoneal seeding the progression of the disease is dramatic and symptoms such as ascites formation and intestinal obstruction deteriorate the quality of life substantially. Thus far, there is no standard therapy for peritoneal tumor spread in solid gastrointestinal tumors. The application of systemic or i.p. chemotherapy and radiation has always been experimental and limited in dose because of severe unspecific side effects. Therefore, new therapeutic strategies are urgently needed for this type of cancer.

With the description of tumor-specific E-cad mutations and the development of MAbs targeting these mutant E-cad, the basis for a selective targeted tumor therapy has been prepared (5, 6, 7). Because the specific antibody investigated in this study showed a high specific tumor binding to tumor cells expressing mutant E-cad, coupling of cytotoxic substances such as α-emitters should improve therapeutic efficacy. Thus far, only one other tumor specific MAb has been described. This MAb recognizes a mutant form of the epidermal growth factor receptor (EGFR v III) and has been labeled with the α-emitter 211At for RIT of malignant glioma (14).

α-Particles are of much higher energy, much shorter range of only a few cell diameters and considerably higher linear energy transfer compared with commonly used β-particles for RIT. Therefore, they seem to be particularly effective in the treatment of single tumor cells of hematological diseases or compartmentally spread malignancies combined with a reduction of nonspecific irradiation of normal tissue around the target cells. α-Emitter immunoconjugates have proven to be powerful therapeutic agents in animal experiments after i.v. or intracavitary application (15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25) and also in two clinical trials (2, 26).

The α-emitter 213Bi was chosen for our therapeutic studies because of its availability from a 225Ac/213Bi generator system (11). Coupling of 213Bi to the tumor specific antibody d9MAb-targeting mutant E-cad of HSC45-M2 gastric cancer cells therefore should result in a highly effective tool to selectively eliminate cancer cells in the peritoneal cavity.

The study described here addressed the following questions in a mouse model for i.p. tumor cell dissemination:

Can 213Bi-immunoconjugates significantly increase survival compared with untreated controls, and if so, is there a survival benefit and a chance of reduction of the administered activity for the group of animals treated with the mutation-specific antibody 213Bi-d9MAb compared with animals treated with the unspecific 213Bi-d8MAb? Is the toxicity of 213Bi-immunoconjugates low enough to justify the i.p. application of 213Bi-immunotherapy in human gastric carcinoma?

To answer these questions, nude mice were i.p. inoculated with HSC45-M2 cells and injected i.p. with different activities of specific 213Bi-d9MAb, unspecific 213Bi-d8MAb, or nonlabeled d9MAb 24 h after tumor cell inoculation. This time interval between tumor cell inoculation and treatment seems promising to destroy single tumor cells because at that time point most of the tumor cells are still free floating in the peritoneal cavity. At later time points, the cells migrate into the serosa and are no more accessible for the MAb as shown by histology (8). First RIT experiments with injection of 213Bi-d9MAb 8 d after tumor cell inoculation indeed indicate less therapeutic efficacy.

Locoregional application of a labeled MAb as opposed to i.v. injection can improve rapid targeting of cancer cells. The short half-life of 213Bi ensures that most of the injected activity decays at the tumor cells in the peritoneal cavity with a high localized energy deposition reducing the risk of systemic toxicity. Doses of β-particles from decay daughters of 213Bi, as calculated by Kennel et al. (27), were <1% of the α-particle doses and thus should not contribute to toxicity and therapeutic efficacy.

The biodistribution data after i.p. injection of the 213Bi-MAb demonstrate only a slow release of the activity from the peritoneal cavity into the blood pool. Therefore, low activity concentrations in all tissues investigated were found after four half-lives of 213Bi (3 h). A slight uptake of 213Bi was only found in the kidneys probably because of a slight release of 213Bi from the immunoconjugate. After injection of free bismuth, however, the activity accumulation in the kidney is far higher (∼100% ID/g) as shown in other experimental studies (19, 28).

The treatment of animals with nonlabeled d9MAb did not have any effect on survival compared with untreated controls. 213Bi-immunoconjugates, however, significantly prolonged survival depending on antibody specificity and 213Bi-activity (P < 0.0001). Because the application of 14.8 MBq of a 213Bi immunoconjugate injected i.p. has been reported to have no toxic effect whereas the administration of 54 MBq was above the maximum-tolerated dose for mice (28), we decided to carry out first therapeutic trials with the injection of 22.2 MBq 213Bi-labeled MAbs.

Mean survival of mice without 213Bi-immunotherapy was 23 or 26 days after tumor cell inoculation. Application of 22.2 MBq of 213Bi-immunoconjugates caused mean survivals of 143 days with specific d9MAb and of 130 days with unspecific d8MAb. A significant growth delay also with only slight difference between a specific and an unspecific MAb has been reported after intratumoral injection of a 211At-labeled MAb and after i.v. injection of a 213Bi-labeled antitumor single-chain fragment (29, 30). Animals without tumor cell inoculation injected with 22.2 MBq of 213Bi-d9MAb showed similar mean survivals as those treated after tumor cell inoculation. Both animal groups developed tumor cell free ascites and showed pathological affections of the kidneys. These observations suggest tumor independent toxicity of 22.2 MBq of 213Bi-d9MAb.

The major argument for the concept of high tumor specificity of a 213Bi-labeled d9MAb and low side effects was found in our study after the reduction of the injected activity to 7.4 MBq of the 213Bi-immunoconjugates 24 h after tumor cell inoculation. The survival rates significantly increased up to 232 days using the specific d9MAb compared with 172 days using the unspecific d8MAb.

Toxicity of 213Bi-immunoconjugates was monitored via WBC counts in the peripheral blood and chromosomal aberrations in bone marrow cells because the applied doses neither caused lethal complications nor other major detectable side effects. Furthermore, chromosomal aberrations are known to be the predominant type of damage caused by high linear energy transfer α-irradiation.

WBC counts were reduced within 2 days after therapy depending on the 213Bi-activity but recovered to pretreatment values approximately at day 21. Reduction was maximal after application 22.2 MBq and minimal after 1.85 MBq. Chromosomal aberrations in bone marrow cells induced by 213Bi-MAb were not persistent. At day 1 after 213Bi-therapy, 48% of cells showed aberrations following the application of 22.2 MBq, whereas only 17% were damaged by 7.4 MBq. Independent of the extent of chromosomal aberrations in the bone marrow cells aberrations could only be observed at day 1 after therapy. Already 2 days after 213Bi-treatment, cells containing damaged chromosomes were no longer found in the bone marrow. This means that severely damaged cells are eliminated rapidly from the bone marrow. Cells with minor DNA adducts should survive because of efficient DNA repair mechanisms.

Therapeutic efficacy of 213Bi-immunotherapy increased with reduction of 213Bi-activity. In addition the reduction of the injected activity allowed a discrimination in the survival between the groups of animals treated with the specific and the unspecific radioimmunoconjugate. At higher activities, even the unspecific freely diffusing but not binding 213Bi-d8MAb in proximity to the tumor cells has a therapeutic efficacy through the crossfire effect as also found in vitro(31). Our results confirm the concept that specific binding of the 213Bi-labeled d9MAb results in a higher therapeutic effect and a lower toxicity because of the reduction of unspecific crossfire effect after locoregional therapy. To establish the optimal correlation between the lowest toxicity and the highest specific therapeutic efficacy studies with further reduction of the injected activity are on the way.

In conclusion, the high therapeutic efficacy of locoregional 213Bi-therapy of peritoneal tumor cell spread compared with its toxicity in the mouse model supports the initiation of therapeutic clinical trials for diffuse-type gastric carcinoma expressing d9-E-cad.

2

To whom requests for reprints should be addressed, at Nuklearmedizinische Klinik d. Technischen Universität München, Ismaninger Str. 22, 81675 Munich, Germany. Phone: 49-89-41404550; Fax: 49-89-41404897; E-mail: senekowitsch@lrz.tu-muenchen

3

The abbreviations used are: MAb, monoclonal antibody; E-cad, E-cadherin; i.p., intraperitoneal; RIT, radioimmunotherapy; % ID/g, percentage of the injected dose/gram; WBC, white blood cell.

Fig. 1.

Survival of nude mice after 213Bi-therapy. Animals were inoculated with 1 × 107 HSC45-M2 gastric cancer cells. They were injected with saline or nonlabeled d9MAb (untreated control) or treated with two different activities of specific 213Bi-d9MAb or unspecific 213Bi-d8MAb 24 h after tumor cell inoculation. Mice that had survived longer than 245 days were sacrificed.

Fig. 1.

Survival of nude mice after 213Bi-therapy. Animals were inoculated with 1 × 107 HSC45-M2 gastric cancer cells. They were injected with saline or nonlabeled d9MAb (untreated control) or treated with two different activities of specific 213Bi-d9MAb or unspecific 213Bi-d8MAb 24 h after tumor cell inoculation. Mice that had survived longer than 245 days were sacrificed.

Close modal
Fig. 2.

Alterations in WBC counts after 213Bi-d9MAb treatment. After injection of 1.85, 7.4, or 22.2 MBq of 213Bi-d9MAb or nonlabeled (native) d9MAb, 50 μl of blood were taken from the jugular vein at different time points postinjection and analyzed for WBC. Counts are expressed as percentage of pretreatment values.

Fig. 2.

Alterations in WBC counts after 213Bi-d9MAb treatment. After injection of 1.85, 7.4, or 22.2 MBq of 213Bi-d9MAb or nonlabeled (native) d9MAb, 50 μl of blood were taken from the jugular vein at different time points postinjection and analyzed for WBC. Counts are expressed as percentage of pretreatment values.

Close modal
Fig. 3.

Karyograms of murine bone marrow cells demonstrating different types of lesions after i.p. injection of 213Bi-d9MAb. a, controls after injection of nonlabeled d9MAb showing normal chromosomes. b, heavy aberrations after 213Bi-therapy: telomeric fragments. c, multiple aberrations after 213Bi-therapy: severe fragmentation showing an appearance like PCC (premature chromosome condensation).

Fig. 3.

Karyograms of murine bone marrow cells demonstrating different types of lesions after i.p. injection of 213Bi-d9MAb. a, controls after injection of nonlabeled d9MAb showing normal chromosomes. b, heavy aberrations after 213Bi-therapy: telomeric fragments. c, multiple aberrations after 213Bi-therapy: severe fragmentation showing an appearance like PCC (premature chromosome condensation).

Close modal
Fig. 4.

Percentage of chromosomal aberrations in bone marrow cells of nude mice at day 1 after treatment with 213Bi-d9MAb or nonlabeled d9MAb. Animals received injections of 7.4, 14.8, or 22.2 MBq of 213Bi-d9MAb. Bone marrow cells were rinsed from the femur after ConA and Demecolcine injection. After Giemsa staining chromosomes were analyzed microscopically.

Fig. 4.

Percentage of chromosomal aberrations in bone marrow cells of nude mice at day 1 after treatment with 213Bi-d9MAb or nonlabeled d9MAb. Animals received injections of 7.4, 14.8, or 22.2 MBq of 213Bi-d9MAb. Bone marrow cells were rinsed from the femur after ConA and Demecolcine injection. After Giemsa staining chromosomes were analyzed microscopically.

Close modal
Table 1

Biodistribution of 213Bi-d9MAb in nude mice 45 min and 3 h after i.v. or i.p. injection, expressed as % ID/g tissue; mean ± SD, n = 6

Organi.v. injectioni.p. injection
45 min3 h45 min3 h
Blood 30.7 ± 4.1 24.2 ± 1.7 2.4 ± 0.6 5.2 ± 1.6 
Heart 13.9 ± 2.4 8.1 ± 1.1 0.8 ± 0.2 1.8 ± 0.8 
Lung 13.5 ± 1.3 11.3 ± 2.4 0.8 ± 0.2 1.6 ± 0.4 
Spleen 11.1 ± 0.9 9.1 ± 0.9 1.4 ± 0.4 1.8 ± 0.5 
Stomach 5.4 ± 0.8 5.2 ± 1.3 3.1 ± 0.9 3.7 ± 1.3 
Kidney 16.1 ± 3.9 19.8 ± 3.7 3.2 ± 1.2 5.4 ± 1.9 
Liver 3.6 ± 0.9 5.3 ± 0.8 1.8 ± 0.3 3.2 ± 0.9 
Muscle 1.0 ± 0.3 1.0 ± 0.2 0.3 ± 0.1 0.6 ± 0.2 
Organi.v. injectioni.p. injection
45 min3 h45 min3 h
Blood 30.7 ± 4.1 24.2 ± 1.7 2.4 ± 0.6 5.2 ± 1.6 
Heart 13.9 ± 2.4 8.1 ± 1.1 0.8 ± 0.2 1.8 ± 0.8 
Lung 13.5 ± 1.3 11.3 ± 2.4 0.8 ± 0.2 1.6 ± 0.4 
Spleen 11.1 ± 0.9 9.1 ± 0.9 1.4 ± 0.4 1.8 ± 0.5 
Stomach 5.4 ± 0.8 5.2 ± 1.3 3.1 ± 0.9 3.7 ± 1.3 
Kidney 16.1 ± 3.9 19.8 ± 3.7 3.2 ± 1.2 5.4 ± 1.9 
Liver 3.6 ± 0.9 5.3 ± 0.8 1.8 ± 0.3 3.2 ± 0.9 
Muscle 1.0 ± 0.3 1.0 ± 0.2 0.3 ± 0.1 0.6 ± 0.2 

We thank Dr. Martin W. Brechbiel at the NIH for providing SCN-CHX-A“-DTPA chelate. We also thank Dr. Kazuyoshi Yanagihara at the National Cancer Center Research Institute (Tokyo, Japan) for providing the HSC45-M2 stomach cancer cell line. We thank Ramon Carlos-Marquez and Roger Molinet from the Institute of Transuranium Elements (Karlsruhe, Germany) for the preparation of the 225Ac/213Bi generator systems and Claudia Bodenstein and Ulrike Schwaiger for their technical assistance.

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