mouse chimeric monoclonal antibody: a long-term survival study with histologic analysis

Int. J. Radiation Oncology Biol. Phys., Vol. 45, No. 2, pp. 491– 499, 1999 Copyright © 1999 Elsevier Science Inc. Printed in the USA. All rights reserved 0360-3016/99/$–see front matter

PII S0360-3016(99)00206-0

BIOLOGY CONTRIBUTION

RADIOTOXICITY OF SYSTEMICALLY ADMINISTERED 211At-LABELED HUMAN/MOUSE CHIMERIC MONOCLONAL ANTIBODY: A LONG-TERM SURVIVAL STUDY WITH HISTOLOGIC ANALYSIS

GAMAL

ROGER E. MCLENDON, M.D.,* GARY E. ARCHER, PH.D.,† ROY H. LARSEN, PH.D.,‡ AKABANI, PH.D.,‡ DARELL D. BIGNER, M.D., PH.D.,* AND MICHAEL R. ZALUTSKY, PH.D.*‡

Departments of *Pathology, †Surgery (Division of Neurosurgery), and ‡Radiology, Duke University Medical Center, Durham, NC Purpose: The antitenascin human/mouse chimeric monoclonal antibody labeled with the ␣-particle-emitting radionuclide 211At is of interest as an endoradiotherapeutic agent for the treatment of brain tumors. To facilitate the investigation of 211At-labeled chimeric 81C6 in patients, the long-term radiotoxicity of this radiopharmaceutical has been evaluated. Methods and Materials: Antibody labeling was performed using N-succinimidyl 3-[211At]astato-benzoate. After an initial dose-finding experiment, a second toxicity study was carried out at 4 dose levels in groups of 30 nonthyroid blocked B6C3F1 mice per group (15 males, 15 females). Male mice received either saline or 15– 81 kBq/g and females received either saline or 16 – 83 kBq/g of 211At-labeled antibody. Ten animals (5 males, 5 females) were followed for 6 months and the remainder for 1 year. Results: The lethal dose in 10% of animals (LD10) for 211At-labeled chimeric 81C6 was 46 kBq/g in females and 102 kBq/g in males. Toxic effects—perivascular fibrosis of the intraventricular septum of the heart, bone marrow suppression, splenic white pulp atrophy, and spermatic maturational delay— generally were confined to a few animals receiving the highest doses of labeled antibody. Conclusions: The LD10 of 211At-labeled chimeric 81C6 in this mouse strain was about half that of [211At]astatide. These results establish the preclinical maximum tolerated dose of 211At-labeled chimeric 81C6 and define in the mouse the target organs for toxicity. These studies will be useful for determining starting doses for clinical studies with 211At-labeled chimeric 81C6. © 1999 Elsevier Science Inc. Astatine-211, Radioimmunotherapy, Monoclonal antibodies, Glioma, Radiotoxicity.

␤-emitter 131I (5). A number of 211At-labeled compounds have been investigated in mouse and rat xenograft models and the results obtained have been highly encouraging (7, 10, 11). Taken together, this work has provided a compelling rationale for clinical investigations with 211At-labeled endoradiotherapeutic agents. One clinical application that has been predicted to be well suited for treatment with 211At-labeled compounds is cystic gliomas (12). Currently, we are investigating the efficacy of 131 I-labeled monoclonal antibodies (mAbs) administered directly into spontaneous cystic gliomas and into surgically created glioma resection cavities, with encouraging responses seen in some patients (13, 14). Most of these studies have utilized 81C6, a murine IgG2b mAb reactive with tenascin, an extracellular matrix glycoprotein that is significantly overexpressed on gliomas relative to normal tissues of the central nervous system (15). In anticipation of multidose radioimmunotherapy protocols, a human/mouse chi-

INTRODUCTION A variety of endoradiotherapeutic agents labeled with the ␣-emitting radionuclide 211At are being developed for use in cancer treatment (1–9). Astatine-211 has a longer half life, 7.2 h, than other ␣-emitters of therapeutic interest, and each decay of 211At yields an ␣-particle. The short tissue range (55–70 ␮m) and high linear energy transfer (mean LET, ⬃100 keV/␮m) of 211At ␣-particles offers the possibility of achieving highly efficient and focal irradiation of tumor cells while minimizing damage to neighboring normal tissue. Astatine-211 has been shown to be exquisitely cytotoxic when targeted to malignant cell populations in vitro. With a variety of molecular carriers, reduction in survival to 37% (D37) has been achieved with only 1 to 10 211At atoms bound per cell (4 – 6). Under single cell conditions, the cytotoxicity of 211At has been shown to be about 1000 times higher than the equivalent compound labeled with the Reprint requests to: Roger E. McLendon, M.D., Department of Pathology, DUMC 3712, Durham, NC 27710. Acknowledgments—Supported in part by NIH Grants CA42324 and NS20023, and DOE Grant 95ER62021.

Accepted for publication 14 May 1999.

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meric mAb consisting of the variable regions of murine 81C6 and the human IgG2 constant region domains was generated (16). More favorable tumor targeting characteristics were shown with chimeric 81C6 mAb compared with its murine parent (17). A number of studies have been performed evaluating chimeric 81C6 as a vehicle for targeting 211At to gliomas. The cytotoxicity and microdosimetry of 211At-labeled chimeric 81C6 have been evaluated in two human tumor microcolony models (4). Reduction in survival to 37% (D37) was achieved with 0.27 Gy, equivalent to 1.2 to 1.5 ␣-particle hits per cell. Tissue distribution studies performed in athymic mice bearing subcutaneous D-54 MG human glioma xenografts exhibited a high degree of tumor uptake and in vivo stability for 211At-labeled chimeric 81C6 (8). Furthermore, radiation-absorbed doses calculated based on these data support the subsequent evaluation of this agent in patients, particularly with direct intraparenchymal rather than systemic intravenous routes. However, preclinical evaluation of 211At-labeled compounds requires an understanding of the acute, subacute, and chronic systemic toxicities of each potential therapeutic agent. With this goal in mind, the current study was undertaken to investigate the histopathological lesions in male and female mice exposed to sublethal and lethal doses of intravenously administered 211At-labeled chimeric 81C6 mAb. METHODS AND MATERIALS Preparation of 211At-labeled chimeric 81C6 mAb The human/mouse antitenascin chimeric mAb, generated by genomic cloning, was constructed by combining the human IgG2 constant region domain genes with those encoding the variable region domains of murine 81C6. Details concerning the generation, characterization, and purification of chimeric 81C6 can be found in a previous publication (16). Astatine-211 was produced on the Duke University Medical Center cyclotron via the 209Bi(␣,2n)211At reaction by irradiating a natural bismuth internal target with 28 MeV ␣-particles (18). The 211At was distilled into a small volume of chloroform, and reacted with N-succinimidyl 3-(tri-nbutylstannyl)benzoate and t-butylhydroperoxide using a previously described procedure (19). The resultant acylation agent, N-succinimidyl 3-[211At]astatobenzoate, was reacted with chimeric 81C6 (2– 6 mg/ml) in pH 8.5 borate buffer. The labeled mAb was purified by elution through a Sephadex G-25 column and had a specific activity of 40 MBq/ mg. The immunoreactive fraction of 211At-labeled chimeric 81C6 was determined by incubation with antigen-positive D-54 MG human glioma and antigen-negative rat brain homogenate (19). The immunoreactive fractions of the preparations used in this study were 80 –90%. Animal experiments These studies were performed in male and female B6C3F1 mice aged 3 to 4 weeks weighing 20 –22 g (mean,

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21.1 g) and 18 –20 g (mean, 18.8 g), respectively. This mouse strain was selected because it is the standard strain used by the National Toxicology Program for carcinogenesis testing (20), thus allowing for both toxicity and shortterm carcinogenesis analysis. Animals were housed in separate cages in the Duke University Cancer Center Isolation Facility, and allowed free access to water and rodent chow. Thyroid blocking protocols were not utilized. Animals were randomized into groups using a random number generator. The variation in mean weights among the groups was less than 5% in each experiment. Astatine-211 activity levels were determined in a Capintec CRC-7 dose calibrator that had previously been calibrated for 211At counting by comparison to an efficiency-calibrated germanium semiconductor detector. Groups of mice were injected in the lateral tail vein with either 211At-labeled chimeric 81C6 in phosphatebuffered saline (PBS) or as a control, PBS alone. A t- test assuming equal variances was used to analyze weight gain, and probit analysis was employed to assess lethal toxicity (21). Values of the lethal dose to 10% of animals (LD10) and 95% confidence intervals were determined by a regression fit of the data using the Sigma Plot computer program (Jandel Scientific, San Rafael, CA). Protocols were designed based on discussions with the Food and Drug Administration in order to obtain the data necessary to obtain an Investigational New Drug permit for 211 At-labeled chimeric 81C6. In the first experiment, groups of 8 –10 male B6C3F1 mice were injected with a single dose of 14 kBq/g, 29 kBq/g, 63 kBq/g, or 110 kBq/g of 211Atlabeled chimeric 81C6 or PBS, and the animals were followed over a 60-day observation period. The second toxicity study was carried out at four dose levels in groups of 30 animals per group, each containing 15 males and 15 females. Male groups received saline, 15 kBq/g, 16 kBq/g, 51 kBq/g, or 81 kBq/g of 211At-labeled chimeric 81C6, and females received saline or 16 kBq/g, 17 kBq/g, 55 kBq/g, or 83 kBq/g of labeled mAb. Five mice from each group were randomly selected to be killed at 6 months and the remaining 10 mice were followed for 1 year. The weight of each animal was checked daily and all animals were monitored twice a day for their general health and physical status. Upon completion of the observation period, all surviving animals were euthanized by halothane overdose and autopsied for histopathological examination. Histology All animals, whether dying spontaneously or euthanized, were autopsied within 12 hours of death. Internal organs were weighed and fixed by immersion in 10% buffered formalin, with the exception of the intestines which also were perfusion fixed through the lumen. Representative tissues of the formalin-fixed abdominal and thoracic viscera were submitted for histologic sectioning. Organ samples routinely included the liver, spleen, lungs, heart, small and large intestines, stomach, testes or ovaries, and sternal bone marrow; additional representative sections were submitted from all gross abnormalities. The neck block was dissected,

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Fig. 1. Mean group weight curve (a) and survival curve (b) for B6C3F1 mice exposed to single doses of 14, 29, 63, and 110 kBq/g of intravenously administered 211At-labeled chimeric 81C6 monoclonal antibody; 8 –10 animals per group.

cut into three horizontal slices, and submitted in toto for examination of thyroid. Tissue samples were embedded in paraffin, sectioned at 6 ␮m, and stained with hematoxylin and eosin (H&E). Sections were then evaluated by light microscopy for evidence of histologic abnormalities with the pathologist blinded as to the treatment groups. RESULTS Weight gain and survival data for the initial dose escalation study are illustrated in Figs. 1a and 1b, respectively. All of the animals in the 110 kBq/g group exhibited precipitous weight loss and died between 5 and 8 days after receiving the 211At-labeled chimeric 81C6 mAb. All of the animals in the other treatment groups as well as the PBS controls survived through the 60-day observation period. Marrow elements were ablated in all eight animals in the 110 kBq/g group, consistent with marrow toxicity as the cause of death in these animals. Similarly, atrophy of the white pulp of the spleen was seen in six out of eight animals in this high-dose group. In this group, dysplastic squamous epithelial changes in the epidermoid forestomach were noted in four out of eight animals. Testicular atrophy was confined to the animals exposed to 74 kBq/g and living for greater than 5 days. In the second experiment, 5 males and 5 females were followed for a period of 6 months and the remaining 10 animals of each gender were followed for 1 year. Mouse weight gain results for the 6-month and 1-year groups are presented in Figs. 2a and 2b, respectively. The only death observed in the 6-month groups was 1 male who died 10 days after receiving 81 kBq/g of 211At-labeled chimeric 81C6 antibody. Survival data from this experiment for the 1-year female and male groups are presented in Figs. 3a and 3b, respectively. Perivascular fibrosis of the interventricular septum of the heart was potentially the most significant histologic finding in long-term survivors with regard to clinical implications. This abnormality was observed among animals in the 1-year

survival group exposed to 17 kBq/g to 83 kBq/g of 211Atlabeled chimeric 81C6 but in none of the animals in the 6-month groups or in the preliminary 6-week duration dose finding study. Females seemed to be slightly more sensitive than males, with perivascular fibrosis evident in 3/10 (83 kBq/g) and 4/10 (55 kBq/g) females, while being identified in only 1/10 (81 kBq/g), 1/10 (51 kBq/g), and 1/9 (16.2 kBq/g) male animals. Thyroid atrophy was not encountered as all specimens exhibited size and histologic features found also in animals receiving saline. Lesions noted in the thyroid consisted of mild follicular atrophy and an increase of interstitial inflammatory cells. These features were noted at 6 months (Fig. 4) but were not evident at 12 months (Figs. 5 and 6). Neoplastic changes, predominately papillary carcinomas, were found in the thyroid of 1 female (1-year survivor, 83 kBq/g) (Fig. 7), the kidney of 1 male (1-year survivor, 14.9 kBq/g), the lungs of 1 female (1-year survivor, 16.9 kBq/g) and of 2 males (1-year survivor, 16.2 kBq/g; 1-year survivor, 14.9 kBq/g), and the gynecological tract in 1 female (1-year survivor, 55 kBq/g). Because all of these organs may exhibit papillary cancers and the cancers were confined to those organs, we did not attribute these neoplasms to a metastasis from a subtle, or regressed, thyroid source. Testicular atrophy was found in 1 male (a death in the 1-year survival group, 81 kBq/g), a rate consistent with our previous evaluation of the radiotoxicity of [211At]astatide (22) which indicated that testicular atrophy was confined to the animals exposed to 74 kBq/g and living for greater than 5 days. Atrophy of the white pulp of the spleen was seen in only rare animals in the 80 kBq/g dose groups of both sexes and in both long-term survival studies and was present in 6/8 animals receiving 110 kBq/g in the initial dose escalation study. Marrow toxicity was the cause of death in the one male within the 6-month 81 kBq/g exposure group and of all 8 animals in the 110 kBq/g dose group in the initial dose escalation study. The cause of death in the four males within

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Fig. 2. Mean group weight curves for female (a) and male (b) mice followed for 6 months, and female (c) and male (d) mice followed for 1 year, after exposure to single doses of intravenously administered 211At-labeled chimeric 81C6 monoclonal antibody; 5 males and 5 females in 6-month groups, and 10 males and 10 females in 1-year groups.

the 81 kBq/g 1-year survival study group and the five females in the 83 kBq/g 1-year survival study group was not obvious. One male exhibited mild marrow hypoplasia and one male exhibited gonadal atrophy indicating chronic 211At effects. All four early male deaths in the 81 kBq/g 1-year survival group exhibited focal hepatic necrosis suggestive of agonal effects, though the contribution of 211At to exacerbating the effects of agonal hypoxemia is not known. Among the females, hepatic necrosis was only seen in 2/5 deaths indicating that it alone was not the sole cause of death in these animals. Changes were noted in the sternal bone sample of female mice with dose-related effects seen in the microscopic assessment of trabecular bone density and osteoblastic activity that were not evident in males (Table 1, Figure 8). While the effect does not appear to be chronic, with little effect on bone density noted at 6 or 12 months, a long-term effect on bony remodeling is evident at all dose levels, suggestive of altered calcium metabolism, a possible link to altered para-

thyroid function. Another sexual difference was the presence of cytologic atypia in the bladder. Cytologic atypia, characterized by transitional epithelial cells with nuclear pleomorphism and cytomegaly, a finding common to radiation-exposed tissues, was noted in the bladder epithelium of 2/5 females (1-year survivors, 83 kBq/g group), but not in any males. Squamous epithelial changes in the epidermoid forstomach were not evident in these long-term studies in contrast to the previous study with [211At]astatide (22) and to the initial dose escalation experiment in which this change was noted in 4/8 animals in the 110 kBq/g group. The preservation of the squamous cytology is consistent with the lower doses administered in the present study. Furthermore, gastric glandular atrophy was not found in any animal in any of the three studies. Benign lymphocytic infiltrates were again localized in the kidneys in a minority of animals (both sexes) at all dose levels. Among the females, lymphocytic infiltrates were

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Fig. 3. Survival curves for female (a) and male (b) mice followed for 1 year after exposure to single doses of intravenously administered 211At-labeled chimeric 81C6 monoclonal antibody; 10 males and 10 females in 1-year groups.

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Fig. 4. At 6 months, the thyroid of a male receiving 81 kBq/g exhibits moderate variation in follicular size and shape. The nuclei are mildly enlarged and exhibit punctate nucleoli. An inflammatory infiltrate is slightly increased in the interstitium. There is no increase in fibrous connective tissue evident (H&E, ⫻250).

also localized in the gynecological tract of a minority of all animals in all dose levels. DISCUSSION Oncophilic compounds labeled with 211At have exhibited high cytotoxicity for human cancer cells in vitro and promising therapeutic efficacy in rodent tumor models (1–11). Translation of these potential endoradiotherapeutic agents into the clinical domain has been hindered by a number of problems including the limited information available concerning the toxicities of these compounds to normal tissues. This is reflected by the fact that the clinical literature for

Fig. 5. At 12 months, the thyroid of a male receiving 51 kBq/g exhibits mild variation in follicular size with abundant colloid evident. The nuclei now exhibit a quiescent appearance with only a slight irregular mononuclear inflammatory infiltrate evident in the interstitum. Again, no increase in fibrous connective tissue is evident (H&E, ⫻250).

Fig. 6. Same as Fig. 5 except at lower power (H&E, ⫻52). The entire thyroid has a normal size and shape with mild variation in follicular size and no increase in fibrous connective tissue. A parathyroid gland is evident (arrow).

Fig. 7. Thyroid from 1-year female survivor receiving 83 kBq/g (H&E, ⫻400). Mitotically active epithelial cells exhibiting high nuclear to cytoplasmic ratios and arranged in poorly formed papillae and glands in the growth pattern of papillary adenocarcinomas were the most common forms of cancer encountered in these animals. However, no group of animals exhibited a common organ of involvement and no group of animals exhibited a higher rate of malignancy than found in historical control averages for the species.

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Table 1. Skeletal system lesions in female mice Osteoporosis Dose (kBq/g) 83 55 17 16 saline

6 months 2/5 2/5 1/5 1/5 0/5

1 year 3/10 0/10 0/10 0/6 0/10

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Osteoblastic hyperplasia 6 months 4/5 0/5 2/5 3/5 3/5

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At-labeled compounds consists of a single case report involving direct injection of 211At-labeled microspheres into a lingual carcinoma (23). With 211At, by far the most information is available concerning the subacute, acute, and chronic toxicity of the free halide, [211At]astatide (24 –26). The LD10 at 360 days in B6C3F1 mice, the strain used in the current study, was found to be 15.1 ␮Ci, equivalent to 28 kBq per gram body weight (22). The toxic effects observed with [211At]astatide were consistent with the tissue distribution of this radiohalide: selective accumulation in stomach, spleen, thyroid, and lungs (27). A number of observations have been reported concerning the radiotoxicity of low-molecular-weight compounds labeled with 211At. Link and coworkers (9) have reported that the maximum tolerated dose of 211At-labeled methylene blue in the mouse during a 3.5-month duration therapy experiment was about five times higher than that reported by Cobb for [211At]astatide (24). With the napthoquinol diphosphate 6-[211At]astato-MNDP, the LD50 at 360 days in cured, tumor-bearing C57B1/10 mice was 14.6 kBq/g body weight (1). The extent to which the tissue distribution accounted for the toxicity of these reagents cannot be discerned from these studies. In the current study, the LD10 for 211At-labeled chimeric 81C6 mAb in B6C3F1 mice at 360 days was 46 kBq/g in

Fig. 8. Sternal bone samples exhibited partially mineralized osteoid that was growing in a densely meshed arrangement indicative of osteoid hyperplasia in this female animal receiving 55 kBq/g and surviving 52 weeks (H&E, ⫻130).

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1 year 0/10 8/10 9/10 5/6 1/10

211

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At-labeled chimeric 816C*

[211At]astatide†

Liver Spleen Lungs Heart Kidneys Small intestine Stomach Blood

0.49 0.04 0.25 0.09 0.19 0.25 0.13 2.49

0.17 0.05 0.12 0.02 0.10 0.31 0.67 0.17

* Based on tissue distribution data in Reference 28. † Based on tissue distribution data in Reference 27.

females and 102 kBq/g in males, values about two- and fourfold higher than the LD10 in this mouse strain with [211At]astatide (22). This observation seems counterintuitive given the relatively slow clearance of intact mAbs from most normal tissues following intravenous administration. Using previously published tissue distribution data obtained in athymic mice (27, 28), we have calculated the residence time (cumulative activity concentration in organ divided by the total activity administered) for both 211At-labeled chimeric 81C6 and [211At]astatide. As summarized in Table 2, the residence time for 211At-labeled mAb was greater than [211At]astatide in liver, lungs, heart, kidneys, and blood, comparable in spleen, and less in both the stomach and small intestine. The difference in residence time for the two labeled compounds is consistent with the fact that gastric changes were not found with labeled mAb but were observed in previous long-term evaluations of the radiotoxicity of free [211At]astatide (22). The shorter gastric residence time for labeled mAb versus [211At]astatide at similar doses correlates with the absence of gastric changes found in this study. On the other hand, the lower toxicity of 211At when coupled to mAb would not be predicted based on the longer residence time for labeled mAb in most normal tissues, particularly blood. Because of the short range of ␣-particles in tissue, the homogeneity of radiation dose deposition could be a critical factor. The diffusion coefficient in tissue for an intact mAb such as 211At-labeled chimeric 81C6 has been calculated to be 6.3 ⫻ 10⫺9 cm/sec, about 1000 times higher than that for a small molecule such as [211At]astatide (29). Given the 7.2 hr half life of 211At, this slower diffusion would suggest that the toxic effects of 211At-labeled mAb might be confined to regions within a few ␮m of vascular structures. Blood pool is generally considered to be a source organ for the calculation of marrow dosimetry (30, 31). The threshold for marrow toxicity in the current study was higher than that seen previously with [211At]astatide even though the residence time in blood for the labeled mAb was more than an order of magnitude higher. However, because of the short range of ␣-particles in tissue, the distribution of

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decay sites on a cellular level becomes important in determining marrow dosimetry (32). Indeed, a better understanding of the toxic effects of 211At-labeled compounds apparently will require a knowledge of their distribution within an organ of interest at the histologic or cellular level, with the calculation of pertinent microdosimetric parameters (32, 33). The significant differences in LD10 between the sexes is enigmatic. The only histologic differences noted between males and females related to osteoporosis at 6 months in a minority of females and foci of increased osteoblastic activity in the sternal bone samples in a majority of females at 12 months (Table 1). The effects on calcium metabolism are unknown as serum calcium measurements were not performed. The results indicate altered bone turnover in females but cannot relate the effect to a specific cause. Possible explanations include an effect of the mAb, of 211At, or 211 At-labeled chimeric 81C6 on osteoclasts, osteoblasts, parathyroid, or other tissues involved in calcium homeostasis or bone modeling. Regardless, these results indicate that further studies are needed to elucidate the mechanisms involved in increased mortality and to bone modeling in female mice. Tumor induction in rats has been documented in some long-term investigations with 211At-labeled compounds. A high incidence of mammary tumors and pituitary adenomas has been observed in rats exposed to 19 kBq/g [211At]astatide and followed for 1 year (34). A subsequent study performed in rats at a dose of 11 kBq/g [211At]astatide reported a similar incidence of pituitary and mammary tumors (12/13 were fibroadenomas) with most appearing 14 –26 months later (35). Because both series were performed at thyroid-ablative doses of [211At]astatide, the authors of both studies point out that it is difficult to discern the extent to which tumor induction was a direct effect of radiation. The tumorogenicity of 211At has also been investigated in mice including the B6C3F1 strain used in the current study. Brown and Mitchell (1) have reported a 13% incidence of plasmacytoma in C57B1/10 tumor-bearing mice 13 to 21 months after treatment with 200 –750 kBq of 6-[211At]astato-MNDP. In our previous 1-year follow-up study evaluating the radiotoxicity of [211At]astatide in B6C3F1 mice, only three neoplastic lesions were noted: one hepatocellular carcinoma (12 ␮Ci; 22 kBq/g) and two renal tubular ade-

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nomas (18 ␮Ci; 33 kBq/g) (22). The incidence of hepatic lesions was within historical control ranges (20) but that for renal lesions was not (36). Neoplastic lesions were not observed in BALB/c (nu/nu) mice receiving similar doses of [211At]astatide (22). In this study, papillary carcinomas were found in several animals surviving for the 1-year observation period. These tumors were located in the thyroid (1 female; 83 kBq/g), kidney (1 male; 15 kBq/g), lungs (1 female; 17 kBq/g; 1 male; 16 kBq/g; 1 male; 15 kBq/g), and the gynecological tract (1 female; 55 kBq/g). Neoplastic lesions were not significantly elevated over historical averages (36). Previous studies utilizing unconjugated astatide highlighted perivascular fibrosis in the heart as being a histologic finding of potential clinical significance in long-term survivors (those living greater than 55 days) among those animals exposed to 18 to 37 kBq/g. In the previous study utilizing [211At]astatide, perivascular fibrosis affected 10/10 animals in the 18 kBq/g dose group and 8/10 animals in the 37 kBq/g dose group. The conclusion of that study was that time following exposure was a significant factor in the onset of these histologic changes. The present study further supports this conclusion by identifying this abnormality among animals in the 1-year survival group exposed to 17 kBq/g to 83 kBq/g but in none of the animals in the 6-month or 6-week studies. CONCLUSION The LD10 determined in B6C3F1 mice for intravenously administered 211At-labeled chimeric 81C6 mAb was 46 kBq/g body weight in females and 102 kBq/g in males. Gender-related toxicity for 211At-labeled compounds has not been reported previously and its cause is not known at this time. Nonetheless, even in females, the LD10 for the 211 At-labeled mAb was twice that observed in this mouse strain for [211At]astatide. Use of direct intratumoral, intracystic, intrathecal, or other compartmental injection routes should increase the opportunity for achieving therapeutic efficacy without significant normal tissue toxicity or systemic exposure. A Phase I trial, in which 211At-labeled chimeric 81C6 is injected directly into the surgically created resection cavity, has been initiated to investigate the feasibility of this strategy for the treatment of patients with recurrent glioma.

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1. Brown Mitchell JS. The development of a [ At]-astatinated endoradiotherapeutic drug: Part IV—Late radiation effects. Int J Radiat Oncol Biol Phys 1998;40:1177–1183. 2. Bloomer WD, McLaughlin WH, Lambrecht RM, et al. 211At Radiocolloid therapy: Further observations and comparison with radiocolloids of 32P, 165Dy, and 90Y. Int J Radiat Oncol Biol 1984;10:341–348. 3. Larsen RH, Hoff P, Vergote IB, Bruland ØS, et al. ␣-Particle radiotherapy with 211At-labeled monodisperse polymer particles, 211At-labeled IgG proteins, and free 211At in a murine intraperitoneal tumor model. Gyn Oncol 1995;57:9 –15.

4. Larsen RH, Akabani G, Welsh P, et al. The cytotoxicity and microdosimetry of 211At-labeled chimeric monoclonal antibodies on human glioma and melanoma cells in vitro. Radiat Res 1998;149:155–162. 5. Strickland DK, Vaidyanathan G, Zalutsky MR. Cytotoxicity of alpha-particle-emitting m-[211At]astatobenzylguanidine on human neuroblastoma cells. Cancer Res 1994;54:5414 –5419. 6. Vaidyanathan G, Larsen RH, Zalutsky MR. 5-[211At]astato2⬘-deoxyuridine, an ␣-particle emitting endoradiotherapeutic agent undergoing DNA incorporation. Cancer Res 1996;56: 1204 –1209.

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