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Clinical Cancer Research 14, 875, February 1, 2008. doi: 10.1158/1078-0432.CCR-07-1250
© 2008 American Association for Cancer Research
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Cancer Therapy: Preclinical

Effective Treatment of Established Human Breast Tumor Xenografts in Immunodeficient Mice with a Single Dose of the {alpha}-Emitting Radioisotope Astatine-211 Conjugated to Anti-HER2/neu Diabodies

Matthew K. Robinson1, Calvin Shaller1, Kayhan Garmestani2, Paul S. Plascjak2, Kathryn M. Hodge1, Qing-An Yuan1, James D. Marks3, Thomas A. Waldmann2, Martin W. Brechbiel2 and Gregory P. Adams1

Authors' Affiliations: 1 Department of Medical Oncology, Fox Chase Cancer Center, Philadelphia, Pennsylvania; 2 National Cancer Institute, NIH, Bethesda, Maryland; and 3 Department of Anesthesiology and Pharmaceutical Chemistry, University of California at San Francisco, San Francisco, California

Requests for reprints: Gregory P. Adams, Department of Medical Oncology, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111. Phone: 215-728-3890; Fax: 215-728-2741; E-mail: gp_adams{at}fccc.edu.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
Purpose: Successful radioimmunotherapy strategies depend on selecting radioisotopes with physical properties complementary to the biological properties of the targeting vehicle. Small, engineered antitumor antibody fragments are capable of rapid, highly specific tumor targeting in immunodeficient mouse models. We hypothesized that the C6.5 diabody, a noncovalent anti-HER2 single-chain Fv dimer, would be an ideal radioisotope carrier for the radioimmunotherapy of established tumors using the short-lived {alpha}-emitting radioisotope 211At.

Experimental Design: Immunodeficient nude mice bearing established HER2/neu–positive MDA-MB-361/DYT2 tumors treated with N-succinimidyl N-(4-[211At]astatophenethyl)succinamate (211At-SAPS)-C6.5 diabody. Additional cohorts of mice were treated with 211At-SAPS T84.66 diabody targeting the carcinoembryonic antigen or 211At-SAPS on a diabody specific for the Müllerian inhibiting substance type II receptor, which is minimally expressed on this tumor cell line.

Results: A single i.v. injection of 211At-SAPS C6.5 diabody led to a 30-day delay in tumor growth when a 20 µCi dose was administered and a 57-day delay in tumor growth (60% tumor-free after 1 year) when a 45 µCi dose was used. Treatment of mice bearing the same tumors with 211At-SAPS T84.66 diabody at the same doses led to a delay in tumor growth, but no complete responses, likely due to substantially lower expression of this antigen on the MDA-MB-361/DYT2 tumors. In contrast, a dose of 20 µCi of 211At-SAPS on the anti–Müllerian-inhibiting substance type II receptor diabody did not affect tumor growth rate, demonstrating specificity of the therapeutic effect.

Conclusions: These findings indicate that diabody molecules can be effective agents for targeted radioimmunotherapy of solid tumors using powerful, short-lived {alpha}-emitting radioisotopes.

Radioimmunotherapy exploits the highly specific antigen specificity of antitumor antibody molecules to selectively target and retain therapeutic radioisotopes in tumors. Radioimmunotherapy has been associated with significant clinical therapeutic outcomes in the treatment of hematologic (diffuse) malignant diseases, which has led to the approval of two anti-CD20 monoclonal antibodies (mAb) for radioimmunotherapy applications (1). However, similar clinical successes have yet to be achieved in the treatment of solid, established malignancies. This is thought to be due to a number of factors related to the size of the antibodies and the properties of commonly used therapeutic radioisotopes (2, 3). These include limited tumor penetration and prolonged circulation of intact mAb, which together impair the ability to treat a tumor and increases the bone marrow toxicity; the requirement for thousands of traversals of β-particles through a single tumor cell to mediate its death; and the disparate biological half-lives of intact mAbs and physical half-lives of the commonly used β-emitting radioisotopes such as iodine-131 (131I) and yttrium-90 (90Y). To address these issues, we have focused on developing smaller antibody-based molecules that are capable of greater tumor-targeting specificity and pairing them based on their biological half-lives with high-energy therapeutic radioisotopes that have complementary physical half-lives.

Noncovalent single-chain Fv (scFv) dimers, known as diabodies, can be formed by producing scFv molecules with short (5 aa) linkers between their variable light (VL) and variable heavy (VH) chains (4). This prevents the VH and VL chains from a single molecule from associating with each other to form a functional scFv. Consequently, the VH from one molecule associates with the VL from a second molecule, and vice versa, to form a divalent protein capable of binding to two antigen molecules. We, and others, have previously reported that these Mr 55,000 diabody molecules exhibit a unique combination of highly specific, durable tumor localization and relatively rapid elimination from normal tissues (5–7).

We have produced the human C6.5 diabody, which is specific for the extracellular domain of HER2/neu and exhibits a 40-fold increase in affinity over that observed with C6.5 scFv (5). The C6.5 diabody displays an exceptional combination of quantitative and selective tumor targeting in scid mice. At 24 h postinjection, 6% injected dose per gram (%ID/g) of radio-iodinated C6.5 diabody was retained in SK-OV-3 tumor xenografts in mice and tumor to blood ratios of 10:1 were observed. Diabodies thus represent an improved strategy for selective tumor targeting compared with scFv, Fab, or IgG molecules. Furthermore, as decreasing the size of the molecule increases both its diffusion rate into tumor (8) and its rate of elimination from circulation, the degree of penetration and the specificity of retention in the tumor are enhanced.

We have previously reported the observation that effective radioimmunotherapy of established s.c. human tumor xenografts growing in immunodeficient mice can be accomplished using a radioimmunoconjugate of 90Y and the C6.5 diabody (9). However, in that study, the relatively low linear energy transfer (LET) associated with the β-emission of necessitated doses of 90Y equal to the LD10 to achieve significant tumor growth delays and doses equivalent to the LD20 before two of the eight treated mice (25%) exhibited durable complete responses. Radioimmunotherapy with 131I-conjugated C6.5 diabody was also suboptimal in the same mouse model.4 The β-emissions from 90Y and 131I, the other commonly used radioimmunotherapy radioisotope, have a LET of 0.2 keV/µm (10). An attractive alternative approach is to incorporate {alpha}-emitting radioisotopes with significantly higher LET emissions (reviewed in refs. 10, 11) into diabody-based radioimmunotherapy strategies. We initially assessed the therapeutic potential of the {alpha}-emitting radioisotope bismuth-213 (213Bi) conjugated to the C6.5 diabody and found that the radioisotope physical half-life of 45.6 min was too short to allow systemically administered diabody to specifically localize in an established solid tumor (12). In the current study, we evaluate the utility of pairing astatine-211 (211At), an {alpha}-emitting radioisotope (T1/2 = 7.2 h, LET 97-99 keV/µm), with the C6.5 diabody. The longer physical half-life of 211At is complimentary to the relatively rapid tumor targeting and systemic clearance of the C6.5 diabody.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 Conclusion
 References
 
Antibodies and cell lines
The anti-HER2 C6.5 diabody and the GM17 diabody that is specific for the human Müllerian inhibiting substance type II receptor (MISIIR) were each expressed from Escherichia coli and purified as previously described (5, 13). The anti–carcinoembryonic antigen (CEA) T84.66 diabody was a kind gift of Dr. Anna Wu (Beckman Research Institute, City of Hope and University of California, Los Angeles, CA; ref. 14). The MDA-MB361/DYT2 cell line was a kind gift of Dr. Dajun Yang (Georgetown University, Washington, DC). Cells were expanded in culture as described (9). In vitro expression of HER2, CEA, and MISIIR were determined by flow cytometry as previously described (9) using the anti-HER2 mAb 9G6.10 (NeoMarkers), the anti-CEA mAb CB30 (Cell Signaling Technology), and the anti-MISIIR mAb 12G4 (a kind gift of Dr. Isabelle Teulon of the Centre de Recherche en Cancérologie de Montpellier, Montpellier, France) as primary antibodies and a fluorochrome-conjugated goat anti-mouse mAb (ICN Immunobiologicals) as a secondary antibody. The degree of fluorescence was determined using a FACScan flow cytometer (Becton Dickinson) and was analyzed using the CELLQuest software (Becton Dickinson; data not shown). In these studies, the MDA-MB361/DYT2 cells exhibited a {Delta}MFI of 38 when analyzed the 9G6.10 mAb (NeoMarkers). This corresponds well with previous reports that these cells express 3.8 x 105 copies of HER2 per cell (15). MDA-MB361/DYT2 cells exhibited a low level of both CEA ({Delta}MFI = 2.9) and MISIIR ({Delta}MFI = 2.7) when analyzed by flow cytometry.

Radiolabelings
Succinimidyl N-{4-[125I]iodophenethyl}succinamate and N-{4-[211At]astatophenethyl}succinamate preparation. Iodine-125 was purchased from Perkin-Elmer. Astatine-211 was produced as described at the NIH (16). Succinimidyl N-{4-[125I]iodophenethyl}succinamate (SIPS) and succinimidyl N-{4-[211At]astatophenethyl}succinamate (SAPS) were prepared as previously described (17). We have previously shown that this radioisotope conjugate is extremely stable in both in vitro and in vivo settings (17).

In brief, SAPS was prepared by mixing 211At (5-10 mCi in 70 µL methanol) with its tributylstannyl precursor as previously reported (17). Labeling with 211At was done by mixing 5 µL of 2 mg/mL N-chlorosuccinimide in methanol with 20 µL of 25 mg/mL of the tributylstannyl precursor in methanol containing 1% acetic acid. After 30-min incubation at room temperature, the reaction mixture was air dried and the residue was dissolved in 80 µL of ethyl acetate/methanol 50:50 v/v. The 211At-labeled active ester was isolated by normal phase high-performance liquid chromatography using the following gradient: 90% hexane (10% ethyl acetate) for 1 min and followed by a 9-min linear gradient to 100% ethyl acetate; a retention time of 8.4 for SAPS was routine. The flow rate for all elutions was 3 mL/min. The high-performance liquid chromatography fractions containing the product were combined, dried in an airstream, and used in the next step. Typical labeling efficiencies were 45 ± 5%. SIPS was also prepared as previously reported (16). In brief, 20 µL of 25 mg/mL of tributylstannyl precursor in methanol, 100 µL of CH3COOH/CH3OH (9/91 v/v), and 5 µL of 2 mg/mL N-chlorosuccinimide in methanol were added to 125I (500 µCi). After 30 min at room temperature, the reaction mixture was dried under an air stream, redissolved in 80 µL of ethyl acetate/methanol 50:50 v/v, and purified by normal phase high-performance liquid chromatography, using the same elution protocol described above for purification of SAPS; Rt for SIPS was 8.40 min routinely. Typical labeling efficiencies were 65 ± 5%.

Conjugation to diabodies. 125I-SIPS was conjugated to the C6.5 and T84.66 diabodies and 211At-SAPS was conjugated to the C6.5, T84.66, and GM17 anti-MISIIR diabodies using methods similar to those previously reported (18).

Briefly, 2.0 mL of absolute methanol were added to the 125I-SIPS (0.26 mCi in 1.0 mL methylene chloride) or 211At-SAPS (1.4-2.0 mCi in 1.0 mL methylene chloride) and the mixture was dried completely under a gentle stream of nitrogen. The diabody was concentrated and diluted in 0.5 mol/L borate buffer to a final concentration of ~1 mg/mL for the C6.5 and GM17 diabodies and 5.0 mg/mL for the T84.66 diabody, and added directly to the tube containing the dried 125I-SIPS or 211At-SAPS. Each diabody was added at a ratio of 5 mg diabody to 1 mCi of 211At. After a 15-min incubation at room temperature, 2.0 mL of PBS (pH 7.2) were added to the reaction vial and the reaction was chromatographed over a PD10 column (Bio-Rad) that was equilibrated with PBS (pH 7.2). The column was eluted with an additional 5.0 mL of PBS and 0.5 mL fractions were collected. Each fraction was assayed in a dose calibrator for radioactivity and the fractions containing the radiolabeled diabody were pooled for quality control.

Radiolabeled diabodies were assayed for radiochemical purity and function in instant TLC assays and live cell binding studies as previously described (9). In the instant TLC assay, 1 µL from each the reaction mixture and the final product were applied to silica instant TLC strips (Biodex Medical Systems) and allowed to migrate using normal saline as a mobile phase. The strips were cut at the midpoint and the two halves were counted in a {gamma} well counter (Cobra Quantum, Packard Instruments). Each radiolabeled diabody exhibited a radiochemical purity of >94%. The immunoreactivity of the radiopharmaceutical was determined in a live cell binding assay using SK-OV-3 cells (~1 x 106 copies HER2/neu per cell) as is standard in our laboratory (19). The high antigen density provides the required condition of antigen excess with limited numbers of cells. Briefly, 10 ng of labeled diabody in 100 µL of medium were added in triplicate into 15 mL polypropylene centrifuge tubes containing 3 x 106 SK-OV-3 cells. The cells were allowed to incubate for 30 min at room temperature. One milliliter of PBS, at 4°C, was added to each tube and they were centrifuged for 5 min at 500 x g at 4°C. Supernatants were separated from the cell pellets, both were transferred to 12 x 75 mm counting tubes, and the percentage of radioactivity associated with the cell pellet was determined by counting in a {gamma} counter. Live cell-binding assays routinely revealed that the 125I-SIPS C6.5 diabody and 211At-SAPS C6.5 diabody each exhibited an immunoreactivity >40% on SK-OV-3 tumor cells.

Biodistribution studies
Four- to 6-week-old inbred C.B17/Icr-scid mice were obtained from the Fox Chase Cancer Center Laboratory Animal Facility. MDA-361/DYT2 (5 x 106 in 0.1 mL PBS) were injected s.c. into the abdomen of each mouse. Approximately 3 weeks after the implantation, tumors had achieved a size of ~100 mm3 and the distribution studies were initiated.

Twenty micrograms of 125I-SIPS C6.5 diabody or 125I-SIPS T84.66 diabody (0.2 µCi/µg) were administered to each mouse by tail vein injection. Total injected doses were determined by counting the mice on a Series 30 multichannel analyzer/probe system (probe model 2007, Canberra). Blood sample collection and whole-body counts of the mice were done immediately after injection and just before euthanasia. Groups of five or six mice were euthanized at 4 and 48 h after injection; tumor, organ, and blood retentions were determined as previously described (5, 19). The mean and SE for each group of data were calculated, and tumor to organ ratios were determined.

Radioimmunotherapy studies
Male BALB/c nude mice were obtained at 8 to 12 weeks old from Taconic Labs. Human tumor xenografts were established by implanting 5 x 106 MDA-MB361/DYT2 tumor cells s.c. on the abdomen as previously described (19). Tumor volumes were determined using the following ellipsoidal formula: length (mm) x width (mm) x height (mm) x 0.52 (derived from {pi}/6; ref. 20). After ~3 weeks, the tumors were well established and the therapy studies were initiated. Cohorts of five to seven mice bearing established s.c. tumors were treated with 20, 30, or 45 µCi of 211At-SAPS C6.5 diabody or 211At-SAPS T84.66 diabody or 20 µCi of 211At-SAPS anti-MISIIR GM17 diabody at a dose of ~1 mCi/mg diabody or left untreated. The mean tumor sizes in the 211At-SAPS C6.5 diabody study at the time of treatment were 344 ± 8, 400 ± 78, 221 ± 22, and 423 ± 50 mm3, respectively, for the 20 µCi, 30 µCi, 45 µCi, and control treatment groups. The mean tumor sizes in the 211At-SAPS T84.66 diabody study at the time of treatment were 526 ± 100, 427 ± 60, 358 ± 57, and 504 ± 66 mm3, respectively, for the 20 µCi, 30 µCi, 45 µCi, and control treatment groups. The mean tumor sizes in the anti-MISIIR GM17 diabody study were 534 ± 122 and 467 ± 52 mm3, respectively, for the treated and control groups. Following treatment, the mice were observed and weighed and their tumors were measured with calipers every 3 days. All studies described in this article were done under approved protocols following Fox Chase Cancer Center Institutional Animal Care and Use Committee guidelines. Mice were euthanized when tumor volumes exceeded 10% of the animal's body weight or at 60 days postinitiation of treatment, whichever occurred first. The only exception to this was that the mice that exhibited complete responses were observed for 1 year following treatment after which they were euthanized and examined for signs of remaining tumor.

Renal toxicity and histologic studies
The diabody is primarily eliminated through the kidneys; therefore, a possible outcome of diabody-based radioimmunotherapy could be renal damage. As this type of damage would appear after a significant delay following therapy, the long-term survivors were euthanized at 1 year after the treatment date, their kidneys were fixed in formalin, and sections were obtained for histopathologic examination. Sections were stained with H&E and were then examined by the Fox Chase Cancer Center Histopathology Facility for abnormalities.

Statistics
Treatment cohorts were analyzed by one-way ANOVA using the GraphPad InStat 3 software package (GraphPad Software).


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 Conclusion
 References
 
Biodistribution studies. As the distribution of 211At-conjugated agents is difficult to follow in vivo, biodistribution studies were done using 125I-SIPS–conjugated diabodies as surrogates for the 211At-conjugated diabodies. The 4 and 48 h, biodistributions of the 125I-SIPS C6.5 diabody and 125I-SIPS T84.66 diabody were determined in scid mice bearing s.c. MDA-361/DYT2 tumor xenografts. At 4 h postinjection, the tumor retention of the 125I-SIPS C6.5 diabody was 3.6%ID/g, 2-fold or more greater than that retained in every major organ except the kidneys (19.4% ID/g), which are the site of elimination for this molecule (Fig. 1 ). The rapid blood clearance was also reflected by the ~3:1 tumor to blood ratio at 4 h postinjection. By 48 h after administration, the quantity retained in the tumor had dropped to ~0.6%ID/g, 6-fold or more greater than that retained in blood and all major organs except the kidneys (1.3%ID/g). At both 4 and 48 h postinjection, 125I-SIPS-T84.66 diabody failed to target to tumor to the same level as 125I-SIPS-C6.5 diabody (Fig. 1). The lower level of tumor uptake (1.8 and 0.17%ID/g at 4 and 48 h, respectively) combined with the slower blood clearance prevent 125I SIPS-T84.66 diabody from achieving levels in tumor above that seen in blood or the majority of the organs analyzed (Fig. 1B). Despite clearance through the kidney, as signified by the 17.5%ID/g uptake at 4 h, 125I SIPS-T84.66 was maintained in blood at a level of 3.7% ID/mL at 4 h. This is ~3-fold higher than what was seen with 125I SIPS-C6.5 diabody at the same point and is potentially indicative of circulating CEA, shed from the MDA-361/DYT2 tumors. 125I SIPS-T84.66 levels in normal organs trended higher than levels of 125I SIPS-C6.5 diabody, again consistent with the slower blood clearance observed for 125I SIPS-T84.66 diabody.


Figure 1
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  		Fig. 1. Biodistibutions of 125I-SIPS diabodies. The biodistributions of C6.5 diabody (A) and T84.66 diabody (B) radioiodinated with 125I-SIPS were evaluated at 4 h (solid black columns) and 48 h (hatched columns) in cohorts of five scid mice bearing s.c. MDA-MB361/DYT2 tumors. Average tumor and organ uptake are presented as percentage of the injected dose localized per gram of tissue or milliliter of blood. Columns, mean; bars, SE.

 
Radioimmunotherapy studies. The efficacy and specificity of a single dose of 211At-conjugated diabody was assessed in nude mice bearing established MDA-MB361/DYT2 tumors. The 211At-SAPS–conjugated diabodies were well tolerated at all doses studied. In each group, maximum weight losses occurred at ~1 week following treatment and were typically <15% of the body weight for the 45 µCi groups (Fig. 2 ). No fatalities occurred as a result of the treatment, but the mean body weights in the groups that received 45 µCi were very slow to recover to their initial weights, suggesting that the peak doses used were close to the acute maximum tolerated dose in this model system.


Figure 2
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  		Fig. 2. Weight loss is an indication of acute toxicity following radioimmunotherapy. The mean weights of the groups of nude mice treated with 211At-SAPS-C6.5 diabody (A), 211At-SAPS-T84.66 diabody (B), and 211At-SAPS–anti-MISIIR diabody (C) are shown for the untreated control groups ({blacklozenge}), 20 µCi (bullet), 30 µCi ({blacktriangleup}), and 45 µCi ({blacksquare}) treatment groups.

 
A single dose of 211At-SAPS-C6.5 diabody administered to mice bearing established MDA-361/DYT2 tumors was sufficient to result in a significant antitumor response compared with tumors in the untreated mice, which grew rapidly throughout the study (Fig. 3 ). A single treatment with 20 µCi of 211At-SAPS C6.5 diabody resulted in a 10-day delay, compared with the untreated control group, in the time it took for the mean tumor volumes to double from the volumes measured at the time of treatment (Fig. 3A). Similar delays in tumor growth were observed in two other studies in which doses ranging from 10 to 25 µCi were administered (data not shown). Doses of 30 and 45 µCi of 211At-SAPS C6.5 diabody delayed tumor growth significantly compared with the untreated controls, such that the mean tumor volume did not double during the course of the study (Fig. 3, significance of at least P < 0.05 from day 30 to the end of the study for both the 30 and 45 µCi dose groups). Three of the five mice that received the highest treatment dose (45 µCi of 211At-SAPS C6.5 diabody) exhibited a full remission. These animals were tumor-free for 1 year following treatment, at which time they were euthanized and histopathologic examination revealed no signs of tumors (data not shown). As the mean tumor volume of the mice in this group was smaller at the time of treatment than that of the other groups, the effect of smaller tumor size on the rate of complete responses cannot be discounted.


Figure 3
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  		Fig. 3. 211At-SAPS-C6.5 diabody radioimmunotherapy of nude mice bearing established MDA-361/DYT2 human breast tumors. Tumor volume at time of treatment initiation was set at 100%. Change in tumor volume for cohorts of five mice treated with a single dose of 20 µCi (bullet), 30 µCi ({blacktriangleup}), and 45 µCi ({blacksquare}) of 211At-SAPS-C6.5 diabody and a cohort of seven untreated mice ({blacklozenge}) are presented. Points, mean; bars, SE. Significance between treatment and control groups are indicated as follows: *, P < 0.05; **, P < 0.01.

 
As with the C6.5 diabody above, treatment of mice bearing established MDA-MB361/DYT2 tumors with a single dose of 211At-SAPS T84.66 diabody resulted in an antitumor response (Fig. 4 ). A 20 µCi dose of 211At-SAPS T84.66 diabody resulted in a 6-day delay in the mean tumor volume doubling time compared with the control group (Fig. 4), whereas 30 and 45 µCi doses of 211At-SAPS T84.66 diabody further increased the delay in tumor growth compared with the untreated controls (Fig. 4; significance of at least P < 0.05 from day 42 to the end of the study for both the 30 and 45 µCi dose groups). However, all of the tumors in the mice treated with 211At-SAPS T84.66 diabody eventually grew to 10% of the animal's total body weight, requiring euthanasia as per institutional guidelines.


Figure 4
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  		Fig. 4. 211At-SAPS-T84.66 diabody radioimmunotherapy of nude mice bearing established MDA-MB361/DYT2 human breast tumors. Tumor volume at time of treatment initiation was set at 100%. Change in tumor volume of cohorts of five mice treated with a single dose of 20 µCi (bullet), 30 µCi ({blacktriangleup}), and 45 µCi ({blacksquare}) of 211At-SAPS-T84.66 diabody and a cohort of 11 untreated mice ({blacklozenge}) are presented. Points, mean; bars, SE. Significance between treatment and control groups are indicated as follows: *, P < 0.05; **, P < 0.01.

 
In contrast to the results seen above with 20 µCi of 211At-SAPS conjugated C6.5 and T84.66 diabodies, treatment of mice bearing established MDA-MB361/DYT2 tumors with 20 µCi of 211At-SAPS MISIIR diabody resulted in no delay in tumor growth over that observed in mice left untreated (Fig. 5 ), likely a result of a combination of both the low expression levels of this antigen and the low affinity of GM17 diabody for MISIIR.


Figure 5
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  		Fig. 5. 211At-SAPS–anti-MISIIR GM17 diabody radioimmunotherapy of nude mice bearing established MDA-MB361/DYT2 human breast tumors. Tumor volume at time of treatment initiation was set at 100%. Change in tumor volume of a cohort of five mice treated with a single dose of 20 µCi (bullet) of 211At-SAPS–anti-MISIIR diabody or five untreated mice ({blacklozenge}) are presented. Points, mean; bars, SE. The differences between the values of the treatment and control groups were not significant.

 
Long-term toxicity. To assess long-term renal toxicity associated with 211At-SAPS diabody therapy, we compared the kidneys of mice 1 year following treatment with those of untreated age-matched control mice. The three mice that exhibited complete responses to 211At-SAPS C6.5 diabody therapy above and three control mice were euthanized, and their kidneys were excised and processed for histopathologic examination. Although the kidneys of the untreated control animals and one of the treated animals appeared normal (Fig. 6A ), two of the three treated mice exhibited signs of renal damage. In these animals, both normal regions of their kidneys and regions with varying degrees of damage, including edema and mild fibrosis in one mouse and focal cortical fibrosis and atrophy in the second mouse, were present (Fig. 6B).


Figure 6
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  		Fig. 6. Renal toxicity. The kidneys of mice treated with 45 µCi of 211At-SAPS C6.5 diabody and untreated age-matched control mice were examined 1 year following therapy. Untreated age-matched control mice (A) and one of the three treated mice exhibited no signs of renal damage, whereas two of the treated mice presented with renal damage. B, a section revealing both normal kidney regions and fibrosis seen in one of the treated mice.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
Conclusion
 References
 
Radioimmunotherapy-based strategies for the treatment of diffuse malignancies have exhibited significant successes in the clinical setting (21) and significant advances have been made in the preclinical treatment of solid malignancies (1). Typically, these strategies are based on the delivery of β-emitting radioisotopes to tumors via intact monoclonal antibodies. The two most frequently used β-emitters are 90Y and 131I. As a result of their decay, both emit β-particles with relatively long track lengths ranging up to several millimeters associated with a relatively low LET. These long track lengths extend the range of β-particle–based radioimmunotherapy well beyond the penetration range of the antibody carrier. This also allows for a "crossfire effect" in which tumor cells that lack the target antigen can still be effectively treated. However, the low LET associated with β-emitting radioisotopes necessitates up to 106 cell surface decays or "hits" on a given tumor cell before a cytotoxic event occurs (22).

In contrast with β-particles, {alpha}-particles have relatively short track lengths and high LET (a few, 3-7, can kill a cell; ref. 19). The radioisotope we used in the present study, 211At, has a LET of 97 to 99 keV/µm, ~200 times greater than that associated with 90Y (10). Although the short track length of {alpha} emissions significantly reduces the ability to kill tumor cells located distant from the decay event and limits the crossfire effect described above, their high potency can overcome these limitations if sufficient tumor penetration is achieved through conjugation to smaller antibody fragments, such as diabodies, that are capable of penetrating deeper into solid tumors. In addition, the high LET should translate into a need for fewer antibody molecules targeted to a tumor for effective killing. Localization of these {alpha}-particle–emitting radioimmunoconjugates to perivascular tumor calls could also mediate an antivascular effect (23).

The relatively short physical half-lives of the commonly available, medically relevant, {alpha}-emitting radionuclides limit the ability of {alpha}-emitter–based radioimmunoconjugates to be efficiently and safely administered by systemic routes for the treatment of solid tumors. As a result, the greatest successes using {alpha}-emitting radioisotopes conjugated to intact antibodies have resulted from i.p. delivery for the treatment of tumors that are localized in the peritoneal cavity (24, 25) and intrathecal delivery for the treatment of brain tumors (26).

An alternate strategy used to address the limitations that short physical half-lives impose on radioimmunotherapy with {alpha}-emitters such as 213Bi is the targeted delivery of one of the longer-lived parent radioisotopes of 213Bi, actinium-225 (225Ac), which has a T1/2 of 10 days (27). This approach allows accumulation of 225Ac-conjugated mAbs in the tumor over a longer period of time and then depends on limited diffusion of the 225Ac chain of {alpha}-emitting daughter radioisotopes during their subsequent combined 50-min half-life. As five different decay events occur over this period, releasing a total of four {alpha}-particles, radioimmunotherapy with targeted 225Ac is reported to be associated with impressive therapeutic efficacy (28). However, renal toxicity associated with the release and systemic trafficking of daughter radioisotopes in the 225Ac/213Bi decay chain must be addressed before this methodology can achieve its full potential (29, 30).

In contrast with the work of others, we elected to use smaller antibody-based molecules with biological half-lives that are more complimentary to the short physical half-lives of the medically relevant {alpha}-emitting radionuclides. In selecting a tumor-targeting agent, diabody molecules represent a particularly attractive class of antibody-based molecules. Diabodies are noncovalent dimers of scFv molecules that are held together by the affinity of the variable heavy and variable light chains for each other (4). With a molecular weight of ~55 kDa, diabodies are expected to more efficiently penetrate into tumors than intact IgG molecules that are three times larger. Due to their divalent nature, they also exhibit prolonged retention in tumors compared with monovalent scFv molecules (5).

We have previously reported on the isolation of the C6.5 scFv from a human nonimmune phage display library (31) and the development of the C6.5 diabody from this molecule (5). As the C6.5 diabody is nearly twice the size of the C6.5 scFv molecule, it exhibits a prolonged systemic retention in the mouse model. The C6.5 diabody also exhibits a higher functional affinity for HER2 than the C6.5 scFv (4 x 10–10 versus 1.6 x 10–8 mol/L, respectively). Taken together, these factors confer the C6.5 diabody with a selective targeting advantage over the C6.5 scFv (5).

We have recently shown that the C6.5 diabody is an effective vehicle for 90Y for the radioimmunotherapy of established solid tumors in the preclinical setting (9). In that study, 20% of the animals treated with the highest dose of 90Y CHX-A''–conjugated C6.5 diabody exhibited durable complete responses. However, the same dose was also associated with a 20% fatality rate due to treatment-related acute toxicity. In contrast, radioimmunotherapy of solid tumor xenografts growing in nude mice using the extremely short-lived {alpha}-emitting radioisotope 213Bi (T1/2 = 46 min) conjugated to the C6.5 diabody revealed no delay in tumor growth, likely due to the inability of the diabody to localize in the tumor before the majority of the radioactive decay events (12). Based on these results, we postulated that the most effective radioimmunotherapy could be achieved through rational pairing of the biological half-life of the delivery agent and the physical half-life of the radioisotope. As the C6.5 diabody exhibits an elimination half-life (T1/2 β) of 6.4 h in the circulation and a biological half-life of ~30 h in tumor xenografts in the mouse model (5), we predicted that it would exhibit excellent antitumor efficacy when paired with the potent {alpha}-particle–emitting radioisotope 211At (T1/2 = 7 h).

A major drawback with using a short-lived radioisotope, such as 211At, is the difficulty associated with its acquisition, purification, conjugation, and transport. In the current study, over two physical half-lives passed during these steps, severely limiting the doses we were able to administer to the animals. It is likely that the acute maximum tolerated dose was not achieved in our treatment studies. However, the slow recovery of weight loss by the cohorts of mice that received the highest treatment dose suggests that that it was close to the maximum tolerated dose. Unfortunately, the difficulty in production and transport of the 211At limited the dose used in the study with the anti-MISIIR diabody to a single dose group that received 20 µCi.

In the studies described here, we observed that 211At can be an extremely effective radioisotope for the radioimmunotherapy of solid tumors when it is conjugated to divalent 52 to 55 kDa diabody molecules. A single i.v. treatment with 211At conjugated to both the anti-HER2 C6.5 diabody and anti-CEA T84.66 diabody resulted in dose-dependent delays in tumor growth of MDA-MB361/DYT2 tumor xenografts in nude mice. At the highest dose administered, three of the five mice treated with 211At SAPS-C6.5 diabody exhibited durable complete responses and the remaining mice exhibited prolonged delays in tumor growth. In contrast to the complete responses seen with 211At-SAPS-C6.5 diabody, treatment with 45 µCi 211At-SAPS-T84.66 resulted in significant delays of tumor growth, but no complete responses. It should be noted that tumors treated with a single dose of 45 µCi 211At-SAPS-C6.5 diabody were significantly smaller than tumors in other treatment groups. The smaller size of these tumors may, at least in part, account for the complete responses observed in this treatment group. In addition to tumor size, antigen density and antibody affinity undoubtedly play a role in radioimmunotherapy effectiveness. The lack of complete responses in the 45 µCi 211At-SAPS-T84.66 treatment group likely reflected the significantly greater expression of HER2 on the MDA-MB361/DYT2 tumor cells compared with CEA. Ultimately, the fate of the target antigen could also play a role in the efficacy of {alpha}-emitter radioimmunotherapy. HER2 internalizes into tumor cells, bringing the short track length 211At {alpha} emissions into closer proximity to the nucleus than would be the case with the conjugates targeting the relatively noninternalizing CEA antigen.

As diabodies fall below the threshold for first pass renal elimination, the kidneys often exhibit the greatest degree of retention of radioactivity of any normal organ (Fig. 1), leading to the potential for significant renal toxicity. A recent publication by Jaggi et al. (30) reports that radiation damage to the kidneys following exposure to free {alpha}-emitting radioisotopes is progressive over a 10- to 40-week interval following exposure. In a prior study examining therapeutic efficacy of the β-emitting radioisotope 90Y conjugated to the C6.5 diabody in the same mouse model used in the current study, we observed significant renal damage at 1 year following therapy (9). Accordingly, we decided to perform a preliminary evaluation of renal toxicity 1 year after treatment in the three mice whose tumors were cured with the 45 µCi dose of 211At-SAPS C6.5 diabody. Histopathologic examination of the kidneys revealed that two of the three mice exhibited both regions of fibrosis and healthy regions in their kidneys compared with the untreated, age-matched controls and the third treated mouse. Although the cohort of animals studied is too small to draw significant conclusions, the degree of renal toxicity observed here seemed to be mild in contrast to the significant renal toxicity seen in some of the mice treated with 90Y-conjugated C6.5 diabody. If this observation were repeated when larger cohorts of animals are studied, it would be consistent with the overall lower renal retention seen with halogen-based radioisotopes compared with residualizing radiometals (9).


   Conclusion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 Conclusion
References
 
High LET, short-range {alpha}-emitters offer a promising alternative to lower LET β-emitters. However, to fully fulfill their potential for the systemic treatment of solid tumors and residual disease in an adjuvant setting, it is necessary to deliver these radioisotopes in a rapid and specific manner. Our report here represents the first successful use of small, 52 kDa, diabody molecules as vehicles for {alpha}-emitter radioimmunotherapy of established tumors.


   Acknowledgments
 
We thank Dr. Louis Weiner, Heidi Simmons, and Eva Horak of the Department of Medical Oncology for helpful discussions; Dr. Andres Klein-Szanto and Cass Renner of the Fox Chase Cancer Center Histopathology Facility; and the members of the Fox Chase Cancer Center Laboratory Animal Resources Group for their expert assistance.


   Footnotes
 
Grant support: Department of Energy grant DE-FG02-01ER63190 (G.P. Adams) and the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

4 Unpublished data. Back

Received 5/21/07; revised 9/19/07; accepted 11/ 1/07.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 Conclusion
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