Comparison of therapeutic efficacy and biodistribution of ²¹³Bi- and ²¹¹At-labeled monoclonal antibody MX35 in an ovarian cancer model☆

Article Outline

• Abstract
• 1. Introduction
• 2. Materials and methods
  • 2.1. Radionuclides
  • 2.2. Antibody
  • 2.3. Cell line
  • 2.4. Animals
  • 2.5. Antibody conjugation
  • 2.6. Antibody conjugate labeling
  • 2.7. Immunoreactivity of the radiolabeled antibodies
  • 2.8. Procedures for therapy and toxicity study
  • 2.9. Procedures for biodistribution
  • 2.10. Dosimetric calculations
• 3. Results
  • 3.1. Antibody conjugation, labeling and immunoreactivity
  • 3.2. Therapeutic efficacy
  • 3.3. Toxicity and biodistribution
  • 3.4. Dosimetric calculations
• 4. Discussion
• 5. Conclusion
• Acknowledgments
• References
• Copyright

Abstract 

Introduction

The purpose of this study was to compare the therapeutic efficacy and biodistribution of the monoclonal antibody MX35 labeled with either ²¹³Bi or ²¹¹At, both α-emitters, in an ovarian cancer model.

Methods

One hundred female nude BALB/c (nu/nu) mice were inoculated intraperitoneally with human ovarian cancer cells (OVCAR-3). Two weeks later, 40 of these mice were injected intraperitoneally with ∼2.7 MBq of ²¹³Bi-MX35 (n=20) or ∼0.44 MBq of ²¹¹At-MX35 (n=20). Four weeks after inoculation, 40 new OVCAR-3-inoculated mice were injected with the same activities of ²¹³Bi-MX35 (n=20) or ²¹¹At-MX35 (n=20). Presence of tumors and ascites was investigated 8 weeks after therapy. Biodistributions of intraperitoneally injected ²¹³Bi-MX35 and ²¹¹At-MX35 were studied in tumor-free nude BALB/c (nu/nu) mice (n=16).

Results

The animals injected with ²¹³Bi-MX35 or ²¹¹At-MX35 2 weeks after cell inoculation had tumor-free fractions (TFFs) of 0.60 and 0.90, respectively. The untreated reference group had a TFF of 0.20. The groups treated with ²¹³Bi-MX35 or ²¹¹At-MX35 4 weeks after inoculation both had TFFs of 0.25, and the reference animals all exhibited evidence of disease. The biodistributions of ²¹³Bi-MX35 and ²¹¹At-MX35 were very similar to each other and displayed no alarming activity levels in the investigated organs.

Conclusions

Micrometastatic growth of an ovarian cancer cell line was reduced in nude mice after treatment with ²¹³Bi-MX35or ²¹¹At-MX35. Treatment with ²¹¹At-MX35 provided a non-significantly better result for the chosen activity levels. The radiolabeled MX35 did not accumulate to a high extent in the investigated organs. No considerable signs of toxicity were observed.

Keywords: Radioimmunotherapy, Bismuth, Astatine, MX35, α-Particle

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1. Introduction 

Radioimmunotherapy (RIT) is a promising adjuvant treatment for various types of disseminated cancer. Ovarian cancer, diagnosed in 1–2% of women, is a cancer type believed to be suitable for RIT. Despite initially successful treatments involving surgery and systemic chemotherapy, the majority of ovarian cancer patients will suffer from recurrence, mainly in the peritoneal cavity, with ascites production and bowel obstruction prior to death. The 5-year relative chance of surviving ovarian cancer is only 45% for all stages at diagnosis in American women [1], and novel adjuvant treatment strategies such as RIT are urgently needed to increase this survival rate.

The search continues for radionuclides that are useful in cancer treatment with RIT. Most investigations have involved β-emitters such as ⁹⁰Y and ¹³¹I [2], [3], [4], [5], [6], [7], [8], [9], [10], and, unfortunately, no improvement in overall survival for ovarian cancer patients has thus far been achieved with these radionuclides [2]. β-particles may not be optimal for killing microscopic tumors due to their relatively long path lengths (up to several millimeters) [11], [12]. This long particle range results in a deposition of most of the emitted energy outside the microscopic tumor, a suboptimal situation for both treating the tumor and limiting irradiation of surrounding normal tissues.

α-Emitters such as bismuth-213 (²¹³Bi) and astatine-211(²¹¹At) have several advantages over β-emitters in the treatment of microscopic tumors. α-Emissions have energies of 4–9 MeV [13] that are deposited over a very short distance (∼50–100 μm, i.e., a few cell diameters). The high linear energy transfer of ∼100 keV/μm results in complex DNA damage that is very difficult for the cell to repair.

Because of the favorable features of α-emitting radionuclides, ²¹¹At has been studied extensively in our research group. We previously demonstrated that intraperitoneal injection of ²¹¹At-labeled monoclonal antibodies (mAbs) or F(ab′)₂ fragments is efficient for treatment of microscopic tumor growth in the mouse peritoneal cavity [14], [15], [16], [17], [18], [19], [20], [21]. These promising observations have led to a patient Phase I study [22], and additional clinical studies are planned. ²¹¹At has a rather short half-life (7.21 h) and can be readily produced by the ²⁰⁹Bi(α,2n) ²¹¹At reaction in a cyclotron.

²¹³Bi can be generator-produced from the decay of ²²⁵Ac. The α-particle from the dominating decay chain of ²¹³Bi has a somewhat longer path length (82 μm in tissue) than the two α-particles from the decay chains of ²¹¹At (46 and 68 μm in tissue) [23]. Therapy studies using ²¹¹At treatment of various-sized tumors (30–340 μm) have demonstrated that therapeutic efficacy decreases with increasing tumor size [15]. The longer path length of ²¹³Bi may increase the absorbed dose to the central portions of microscopic tumors and possibly improve the therapeutic outcome when somewhat larger tumors are involved. The half-life of ²¹³Bi is ∼1/9 of that of ²¹¹At (45.6 min), which may make ²¹³Bi more beneficial for the local therapy suitable for ovarian cancer, since almost all disintegrations will occur in the peritoneal cavity before appreciable amounts of radionuclide are transported to the blood and other organs. Thus, there will be less collateral irradiation of bone marrow and other bodily tissues for ²¹³Bi than for ²¹¹At, which may help ameliorate the long-term risks of α-radiation.

The two goals of the present study were to compare the therapeutic efficacies of ²¹³Bi and ²¹¹At in α-RIT of ovarian cancer with the mAb MX35 and to compare the biodistributions of these two radiolabeled immunoconjugates. The dose-limiting organ in RIT is usually the bone marrow [24]; however, local healthy peritoneum may be dose limiting in local therapy in humans with short-lived radionuclides like ²¹³Bi and ²¹¹At [22]. Therefore, we chose ²¹³Bi and ²¹¹At activity levels for our therapeutic efficacy comparison that would result in equal irradiation of the peritoneal lining.

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2. Materials and methods 

2.1. Radionuclides 

²¹³Bi was produced by a ²²⁵Ac/²¹³Bi generator [Institute for Transuranium Elements (ITU), Karlsruhe, Germany] as described previously [25], [26]. The generator was handled according to the ITU standard protocol. Briefly, 600 μl of 0.1 M HCl/0.1 M NaI solution was run through the generator column to elute the ²¹³Bi. The pH of the eluate was adjusted to 5.3–5.5 with a 20% l-ascorbic acid solution and 4 M sodium acetate. l-Ascorbic acid additionally helps to radioprotect the mAb conjugate [27].

²¹¹At was produced at the PET and Cyclotron Unit, Rigshospitalet, Copenhagen, Denmark, as previously described [14]. Briefly, a ²⁰⁹Bi target was irradiated with 28-MeV α-particles with a beam current of ∼18 μA for 4–8 h. The resulting ²¹¹At was isolated by a dry distillation process as previously described [28].

2.2. Antibody 

Murine IgG₁ MX35, the mAb used in these experiments, was developed at the Sloan-Kettering Cancer Center (New York, NY, USA) [29], [30]. MX35 recognizes the sodium-dependent phosphate transport protein 2b (NaPi2b), which is expressed in a number of cancer cell lines, including OVCAR-3. MX35 previously exhibited homogenous reactivity with ∼90% of human epithelial ovarian cancer cells, but with a limited number of normal tissues [31]. The MX35 used here was produced from hybridoma cells kindly provided by the Ludwig Institute for Cancer Research, Zürich, Switzerland.

2.3. Cell line 

The National Institute of Health Ovarian Carcinoma Cell Line 3 (NIH:OVCAR-3) (American Type Culture Collection, Rocksville, MD, USA) was used for in vitro quality control of labeled antibodies and for tumor inoculation in the mice. The cells were grown in RPMI 1640 medium supplemented with 10% fetal calf serum, 1% l-glutamine and 1% penicillin–streptomycin in T-75 culture flasks, and cultivated in a humidified atmosphere of 95% O₂/5% CO₂ at 37°C.

2.4. Animals 

Female nude BALB/c (nu/nu) mice (Charles River Laboratories International, Wilmington, MA, USA) were used in all in vivo experiments. The animals were housed at 22°C in 50–60% humidity with a light/dark cycle of 12 h. They were kept under pathogen-free conditions and were given standard autoclaved pellets and water ad libitum. The animal experiments were approved by the Ethics Committee at the University of Gothenburg.

2.5. Antibody conjugation 

For labeling with ²¹³Bi, MX35 was conjugated with 2-(p-SCN-benzyl)-cyclohexyl-A-diethylenetriaminepentaacetic acid (CHX-A″-DTPA). The conjugation was performed at room temperature (RT) overnight in 0.2 M carbonate buffer, pH 8.5–8.6, containing 1 μM EDTA and 2–4 mg/ml mAb. A solution of 0.018 M CHX-A″-DTPA in DMSO was added to the mAb at 15× molar excess. After conjugation, the buffer was exchanged to 0.1 M citrate buffer, pH 5.5, that had been incubated with Chelex 100 resin (Bio-Rad Laboratories, Hercules, CA, USA) for at least 1 h. The buffer exchange was performed either with a 30-K Microsep Centrifugal Device (PALL Life Sciences, Ann Arbor, MI, USA) or with dialysis. When using a centrifugal device, the Chelex 100 resin was filtered off and the solution was centrifuged at 1680×g until 20 ml of citrate buffer had run through the filter. The dialysis was performed in 1 L of buffer using a 12,000- to 14,000-Da molecular-weight cut-off dialysis membrane (Spectrum Laboratories, Rancho Dominguez, CA, USA). The sample was dialyzed for 2 days with one buffer exchange. All equipment was made as metal free as possible to prevent contamination. The number of chelators on the MX35 conjugate was assessed by an arsenazo III spectrophotometric assay [32].

For labeling with ²¹¹At, MX35 was conjugated with N-succinimidyl-3-(trimetylstannyl)benzoate (m-MeATE) as described previously [33].

2.6. Antibody conjugate labeling 

The ²¹³Bi-labeling of MX35 was performed in the pH-adjusted generator eluate (see Section 2.1). To the ²¹³Bi eluted from the ²²⁵Ac/²¹³Bi generator, 0.1 mg of the conjugated MX35 was added and the reaction solution was incubated with vigorous agitation for 5 min. The reaction was quenched with 10 μl of 1.5 mg/ml DTPA. Product purification and buffer exchange to phosphate-buffered saline (PBS) were performed using a PD-10 column (GE Healthcare, Buckinghamshire, UK). Radiochemical purity was determined by ITLC SG (PALL Life Sciences) with 0.1 M citrate buffer, pH 5.5, as mobile phase. The stability of the radiolabeled antibodies was analyzed by HPLC using an ÄKTA purifier (GE Healthcare) with a Superdex-200 size exclusion column (GE Healthcare).

The ²¹¹At-labeling of MX35 and subsequent quality analyses were performed as described previously [33].

2.7. Immunoreactivity of the radiolabeled antibodies 

Immunoreactivity was assessed in vitro by adding 10 ng of radiolabeled MX35 to 0.5 ml of OVCAR-3 cell suspensions of various concentrations up to 5×10⁶ cells/ml. The samples were incubated with vigorous agitation for 2 h at RT, then centrifuged at 1438×g for 5 min, washed with 1 ml of PBS and centrifuged again. The supernatant was discarded and the activities of the cell pellets were measured in a γ-counter. The measurements were compared to reference solutions of labeled mAbs to estimate the ratio of bound activity to total applied activity (B/T).

2.8. Procedures for therapy and toxicity study 

Therapeutic efficacy was investigated for ²¹³Bi-labeled MX35 and ²¹¹At-labeled MX35 in mice at two different stages of tumor development. One hundred mice were inoculated intraperitoneally with ∼1×10⁷ OVCAR-3 cells. Forty animals were injected intraperitoneally with therapeutic solutions 2 weeks after inoculation: 20 mice with 2.55–2.95 MBq ²¹³Bi-labeled MX35 and 20 mice with 0.35–0.54 MBq ²¹¹At-labeled MX35. Another 40 animals were injected intraperitoneally 4 weeks from inoculation: 20 mice with 2.51–3.08 MBq ²¹³Bi-MX35 and 20 mice with 0.42 MBq ²¹¹At-MX35. The radiolabeled mAbs were administered in 1 ml of PBS. Twenty OVCAR-3-inoculated mice served as the reference group, not receiving any treatment.

The mice were weighed every 7–10 days. For acute myelotoxicity, white blood cell (WBC) counts were measured in 15 animals: five mice treated with ²¹³Bi-MX35, five mice with ²¹¹At-MX35 and five without treatment. WBC counts were measured before the treatment and 5 days and 14 days after the treatment. The animals were sacrificed and dissected 8 weeks after the therapeutic injections were administered. The reference animals were divided into two groups: 10 mice were sacrificed together with the first 40 animals treated (10 weeks after inoculation), and 10 mice were sacrificed with the second group of 40 animals treated (12 weeks after inoculation). After sacrifice, the abdominal cavity was opened and the presence of ascites and macroscopic tumors was investigated. Samples of the abdominal wall and the mesentery were taken from mice without macroscopic tumors or ascites to check for microscopic tumors. The examination of the animals was performed without knowledge of the treatment administered. The statistical significance of the difference in the therapeutic results between ²¹³Bi- MX35 and ²¹¹At-MX35 was calculated with Fisher's Exact Test.

2.9. Procedures for biodistribution 

Sixteen tumor-free mice were divided into four groups of four individuals each. The mice were injected intraperitoneally with a mixture of ²¹³Bi-MX35 and ²¹¹At-MX35 in 1 ml of PBS. The activities of ²¹³Bi-MX35 and ²¹¹At-MX35 per animal were 2.8–3.1 and 0.14–0.16 MBq, respectively. The groups were sacrificed 15, 45, 90 or 180 min after injection, when 20%, 50%, 75% or 94%, respectively, of the ²¹³Bi (t1/2=45.6 min) had decayed. Biodistribution studies of ²¹¹At-MX35 (t1/2 ²¹¹At=7.21 h) with longer times between injection and sacrifice have been performed previously [24]. The animals were dissected directly after sacrifice, and the blood, liver, lungs, kidneys, spleen, stomach, small intestine, throat and salivary glands were removed and measured in a γ-counter.

2.10. Dosimetric calculations 

The injected activities of ²¹³Bi-MX35 and ²¹¹At-MX35 in the therapy study were chosen to result in equal absorbed doses to the peritoneal lining in a patient therapeutic situation. The peritoneum could be assumed to be the dose-limiting organ in a patient receiving intraperitoneal RIT with short-lived radionuclides such as ²¹³Bi and ²¹¹At. Injected fluid was assumed to remain in the peritoneal cavity for 24 h. The absorbed dose to the peritoneum was calculated per activity unit of ²¹³Bi-MX35 and ²¹¹At-MX35, with the assumption that the decay-corrected activity concentration of the fluid was constant during this time and equal to the concentration of the injected solution. The absorbed dose was calculated as 50% of the equilibrium dose in the solution:

where A₀ is the injected activity, λ is the decay constant, V is the injected volume and Δ is the equilibrium dose constant for each of the two radionuclides. Injected activities of the two radionuclides were chosen to result in absorbed peritoneal doses of approximately 8 Gy. It was calculated that 3.0 MBq ²¹³Bi-MX35 and 0.4 MBq ²¹¹At-MX35 should be injected to obtain this peritoneal dose.

The absorbed dose to blood was calculated for ²¹³Bi-MX35 and ²¹¹At-MX35 to estimate the absorbed dose to the bone marrow. Data were used from the biodistribution experiment as well as data for ²¹¹At-MX35 from previous experiments with longer times before sacrifice (6, 12 and 18 h) [24]. A previously obtained [24] bone marrow-to-blood ratio for ²¹¹At-MX35 was used for both ²¹³Bi-MX35 and ²¹¹At-MX35 to estimate the absorbed dose to the bone marrow.

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3. Results 

3.1. Antibody conjugation, labeling and immunoreactivity 

According to the arsenazo III spectrophotometric assay, there were two available CHX-A″-DTPA on average attached to the mAb. The radiochemical yields of the ²¹³Bi-labeling procedure were 20–51% (without correction for decay), the radiochemical purity was 90–97% and the immunoreactivity (B/T) was 58–70%. There was one ²¹³Bi atom in every 1500 mAbs in the ²¹³Bi-MX35 injection solution. For the ²¹¹At-labeling, the radiochemical yields were 60–63% (without correction for decay), the radiochemical purity was 96% and the immunoreactivity was 85–87%. In the ²¹¹At-MX35 solution, there was one ²¹¹At atom in every 500 mAbs.

3.2. Therapeutic efficacy 

The tumor free fractions (TFFs) of the groups treated with ²¹¹At-MX35 or ²¹³Bi-MX35 2 weeks after cell inoculation were 0.90 and 0.60, respectively (Table 1); the difference was not statistically significant (P=.065). The TFFs of the groups treated with ²¹¹At-MX35 and ²¹³Bi-MX35 4 weeks after cell inoculation were both 0.25 (Table 1). No animal that received treatment 2 weeks after cell inoculation developed ascites, but among the animals treated 4 weeks after cell inoculation, four and three animals developed ascites after treatment with ²¹¹At-MX35 or ²¹³Bi-MX35, respectively (Table 1). Animals that developed ascites always had macroscopic tumors as well. The reference group that was sacrificed together with the first 40 animals had a TFF of 0.20, while the reference group that was sacrificed together with the second 40 animals had a TFF of 0 (Table 1).

Table 1. Results of the mouse groups treated with ²¹³Bi-MX35, ²¹¹At-MX35 or receiving no treatment

TFF=Tumor free fraction: the fraction of mice lacking macroscopic or microscopic tumors and ascites. Microscopic tumors were only sought in mice with no macroscopic tumors and/or ascites; thus, “microscopic tumor” refers to microscopic tumors without macroscopic tumor or ascites. Group 5 was sacrificed together with Groups 1 and 2, and Group 6 was sacrificed together with Groups 3 and 4.

3.3. Toxicity and biodistribution 

The WBC counts were reduced by 10% on average after 5 days in animals injected with ²¹³Bi-MX35, while there was a 45% reduction on average 5 days after treatment in animals injected with ²¹¹At-MX35; the WBC counts returned to starting levels 14 days after treatment (Fig. 1). WBC counts increased during the 14-day observation period in five mice that did not receive any treatment (Fig. 1). The weights of the treated and the untreated mice were very similar, increasing by ∼10% on average during the 8 weeks of therapy (data not shown).

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• Fig. 1. 

  White blood cell (WBC) counts before treatment, 5 days after treatment and 14 days after treatment. Each group consisted of five animals. Means and S.E.M. (error bars) are depicted.

Our biodistribution analysis revealed very similar tissue distributions for ²¹³Bi-MX35 and ²¹¹At-MX35 (Fig. 2). The throat (which was taken to represent the thyroid) exhibited slightly higher ²¹¹At-MX35 activities, while the kidneys displayed slightly higher activities of ²¹³Bi-MX35 (Fig. 2); these observations were expected because free ²¹¹At is known to accumulate in the thyroid, while free ²¹³Bi accumulates in the kidneys. No tissue demonstrated alarmingly high activities of any of the nuclides (Fig. 2).

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• Fig. 2. 

  Biodistribution of ²¹¹At- and ²¹³Bi-labeled monoclonal antibody MX35. Mice were injected with a mixture of ²¹¹At-MX35 and ²¹³Bi-MX35, and data were collected 15 min (A), 45 min (B), 90 min (C) and 180 min (D) after injection. Four animals were sacrificed at each time point. Tissue concentrations of radioactivity are expressed as the percentages of injected dose per gram (% ID/g). Means and S.E.M. (error bars) are depicted.

3.4. Dosimetric calculations 

The absorbed doses to a human peritoneum resulting from the activity concentrations administered in our therapeutic efficacy study would be 7.3 and 8.2 Gy for ²¹³Bi-MX35 and ²¹¹At-MX35, respectively. The absorbed dose to blood was on average 1.2 Gy in the case of ²¹³Bi-MX35, which gives an estimated absorbed dose to the bone marrow of 0.7 Gy. For ²¹¹At-MX35, the absorbed dose to blood was on average 1.9 Gy, yielding an estimated absorbed dose to the bone marrow of 1.1 Gy.

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4. Discussion 

The aim of this study was to, in an ovarian cancer model, compare the therapeutic efficacy and biodistribution of RIT with the mAb MX35 bound to the α-emitter ²¹³Bi or ²¹¹At. In the therapeutic efficacy experiment, the nuclide activities that were administered intraperitoneally in nude mice would correspond to an approximately equal dose to the peritoneum in a patient therapeutic situation. The peritoneum is suspected to be the dose-limiting organ in patients receiving RIT intraperitoneally when the radionuclide is as short lived as ²¹³Bi and ²¹¹At, due to low systemic irradiation following slow peritoneal cavity-to-blood transfer in humans.

Our therapy experiments demonstrated that both radionuclides were effective in intraperitoneal α-RIT for microscopic (2-week) tumors (Table 1), with ²¹¹At slightly (but not significantly) more effective under these experimental conditions; intraperitoneal α-RIT was less effective for larger (4-week) tumors (Table 1). The maximum tolerated dose of both ²¹³Bi-MX35 and ²¹¹At-MX35 is expected to be higher than the activities administered in this therapy study, and injection of higher activities might improve the therapeutic efficacy. Animal experiments have shown that there may be a radiation effect following an absorbed dose of approximately 15–16 Gy to the peritoneum (manuscript under preparation). A dose of approximately 7–8 Gy was reached in the experiments described here, which may be reasonable in a clinical situation in which a safe dose level is desirable. In our clinical Phase I study using ²¹¹At-MX35 F(ab′)₂, a maximum of 1.6 Gy to the peritoneum was achieved and no side effects were observed [22].

The injected activity of ²¹¹At-MX35 corresponds to a slightly higher absorbed dose to the human peritoneum than the injected activity of ²¹³Bi-MX35 (8.2 and 7.3 Gy, respectively), but this small difference is assumed to not affect the therapy outcome. Since our model was designed to mimic the therapeutic situation in humans as closely as possible, our dosimetric calculations assumed that the injected fluid stayed in the peritoneal cavity for 24 h, even though lymphatic drainage from the peritoneal cavity is somewhat faster in mice. Taking this faster drainage into consideration, the actual peritoneal dose from ²¹¹At-MX35 would be lower and the absorbed doses (8.2 and 7.3 Gy) may be more equal.

A consequence of the more rapid fluid drainage from the intraperitoneal cavity in mice is that the dose-limiting organ normally is the bone marrow and not the peritoneum, including cases involving short-lived radionuclides. The WBC counts from the therapy study (Fig. 1), as well as from previous studies performed with ²¹¹At-MX35 [14], indicate that the activities injected here are not close to the bone marrow toxicity level for mice. The WBC counts were even less affected by ²¹³Bi-MX35 treatment than by ²¹¹At-MX35 treatment in this study (Fig. 1), suggesting that an additional amount of ²¹³Bi-MX35 could probably be safely administered. Activities of ²¹¹At-MX35 higher than 1.2 MBq are probably not possible to administer intraperitoneally in mice due to severe hematologic toxicity from bone marrow irradiation [14]. Previously, activities of ²¹³Bi-mAbs as high as 14.8 MBq did not cause severely toxic effects when injected intraperitoneally into normal BALB/c mice; these mice exhibited a small weight loss and depression in WBC counts after 1 week, but these effects were reversed after 3 weeks [27]. However, although the peritoneum is not the dose-limiting organ in mice for intraperitoneally injected ²¹¹At-mAbs, it could be dose limiting for ²¹³Bi-MX35 due to the very short half-life of this radionuclide.

The absorbed dose to the bone marrow is possibly overestimated for ²¹³Bi-MX35 and ²¹¹At-MX35; the bone marrow-to-blood ratio for ²¹¹At-MX35 used here, 0.58, is somewhat higher than those found in the literature for other IgGs.

Previous therapeutic experiments with ²¹¹At-MX35 with intraperitoneal injections of 0.4–1.2 MBq demonstrated that TFF was not related with the administered activity of ²¹¹At-MX35 within this range [14]. This observation should be investigated further, since it could be a consequence of saturated antigenic sites due to a relatively low specific activity. However, even if those results are pertinent to ²¹¹At-MX35, they are not necessarily applicable for equivalent activities of ²¹³Bi-MX35.

In previous studies, it has been shown that the cytotoxicity of radiolabeled antibodies strongly depends on their specific activity (or on the ratio of labeled-to-unlabeled mAbs), i.e., the cytotoxicity increases with increasing specific activity [34]. In our study, the fraction of radiolabeled mAbs in the ²¹³Bi-MX35 and ²¹¹At-MX35 solutions differed by a factor of 3, as there was 1 in 1500 mAbs labeled with ²¹³Bi compared to 1 in 500 mAbs labeled with ²¹¹At. More equal fractions of labeled mAbs in the ²¹³Bi-MX35 and ²¹¹At-MX35 solutions could lead to more equal therapy results. Since a larger amount of unlabeled mAbs was injected in the case of ²¹³Bi-MX35, more antigenic sites on the cancer cells were blocked from labeled mAbs.

The biodistributions of ²¹³Bi-MX35 and ²¹¹At-MX35 were very similar (Fig. 2), indicating that the radiochemical purity at the time of injection was high and that the labeled mAbs were stable throughout the duration of the study. Otherwise, free ²¹³Bi would have accumulated in the kidneys and free ²¹¹At would have accumulated in the thyroid.

²¹³Bi and ²¹¹At have different physical properties, e.g., different half-lives and different path lengths of their emitted α-particles. Moreover, the main part of the α-energy of ²¹¹At is emitted by the daughter nuclide ²¹¹Po, which has a relatively long half-life of 0.5 s compared to the α-emitting daughter of ²¹³Bi: ²¹³Po (t1/2=4.2 μs). Because of the relatively long half-life, ²¹¹Po is able to diffuse from the target cell and consequently decrease the irradiation of the cell [35]. The effect can be noticeable especially in irradiating single cells and small cell clusters. However, the possible advantages of ²¹³Bi-MX35 did not yield better therapeutic results than ²¹¹At-MX35. The very short half-life of ²¹³Bi resulting in low systemic irradiation with ²¹³Bi-MX35 is still considerably advantageous, but the shorter half-life of ²¹³Bi also leads to a lower tumor dose relative to the peritoneum dose than ²¹¹At. During cell uptake of the mAbs (lasting 2–4 h [14]), ²¹³Bi decays to a higher extent than ²¹¹At, resulting in a reduction of the absorbed dose to the tumors. If the peritoneum is unambiguously demonstrated to be the dose-limiting organ in humans, increasing the activity of ²¹³Bi cannot compensate for the effect of the decay.

The α-emission following the decay of ²¹²Bi, which can be produced by a ²¹²Pb/²¹²Bi generator, is also interesting for targeted α-therapy. The decay of ²¹²Bi is very similar to the decay of ²¹³Bi in terms of half-life and energy emitted (t1/2 ²¹²Bi=60.6 min vs. t1/2 ²¹³Bi=45.6 min, and mean Eα of both nuclides ≈8–8.5 MeV). Therefore, similar therapeutic results would be expected for the two Bi isotopes. However, while ²¹³Bi can be extracted directly from the ²²⁵Ac/²¹³Bi generator, ²¹²Bi is obtained in situ from the decay of ²¹²Pb. The half-life of ²¹²Pb is 10.6 h, and together with the 1-h decay of ²¹²Bi, the overall half-life is almost 12 h. Compared to intraperitoneal treatment with ²¹³Bi or ²¹¹At (t1/2=7.21 h), more of the radioactive decay ought to occur outside of the peritoneal cavity in a treatment with ²¹²Pb. ²¹²Pb and ²¹²Bi both bind to chelators such as DOTA; however, during the decay of ²¹²Pb bound to a chelator, it has been shown that a fraction of the generated ²¹²Bi may be released as free ²¹²Bi [36].

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5. Conclusion 

Micrometastatic growth of the ovarian cancer cell line OVCAR-3 was reduced in nude mice after treatment with ²¹³Bi-MX35 or ²¹¹At-MX35. Treatment with ²¹¹At-MX35 provided a somewhat better result, but the difference was not statistically significant (P=.065), suggesting that the potentially more suitable physical properties of ²¹³Bi compared to ²¹¹At were not shown to be advantageous in this study. However, a patient therapeutic situation may benefit from a lower toxicity of ²¹³Bi due to lower systemic irradiation. No considerable side-effects were observed after the RIT treatments. WBC counts, reflecting the degree of acute myelotoxicity, were affected after treatment with ²¹¹At-MX35, but were influenced only to a very small extent by treatment with ²¹³Bi-MX35. The biodistributions of ²¹³Bi-MX35 and ²¹¹At-MX35 were very similar, highlighting the stability of the labeled mAbs. The radiolabeled immunoconjugates did not accumulate to a large extent in the investigated organs. Higher administered activities of ²¹³Bi-MX35 and ²¹¹At-MX35 should be possible as toxic levels presumably were not achieved in this study.

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Acknowledgments 

The excellent assistance of Helena Kahu (cell culturing and animal experiments), Elin Cederkrantz, Nicolas Chouin (dosimetric calculations) and Sofia Frost (HPLC and immunoreactivity analyses) is greatly appreciated.

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