Radioimmunotherapy of Breast Cancer Metastases with Alpha-Particle-emitter ²²⁵Ac: Comparing Efficacy with ²¹³Bi, ⁹⁰Y

Abstract

Alpha-particles are suitable to treat cancer micrometastases because of their short range and very high linear energy transfer. Alpha-particle-emitter ²¹³Bi based radioimmunotherapy has shown efficacy in a variety of metastatic animal cancer models, such as breast, ovarian, prostate cancer and leukemia. Its clinical implementation, however, is challenging due to the limited supply of ²²⁵Ac, the high technical requirement to prepare radioimmunoconjugate with very short half-life (T_1/2=45.6 mins) on site and prohibitive cost. In this study, we investigated the efficacy of the alpha-particle-emitter ²²⁵Ac, parent of ²¹³Bi, in a mouse model of breast cancer metastases. A single administration of ²²⁵Ac (400 nCi) labeled anti-rat HER-2/neu monoclonal antibody (7.16.4) completely eradicated breast cancer lung micrometastases in about 67% of HER-2/neu transgenic mice and led to long-term survival of these mice for up to one year. Treatment with ²²⁵Ac-7.16.4 is significantly more effective than ²¹³Bi-7.16.4 (120 μCi) (median survival = 61 days, P=0.001), and ⁹⁰Y-7.16.4 (120 μCi) (median survival = 50 days, P<0.001), as well as untreated control (median survival = 41 days, P=0.0001). Dosimetric analysis showed that ²²⁵Ac treated metastases received a total dose of 9.6 Gy, significantly higher than 2.0 Gy from ²¹³Bi and 2.4 Gy from ⁹⁰Y. Biodistribution studies revealed that ²²⁵Ac daughters, ²²¹Fr and ²¹³Bi, accumulated in kidneys and probably contributed to the long-term renal toxicity observed in surviving mice. These data suggest ²²⁵Ac labeled anti-HER-2/neu monoclonal antibody could significantly prolong survival in HER-2/neu-positive metastatic breast cancer patients.

INTRODUCTION

Radioimmunotherapy of metastatic cancer using alpha-particle-emitter-labeled monoclonal antibodies (mAb) is promising because alpha-particles can deliver highly focused energy along their short path length (1). The high-energy alpha radiation typically causes complex DNA double strand breaks (DSBs) that are difficult to repair, thus leading to effective tumor cell kill. Preclinical studies using ²¹³Bi labeled mAbs have shown substantial efficacy in various tumor models including leukemia (2), prostate (3), ovarian (4), and colon cancer (5). We have also shown that ²¹³Bi labeled anti-HER-2/neu mAb (7.16.4) is effective in prolonging the survival of HER-2/neu transgenic mice that if left untreated develop widespread metastases, including bone and liver metastases (6). Clinical trials using ²¹³Bi labeled anti-CD33 mAb to treat myeloid leukemia have shown feasibility and safety (7,8). However, the short half-life of ²¹³Bi complicates the preparation of the radioimmunoconjugates for clinical use and demands a large amount of ²¹³Bi activity that is limited by worldwide availability of its parent, ²²⁵Ac. As a result, the maximum tolerated dose was not reached in the clinical trial at the largest administered activity of 37 MBq/kg (8).

To overcome the short half-life of ²¹³Bi, McDevitt et al. (9) proposed the concept of an in vivo ²²⁵Ac (T_1/2=10 days) generator which would deliver 4 alpha-particles to the target site per decay of ²²⁵Ac. This compares to one alpha from ²¹³Bi, making, ²²⁵Ac much more potent. Indeed, ²²⁵Ac-labeled mAbs have greatly improved the survival in lymphoma and ovarian cancer models (9,10). More recently, ²²⁵Ac-labeled anti-vascular endothelial cadherin mAb targeting tumor neovasculature has been shown to inhibit tumor growth in a prostate cancer (LNCaP) model especially when it is combined with sequential chemotherapy (11). The in vivo generator concept has also been investigated in other alpha-particle-emitters. ²¹²Pb (T_1/2=10.6 hr, alpha-particle-emitting daughter ²¹²Bi) in vivo generator (²¹²Pb-Trastuzumab) has prolonged survival in a colon cancer xenograft model (12). Dahle et al. showed that a single injection of ²²⁷Th-Rituximab (T_1/2=18.7 days, alpha-particle-emitting daughters ²²³Ra, ²¹³Rn, ²¹⁵Po and ²¹¹Bi) is able to completely eradicate 60% of B-cell lymphoma xenografts (13). Since ²²³Ra, the first daughter of ²²⁷Th has a 10-day half-life and rapidly localizes to bone, alpha-particles are mainly delivered from ²²⁷Th itself and the studies have demonstrated that subsequent daughters do not lead to toxicity.

Several studies have been published to compare the efficacy of alpha and beta-radiation directly in metastatic tumor models. Behr et al. (14) found that ²¹³Bi is therapeutically more effective than the beta emitter, ⁹⁰Y, in a metastatic colon cancer model. Few studies, however, directly compared the efficacy of in vivo alpha-particle generators with that of conventional alpha and beta particle-emitters. In this work, we compare the efficacy of targeted therapy using the ²²⁵Ac in vivo generator with targeted ²¹³Bi and ⁹⁰Y in a syngeneic HER-2/neu metastatic breast cancer model.

HER-2/neu is a tumor cell surface tyrosine kinase associated with aggressive phenotype and poor prognosis (15). Targeting HER-2/neu with Trastuzumab has shown significant clinical benefit in patients with metastatic breast cancer (16). In this study, we demonstrate the efficacy of ²²⁵Ac-7.16.4 in targeting rat HER-2/neu positive pulmonary metastases. Rat HER-2/neu is also expressed on normal lung tissue in this mouse model as determined by Western blot (17). This allows for evaluating efficacy and toxicity of ²²⁵Ac-7.16.4 in a model that closely mimics clinical cases where cross reactivity of tumor antigen expressed on normal organs is common.

MATERIALS AND METHODS

Mice, cell line and monoclonal antibodies

neu-N transgenic mice, age 6 to 8 wk, expressing rat HER-2/neu under the mouse mammary tumor virus (MMTV) promoter were obtained from Harlan (Harlan Lab., Madison, WI). All experiments involving the use of mice were conducted with the approval of the Animal Care and Use Committee of The Johns Hopkins University School of Medicine. NT2.5, a rat HER-2/neu expressing mouse mammary tumor cell line, was established from spontaneous mammary tumors (18). The NT2.5 cells are maintained in RPMI media containing 20% fetal bovine serum, 0.5% penicillin/streptomycin (Invitrogen, Carlsbad, CA), 1% L-glutamine, 1% nonessential amino acids, 1% sodium pyruvate, 0.02% gentamicin, and 0.2% insulin (Sigma, St. Louis, MO) at 37°C in 5% CO₂. 7.16.4, a mouse anti-rat HER-2/neu mAb was purified from the ascites of athymic mice. The hybridoma cell line was kindly provided by Dr. Mark Greene (University of Pennsylvania). Rituximab (IDEC Pharmaceuticals Corp.), an anti-human CD20 monoclonal antibody, was used as a negative control.

Radiolabeling of antibody with ²¹³Bi, ⁹⁰Y and ²²⁵Ac

7.16.4 was conjugated to SCN-CHX-A”-DTPA following published protocol (19). ⁹⁰Y was purchased from PerkinElmer (Waltham, MA) and labeled to 7.16.4-Chx-A”-DTPA (10 mCi/mg) at 37°C for 30 mins in acetate buffer (pH=4.5). ²²⁵Ac/²¹³Bi (Institute for Transuranium Elements) generator was constructed and ²¹³Bi was labeled to 7.16.4-Chx-A”-DTPA (10 mCi/mg) as described previously (6). Both ⁹⁰Y-, and ²¹³Bi-labeled 7.16.4 radioimmunoconjugates were purified by MicroSpin G-25 column (GE BioSciences, Pittsburgh, PA).

²²⁵Ac was purchased from Curative Technologies Corporation (Richland, WA). ²²⁵Ac was labeled to mAb in a two-step reaction following McDevitt et al (20). First, ²²⁵Ac (0.15-0.2 mCi in 20-80μL) was chelated to 1μL (10mg/mL) p-SCN-Bn-DOTA (Macrocyclics, Richardson, TX) at 56°C for 1 hr. Ascorbic acid (1μL, 150mg/mL) was added as a radio-protectant and 2M Sodium acetate (40 to 60 μL) was added to raise the pH to 6.5. The efficiency of ²²⁵Ac chelation to DOTA was determined by Sephadex C-25 column (GE Bioscience). Second, 100μg mAb (~20μL, 5mg/mL) was incubated with p-SCN-Bn-DOTA-²²⁵Ac at 37°C for 45 mins (pH=8.5). ²²⁵Ac-labeled mAb was purified with a Centricon centrifuge filter unit (YM-10, Millipore).

The reaction efficiency and purity of the radioimmunoconjugates were determined with instant thin layer chromatography (ITLC) using silica gel impregnated paper (Gelman Science Inc., Ann Arbor, MI). ITLC paper strips were counted the next day with a gamma counter (LKB Wallac, Perkin-Elmer) to allow ²²⁵Ac to reach equilibrium. ²²⁵Ac-7.16.4 immunoreactivity was determined by incubating 5 ng of ²²⁵Ac-7.16.4 with excess antigen binding sites (1×10⁷ NT2.5 cells) twice on ice for 30 mins each time. Immunoreactivity was calculated as the percentage of ²²⁵Ac-7.16.4 bound to the cells. Stability of ²²⁵Ac-7.16.4 was measured by incubating ²²⁵Ac-7.16.4 in cell culture media containing 20% FBS for 30 days and the fraction of ²²⁵Ac chelated to DOTA was measured with Sephadex C-25 column and ITLC. To determine internalization of 7.16.4, NT2.5 cells (2×10⁶ cells/ml) were incubated with ¹¹¹In labeled 7.16.4 (1μg/ml) at 37 °C. After 0, 10, 20, 30, 60, 120, 240, 360, 480 mins (two samples for each time point) of incubation, reaction was stopped and NT2.5 cells were washed three times with cold PBS. Cell pellet was then incubated with 1 ml NaCl 150 mM/50 mM Glycine (pH=2.0) for 10 mins at room temperature and washed twice with PBS. Both pellet and supernatant were collected and counted with gamma counter. The activity fraction of the cell pellet was the fraction of internalized 7.16.4 (10).

Specific cell kill in vitro by ²²⁵Ac-7.16.4

Specific cell kill in vitro was determined by colony formation assay. NT2.5 cells were seeded into 96 well plates at 2.0×10³ cells/well. NT2.5 cells were then treated with serially diluted ²²⁵Ac-7.16.4 or ²²⁵Ac-Rituximab (from 0.2 to 20 nCi/ml at a specific activity of 0.10 μCi/μg). After incubation for either 3 or 5 days, NT2.5 cells were trypsinized and re-plated on cell culture petri dishes for colony growth. Blocking of cell kill by ²²⁵Ac-7.16.4 using unconjugated 7.16.4 (50 μg/ml) was also tested.

Biodistribution of ²²⁵Ac-, ²¹³Bi- and ¹¹¹In- 7.16.4

neu-N mice (3 mice/group) bearing s.c. tumors in the mammary fat pad were injected intravenously with 400 nCi ²²⁵Ac-7.16.4. At 1, 6, 24, 72, 144, 288 hrs after injection, mice were sacrificed and major organs including blood, heart, lung, liver, spleen, kidney, stomach, intestine, femur, tumor were collected. ²²⁵Ac in each organ was counted the next day using an energy window of 150-500 keV. To determine the distribution of free ²²¹Fr (T_1/2=4.9 mins) and ²¹³Bi that was released after ²²⁵Ac decay, the organs were counted right away repeatedly for ²²¹Fr or ²¹³Bi using the 190-250 keV and the 400-480 keV energy window, respectively. An exponential expression was fitted to the decay curves thus obtained. The fitted activity coefficient at time zero is the activity concentration at the time of sacrifice. Differences in ²²¹Fr and ²¹³Bi activity at the time of sacrifice and the levels from equilibrium of ²²⁵Ac reflect clearance or accumulation of free ²²¹Fr and ²¹³Bi. The time activity curves of free ²¹³Bi and ²²¹Fr in each organ were then constructed with free ²²¹Fr, ²¹³Bi activities at multiple sacrifice time points. Injectates with equivalent injected activity were counted as standards for decay correction. In a separate series of animals, the biodistribution of ²²⁵Ac-, ²¹³Bi-, and ¹¹¹In-7.16.4 to lung metastases were also measured and compared. neu-N mice (3 mice/group) were injected with either ²²⁵Ac-, ²¹³Bi-, or ¹¹¹In-labeled 7.16.4 and sacrificed at 0.5, 1.0, 3.0, and 6.0 hr after injection; ¹¹¹In radionuclide was used as a surrogate for ⁹⁰Y (21). Lung metastases were collected and counted for ²²⁵Ac, free ²²¹Fr and ²¹³Bi.

Efficacy of ²²⁵Ac-, ²¹³Bi-, ⁹⁰Y-labeled anti-rat HER-2/neu mAb to treat lung metastases

The maximum tolerated dose (MTD) was determined as the highest administered activity that allows 100% survival with no significant body weight loss (>15%). Healthy neu-N mice were injected (five mice/group) with 100, 200, 400, 500, 600, 700, or 1,000 nCi ²²⁵Ac-7.16.4. Mice were weighed twice per week for 90 days. To evaluate the efficacy of radiolabeled mAbs to treat early stage micrometastases, three days after neu-N mice were injected with 1×10⁵ NT2.5 cells (i.v.), mice were treated intravenously with (a) 400 nCi ²²⁵Ac-7.16.4, n=12; (b) 120 μCi ²¹³Bi-7.16.4, n=10; (c) 120 μCi ⁹⁰Y-7.16.4, n=5; (d) 200 nCi ²²⁵Ac-7.16.4, n=5; (e) 200 nCi + 200 nCi ²²⁵Ac-7.16.4 (injected one week apart), n=5; (f) 100 μg 7.16.4, unlabeled control, n=5; (g) 400 nCi ²²⁵Ac-Rituximab, nonspecific control, n=5; (h) untreated control, n=10. To evaluate the efficacy of treating late stage lung metastases, at eighteen days after tumor cell inoculation, neu-N mice were treated with (a) 400 nCi ²²⁵Ac-7.16.4, n=5; (b) 120 μCi ²¹³Bi-7.16.4, n=5; (c) 120 μCi ⁹⁰Y-7.16.4, n=5. Mice were monitored and weighed three times a week and were euthanized when significant body weight loss (>15%) or breathing difficulties developed. In survival and long-term toxicity studies, animals were followed for up to one year.

At time of sacrifice, all major organs were collected for histopathological examination. The number of visible lung metastases was counted. Lung metastases were snap frozen in liquid nitrogen and sectioned with a cryotome into 8 μm thick slices. The section was fixed in acetone and immunostained with 7.16.4 and a biotinylated rat anti-mouse IgG2a antibody (BD Pharmingen). The stain was developed with a Vectastain ABC Kit (Vector Lab, Burlingame, CA) according to manufacturer’s instruction.

Dosimetry

Organ and tumor absorbed doses for ²²⁵Ac were calculated based on measured biodistribution data (22). Time activity curves in each organ and in s.c. tumors and small lung metastases were measured and integrated over time to obtain the total disintegration of ²²⁵Ac, free ²²¹Fr and ²¹³Bi. ²¹³Po and ²¹⁷At, with half-lives of 4.2μs and 32.3ms, were assumed to decay at the same position as ²¹³Bi or ²²¹Fr. Absorbed doses for three tumor geometries were calculated: subcutaneous (s.c.) tumor (approximately 1 cm-diameter), small lung metastases (300 μm diameter - i.e., day 18) and single cells (day 3). In the s.c. tumor calculation, the alpha-particle and electron energies of ²²⁵Ac were assumed completely absorbed within the tumor. Photon doses were not included since they typically account for less than 1% of the total absorbed dose. Absorbed doses for normal organs and the s.c. tumor were thus calculated as $D_{\alpha }=\tilde{A}\ast {\Delta }_{\alpha }∕\mathrm{M}$; $D_e=\tilde{A}\ast {\Delta }_{\mathrm{e}}∕\mathrm{M}$; D= D_α + D_e, wherein D_α, D_e and D are the alpha-particle, electron, and summed alpha and electron absorbed dose, respectively, $\tilde{A}$ is the total number of disintegrations in an organ/tumor, Δ_α, Δ_e are the mean energy emitted per nuclear transition for alpha-particles and electrons, and M is the weight of the organ/tumor. The total absorbed doses were calculated as the sum of doses from ²²⁵Ac at equilibrium and its free daughters. If an organ is accumulating or depleting free daughters, the dose from free daughters is added or subtracted from the ²²⁵Ac doses at equilibrium. Absorbed doses for single tumor cells (day 3) and small lung metastases (day 18) were also calculated and compared for ²²⁵Ac-, ²¹³Bi-, and ⁹⁰Y- 7.16.4. Bi-213 and In-111 (surrogate for Y-90) biodistribution data for small lung metastases were used for Bi-213 and Y-90 dosimetry. The absorbed fractions of electron and alpha-particle emissions in small lung metastases were calculated using Monte Carlo; these calculations accounted for the range of all of the betas and alphas (23). Absorbed doses to single cells were obtained using MIRD cellular S values (24,25). In the single-cell calculations, daughter radionuclides resulting from the decay of 7.16.4-conjuagated ²²⁵Ac that was tumor-cell bound but not internalized were assumed to diffuse away and not contribute to the target cell absorbed dose. The fraction of internalized ²²⁵Ac-7.16.4 was obtained from the internalization assay.

Statistical Analysis

Kaplan-Meier survival analysis or statistical comparisons between groups (Student t-test) were performed with MedCalc (MedCalc Software, Belgium). For all studies, the level of statistical significance is set at P <0.05.

RESULTS

Radiolabeling, immunoreactivity and antibody internalization

Both ⁹⁰Y, and ²¹³Bi labeling efficiencies were about 90% and purities reached ~98% after size exclusion purification. The two-step ²²⁵Ac labeling efficiency was 12.0% ± 3.8% (n=12). After purification, the purity of ²²⁵Ac-7.16.4 was 97.0% ± 1.8% (n=12). The specific activity of ²²⁵Ac-7.16.4 was between 0.06 to 0.10 μCi/μg. The immunoreactivity of ²²⁵Ac-7.16.4 was 62.4% ± 5.9% (n=3), worse than that of ¹¹¹In- or ²¹³Bi-7.16.4 (~80%). After incubation in 20% BSA at 37°C for 30 days, purity of ²²⁵Ac-7.16.4 became 80.9% ± 8.2% and 65.9% ± 3.9% as measured by Sephadex C-25 column and ITLC (n=3), respectively. 7.16.4 internalizes after binding to NT2.5 cells. The internalized ¹¹¹In-7.16.4 increased from 1.55 × 10^-2 μg/10⁶ cells at 5 mins after binding started to 3.05 × 10^-2 μg/10⁶ cells at 120 mins and plateaued afterwards to 3.25 × 10^-2 μg/10⁶ cells at 1440 mins.

In vitro cell kill

²²⁵Ac-7.16.4 was very effective in killing rat HER-2/neu expressing NT2.5 cells (Fig. 1A). The effective dose concentration of ²²⁵Ac-7.16.4 that can kill 50% of the NT2.5 cells (ED₅₀) was 2.3 nCi/ml and 6.4 nCi/ml for 5-day or 3-day incubation, respectively. When 50 μg/ml unlabeled 7.16.4 was used to block ²²⁵Ac-7.16.4, the ED₅₀ increased to 9.4 nCi/ml (5-day incubation). The ED₅₀ for ²²⁵Ac-Rituximab was 19.0 nCi/ml and 18.8 nCi/ml when it was incubated with NT2.5 cells for 3 or 5 days. In comparison, the ED₅₀ for ²¹³Bi-7.16.4 was 940 nCi/ml.

Figure 1.

A, In vitro kill of NT2.5 breast cancer cells by ²²⁵Ac-7.16.4. NT2.5 cells were treated with ²²⁵Ac-7.16.4 for 3 days (open circle), 5 days (closed triangle), or with the presence of 50 μg/ml unconjugated 7.16.4 (open diamond). ²²⁵Ac-Rituximab was used as non-specific control to treat NT2.5 cells for 5 (open triangle) or 3 (open square) days. Survival fraction was calculated by dividing the number of colonies in treatment groups over that in the untreated control group.

B, Biodistribution of ²²⁵Ac-7.16.4 in neu-N mice at decay equilibrium; data are presented as percent injected dose per gram (%ID/g±SD). Biodistributions of free ²¹³Bi (C) and ²²¹Fr (D) in major organs (at 1 hr) are shown at the time of sacrifice and decay equilibrium. Star (*) marks the difference that is statistically significant; data are expressed as Bq per gram (Bq/g±SD).

Biodistribution of ²²⁵Ac-, ²¹³Bi- and ¹¹¹In-7.16.4

The biodistribution of ²²⁵Ac-7.16.4 at equilibrium in (s.c. mammary fat-pad) tumor bearing neu-N mice is shown in Fig. 1B. The effective half-life of antibody clearance from blood in the first 72 hr was 26.8 hr, similar to that of ¹¹¹In-labeled 7.16.4, 26.3 hr. ²²⁵Ac-7.16.4 targeting to tumors increased continuously and accumulation peaked at 20.6%/g 6 days after injection. Antibody localization in the tumor was 17.3%ID/g 12 days post-injection. The peak tumor uptake of ²²⁵Ac-7.16.4, however, was lower than that of ¹¹¹In-7.16.4 (~38%ID/g), probably reflecting impaired immunoreactivity of ²²⁵Ac-7.16.4.

The biodistributions of free ²¹³Bi, ²²¹Fr at 1hr after ²²⁵Ac-7.16.4 injection are shown in Fig. 1C and 1D. For ²¹³Bi (Fig. 1C), the activity concentration in blood increased from 2779.9 Bq/g at time of sacrifice to 3502.1 Bq/g at equilibrium (P=0.03), while the kidneys activity decreased from 2449.8 Bq/g at time of sacrifice to 887.0 Bq/g at equilibrium (P=0.005). Similar pattern was observed for ²²¹Fr (Fig. 1D). These data strongly suggest that both free ²¹³Bi and ²²¹Fr cleared from blood through kidneys. The fractions of free ²¹³Bi and ²²¹Fr in blood that ended up in kidneys were 41.1% and 18.7%, suggesting a higher kidney retention rate for ²¹³Bi, which has been attributed to the higher positive charge of ²¹³Bi (Bi³⁺) that interacts with the negatively charged basement membrane of the glomerulus. In biodistribution study of lung metastases, ²²⁵Ac-7.16.4 was found to be 13.7±1.5%ID/g at 144 hr, lower than 30.8±1.5%ID/g of ²¹³Bi-7.16.4 at 6 hrs.

Treating rat HER-2/neu expressing lung metastases with ²²⁵Ac-, ²¹³Bi- and ⁹⁰Y-7.16.4

The maximum tolerated dose of ²²⁵Ac-7.16.4 in neu-N mice was found to be 400 nCi in a single injection. MTD of ²¹³Bi-7.16.4 (120 μCi) and ⁹⁰Y-7.16.4 (120 μCi) were obtained from literature and confirmed in the neu-N model (6,26). Kaplan-Meier survival curves of neu-N mice bearing lung metastases after treatment by radiolabeled anti-rat HER-2/neu mAb are shown in Fig. 2A. All untreated mice (100%) developed pulmonary metastases, showing signs of labored breath and stress at later stages. Median survival improved to 50 days in ⁹⁰Y-7.16.4 group (P=0.013), 61 days in ²¹³Bi-7.16.4 group (P=0.0001) compared to untreated mice (41 days). Eight of twelve mice in ²²⁵Ac-7.16.4 treated group achieved long-term (one-year) survival (P<0.0001). Both ²¹³Bi-(P=0.025) and ²²⁵Ac-labeled (P<0.001) 7.16.4 treated mice lived significantly longer than the mice treated by ⁹⁰Y-7.16.4. ²²⁵Ac-7.16.4 also improved survival of neu-N mice compared to alpha emitter ²¹³Bi-7.16.4 (P=0.001). ²²⁵Ac-Rituximab did not demonstrate efficacy in these models with median survivals of only 33 days (P=0.99). Unlabeled 7.16.4 (single i.v. dose 100μg/mouse) slightly improved median survival to 42 days (P=0.014) and the median survival of neu-N mice treated with 200 nCi ²²⁵Ac-7.16.4 improved to 47 days (P=0.029). Notably, when a second dose of 200 nCi ²²⁵Ac-7.16.4 was administered one week after the first 200 nCi injection, median survival improved to 84 days (P=0.004) with 2 out 5 mice achieved long-term survival for up to one year (Fig. 2B).

Figure. 2.

Therapeutic efficacy of ²²⁵Ac-, ²¹³Bi- and ⁹⁰Y-7.16.4. A, Kaplan-Meier survival curves of neu-N transgenic mice treated 3 days after i.v. injection of 1×10⁵ NT2.5 cells. Neu-N mice were treated with 400 nCi ²²⁵Ac-7.16.4 (open triangle, n=12), 120 μCi ²¹³Bi-7.16.4 (closed circle, n=10), 120 μCi ⁹⁰Y-7.16.4 (open square, n=5) or untreated (closed diamond, n=10). B, Neu-N mice were treated 3 days after tumor cell inoculation with 100 μg unlabeled 7.16.4 (open diamond, n=5), 400 nCi ²²⁵Ac-Rituximab (open triangle, n=5), 200 nCi ²²⁵Ac-7.16.4 (open square, n=5), 200 nCi + 200 nCi ²²⁵Ac-7.16.4 (closed triangle, n=5) or untreated (open circle, n=4) C, neu-N mice were treated 18 days after tumor cell inoculation with 400 nCi ²²⁵Ac-7.16.4 (open triangle, n=5), 120 μCi ²¹³Bi-7.16.4 (closed circle, n=5), 120 μCi ⁹⁰Y-7.16.4 (open square, n=5) or untreated (closed diamond, n=10).

To examine the efficacy of ²²⁵Ac-7.16.4 on later stage metastases, neu-N mice were treated 18 days after tumor cell injection (Fig. 2C), where the average diameter of lung metastases was 295.7 ± 93.6 μm (n=29). ²¹³Bi-7.16.4 treated mice have a median survival of 51 days, not a statistically significant improvement over ⁹⁰Y-7.16.4 with a median survival of 50 days (P=0.76). Although mice treated with ²¹³Bi-7.16.4 at 18 days had a significantly shorter survival compared to those treated at 3 days (P=0.0001), mice treated with ⁹⁰Y-7.16.4 at 18 days and 3 days have about the same median survival of 50 days (P=0.96), suggesting that targeting with the ⁹⁰Y is less sensitive to the size of the metastases. The median survival decreased to 66 days for mice treated with ²²⁵Ac-7.16.4 at 18 days compared to 3 days (P=0.0005). Nevertheless, ²²⁵Ac-7.16.4 treated mice still survived significantly longer compared to ²¹³Bi-7.16.4 (P=0.004).

Number of lung metastases

The numbers of visible metastases on the lungs of neu-N mice and representative images are shown in Fig. 3. All treated mice have reduced number of lung metastases compared to untreated mice. When treatment was initiated at 3 days, mice treated with ²¹³Bi-7.16.4 and ²²⁵Ac-7.16.4 had 8.5±1.8 (P<0.0001) and 2.9±6.1 (P<0.0001) lung metastases, significantly less than ⁹⁰Y-7.16.4 treated mice with 36.7±8.1 metastases. No lung metastases were found in neu-N mice surviving ²²⁵Ac-7.16.4 treatment (Fig. 3A, bottom right). It is also evident that the sizes of the metastases were much larger in ²¹³Bi-7.16.4 treated mice (Fig. 3A, bottom left) than those from ⁹⁰Y-7.16.4 treated mice (Fig. 3A, top right), which suggests ²¹³Bi-7.16.4 is able to eliminate more metastases initiating cells compared to ⁹⁰Y-7.16.4 and it takes longer for the surviving tumor cells to grow to a lethal tumor burden. When treatment was initiated at 18 days, the number of metastases found on ⁹⁰Y-7.16.4 treated mice (32.8±13.2) was similar to that obtained for mice treated with ⁹⁰Y-7.16.4 at 3 days. More metastases (34.0±13.5) were found on mice treated with ²¹³Bi-7.16.4 at 18 days compared to 3 days (P<0.0001), indicating reduced efficacy of ²¹³Bi to treat later stage metastases. ²²⁵Ac-7.16.4 treated mice again presented with the fewest number of lung metastases, 11.5±3.5, compared to the other two radioisotopes (Fig. 3B).

Figure 3.

A, Representative photographs show lungs bearing metastases from neu-N mice after treatment by ²²⁵Ac-, ²¹³Bi-, ⁹⁰Y- 7.16.4. These mice were treated at 3 days after tumor cell injection with untreated (top left), 120 μCi ⁹⁰Y-7.16.4 (top right), 120 μCi ²¹³Bi-7.16.4 (bottom left), 400 nCi ²²⁵Ac-7.16.4 (bottom right). B, Number of lung metastases from mice treated at 3 days (filled column) or 18 (striped column) days after tumor cell inoculation was counted. Data are presented as average number of metastases±SD.

Histopathology and immunohistostaining of rat HER-2/neu

Immunohistostaining showed that the HER-2/neu expression level decreased on the metastases surviving the treatment of ⁹⁰Y-7.16.4 (Fig. 4A, middle) and ²¹³Bi-7.16.4 (Fig. 4A, right), compared to untreated tumors (Fig. 4A, left). At one year after treatment, no residual tumor cells were found on the lungs of mice treated with 400 nCi ²²⁵Ac-7.16.4 (Fig. 4B, left) and a single metastatic tumor nodule was found on one of the lungs from a mouse treated with 200 +200nCi ²²⁵Ac-7.16.4 (Fig. 4B, right). No damage to the normal lung tissue was observed in groups treated with ²²⁵Ac-7.16.4. Examination of kidneys from these mice, however, revealed shrunken and pale kidneys (Fig. 4C, left). H&E staining showed wide-spread loss of tubular epithelium in the kidney cortex (Fig. 4C, middle). In the medulla there is extensive loss of tubules and replacement with marked fibrosis and a mixed inflammatory infiltrate composed mainly of mononuclear cells and lesser numbers of neutrophils. Scattered dilated cystic structures are visible throughout the cortex and medulla. These observations are also present but less evident in kidneys treated with 200 + 200 nCi ²²⁵Ac-7.16.4 (Fig. 4C, right).

Figure 4.

A, Immunohistostaining of rat HER-2/neu expression on lung metastases. Untreated control (left); treated with 120 μCi ⁹⁰Y-7.16.4, (middle) or with 120 μCi ²¹³Bi-7.16.4 (right). B, H&E staining of lungs from mice treated with 400 nCi ²²⁵Ac-7.16.4 that showed no sign of tumor cells or lung tissue damage (left) and of a single metastasis found on the lungs of a mouse treated with 200 + 200nCi ²²⁵Ac-7.16.4 (right). C, left, Representative photographs of kidneys from a neu-N mouse surviving one year after treatment with 400 nCi ²²⁵Ac-7.16.4 (left pair) and a healthy neu-N mouse (right pair). H&E staining of kidneys from neu-N mice surviving one year after treatment with 400 nCi (middle) or 200 nCi + 200 nCi ²²⁵Ac-7.16.4 (right). Arrows point to collapse of cortical tissue due to loss of tubular epithelium in the kidney cortex. Bar indicates 100 μm.

Dosimetry

The absorbed doses to major organs and tumors are shown in Table 1. Reductions or increases in the absorbed dose from ²²⁵Ac at equilibrium with its free daughters are indicated by negative or positive dose contributions. The dose calculations show that free ²²¹Fr and ²¹³Bi are cleared from the blood, lungs, liver and spleen while kidney, stomach, and intestine are accumulating these daughters. A 400 nCi administration of ²²⁵Ac-7.16.4 gives a tumor absorbed dose of 10.49 Gy; no significant depletion or accumulation of free daughters is observed. Absorbed doses of ²²⁵Ac-, ²¹³Bi-, ⁹⁰Y-7.16.4 to single tumor cells and small lung metastases (~300μm in diameter) are shown in Table 2. The ²²⁵Ac absorbed dose to small metastases is 9.6 Gy, much higher than that of 2.0 Gy from ²¹³Bi and 2.4 Gy from ⁹⁰Y. Similarly, the 25.3 Gy ²²⁵Ac dose to single tumor cells, is also higher than that of ²¹³Bi at 2.6 Gy and ⁹⁰Y at 0.6 Gy. When no antibody-receptor internalization or 100% internalization was considered, ²²⁵Ac dose to single cells became 12.0 Gy or 38.6 Gy, respectively.

Table 1.

Absorbed doses (Gy) from ²²⁵Ac (400 nCi) and its free daughters ²¹³Bi and ²²¹Fr

Organs	225Ac at Equilibriuma	Free 213Bib	Free 221Fr c	Total Alpha Dosed	Total Beta Dosed
Blood	2.71	-0.12	-0.25	2.29	0.05
Heart	1.07	0.00	0.00	1.04	0.02
Lung	0.43	-0.01	-0.01	0.39	0.01
Liver	8.25	-0.01	-0.05	8.00	0.19
Spleen	7.42	-0.01	0.00	7.24	0.17
Kidney	1.20	0.54	0.23	1.91	0.07
Stomach	0.17	0.00	0.04	0.20	0.00
Intestine	0.52	0.00	0.01	0.52	0.01
Femur	0.56	0.00	0.00	0.54	0.01
Tumor	10.49	0.00	0.00	10.24	0.25

^aIncludes doses from alphas and electrons of ²²⁵Ac and daughters in equilibrium.

^bIncludes doses from alphas and electrons of ²¹³Bi daughters, ²¹³Po, ²⁰⁹Ti and ²⁰⁹Pb.

^cIncludes alphas from ²²¹Fr daughter, ²¹⁷At.

^dTotal doses are presented as doses from alpha-particle and beta-particle emissions, respectively.

Table 2.

Absorbed doses to lung metastases or single tumor cells from ⁹⁰Y-, ²¹³Bi-, ²²⁵Ac-7.16.4

	Lung metastases (Gy) a	Single tumor cells (Gy) b
90Y-7.16.4	2.4	0.6
213Bi-7.16.4	2.0	2.6
225Ac-7.16.4	9.6	25.3
225Ac-7.16.4, 0% internalized	--	12.0
225Ac-7.16.4, 100% internalized	--	38.6

^aElectron doses are calculated with a sphere of 300 μm in diameter. Alpha doses are assumed to be deposited within the sphere. Cross-fire doses are not included.

^bAbsorbed dose to single cells are calculated with 59.2% internalization fraction and specific activities of 0.1, 10, 10μCi/μg for ²²⁵Ac, ²¹³Bi, ⁹⁰Y, respectively.

DISCUSSION

We demonstrated that alpha-particle-emitter ²²⁵Ac-labeled anti-rat HER-2/neu mAb is effective in eliminating breast cancer lung metastases, leading to long-term animal survival but also renal toxicity most likely caused by the free daughters of ²²⁵Ac (27).

The improved efficacy of alpha emitter ²²⁵Ac over ²¹³Bi and ⁹⁰Y can partially be attributed to the higher radiation doses that micrometastases receive from ²²⁵Ac. ²²⁵Ac, emits four alpha-particles along its decay chain, deposits a total energy of 4.50×10^-12 J/Bq-s, 3.2 and 30.0 times higher than that by ²¹³Bi and ⁹⁰Y. Furthermore, the majority of alpha radiation will be absorbed locally, while most of the beta-particle-energy from ⁹⁰Y will be deposited outside of micrometastases. An important finding from the biodistribution studies is that only a small fraction of ²²¹Fr and ²¹³Bi was released from major organs/tumors, suggesting that these daughters are either retained in the cells due to antibody internalization or there is simply not enough time for a substantial fraction of the daughters to diffuse out of the organs/tumors. The long half-life of ²²⁵Ac also helps to deliver more doses to the lung metastases than ²¹³Bi. The biodistribution studies showed that antibody localization to s.c. tumors peaked at least 24 hrs after injection. For lung metastases, antibody localization is much faster, reaching peak at about 6 hrs after injection. Nevertheless, for ²¹³Bi, a substantial fraction (>90%) of the decays will have already occurred, greatly reducing the number of alpha-particles that can be delivered.

The higher potency and longer half-life of ²²⁵Ac overcame the ~35% lower immunoreactivity of ²²⁵Ac- compared to ²¹³Bi- 7.16.4. The immunoreactivity of ²²⁵Ac-labeled mAbs seems to vary significantly among different antibodies (20), independent of the labeling procedure. When the incubation time in the second step of the reaction was reduced to 30 mins or 15 mins, the immunoreactivity of ²²⁵Ac-7.16.4 did not improve in the biodistribution study, with a 16.2%ID/g and 15.4%ID/g tumor localization at 72 hr after injection (3 mice/group). Furthermore, stability assay suggested slow but significant release of ²²⁵Ac from the antibody, which could also contribute to the lower tumor localization. More studies are needed to understand and improve the immunoreactivity and stability of ²²⁵Ac-7.16.4.

The main concern of radioimmunotherapy with alpha emitter ²²⁵Ac-7.16.4 is the long-term renal toxicity caused by free ²²¹Fr and ²¹³Bi (27). Furosemide or chlorothiazide and competitive metal blockade were found to be effective in reducing renal uptake of the free daughters (22). The extent to which such interventions are needed will likely depend upon the residence time of the antibody in circulation and the internalization properties of the antibody-antigen complex. In a recent update of results from a clinical trial of ²²⁵Ac-HuM195 antibody in leukemia patients, no acute toxicity was seen and no evidence of radiation nephritis has been seen, with follow-up to 10 months in patients receiving up to 4 μCi/kg (28). Liposomal, encapsulation of ²²⁵Ac (29) has also been proposed to trap free ²²⁵Ac daughters. Extracorporeal metal chelation of blood ²²¹Fr, ²¹³Bi could also be used to reduce renal toxicity (30). Interestingly, ²²⁵Ac labeled anti-thrombomodulin mAb (201B, targeting lung vasculature) caused lethal toxicity to the lungs while it was able to inhibit metastatic growth (31). No evident damage to lung tissue was observed when rat HER-2/neu was targeted in this study, demonstrating the importance of selecting tumor antigen for targeting to reduce toxicity.

The reduced efficacy when treating micrometastases at 18 day after cancer cell inoculation was expected and may be partially explained by increased tumor burden and increased tumor heterogeneity leading to non-uniform dose distributions (32). Monte Carlo modeling has shown that, when treating a micrometastases containing 1,000 cancer cells, the tumor control probability could drop from 93% to 0% when a 50% variance of antigen expression was introduced to a uniform expression (33). A recent clinical trial has shown that consolidation of chemotherapy regimen with ²¹³Bi-HuM195 in acute myeloid leukemia patients is highly effective (34). The efficacy data from day 18 and day 3 metastatic models suggest that ²²⁵Ac based radioimmunotherapy should be tested ideally in patients undergoing remission after first line chemotherapy treatments.

In conclusion, we have demonstrated that alpha-particle-emitter ²²⁵Ac-labeled mAb is very effective, better than the alpha emitter, ²¹³Bi, and the beta emitter, ⁹⁰Y, in prolonging the survival of neu-N transgenic mice bearing lung metastases. Long-term renal toxicity was observed and approaches designed to mitigate such toxicity (22) may need to be implemented in clinical studies.