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Radioimmunotherapy of A431 Xenografted Mice with Pretargeted B3 Antibody-Streptavidin and ⁹⁰Y-labeled 1,4,7,10-Tetraazacyclododecane-N,N',N'',N'''-tetraacetic Acid (DOTA)-Biotin

Nuclear Medicine Department of the Warren G. Magnuson Clinical Center [Z. Y., K. J. W., L. P., C. W. P., C. H. P., J. A. C.], Metabolism Branch [M. Z., T. A. W.], Radiation Oncology Branch [K. G., M. W. B.], and Laboratory of Molecular Biology [I. P.], National Cancer Institute, NIH, Bethesda, Maryland 20892, and NeoRx Corporation, Seattle, Washington 98119 [D. B. A., R. W. M., L. J. T., E. K. Y.]

	ABSTRACT

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We investigated the biodistribution of ⁸⁸Y/¹¹¹In-labeled 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA)-biotin and therapy with ⁹⁰Y-labeled DOTA-biotin in tumor-bearing mice after B3-streptavidin antibody conjugate (B3-SA) pretargeting. B3 antibody, recognizing Lewis^y antigen, was conjugated to streptavidin (B3-SA). For pretargeting, 400 µg of the B3-SA was injected i.v. into mice bearing A431 tumor xenografts. After tumor localization of B3-SA, 100 µg of synthetic clearing agent was injected i.v. to clear the unbound B3-SA from the circulation. Four h later, 1 µg of radiolabeled DOTA-biotin was injected i.v. Radioimmunotherapy was performed with doses of 9.25 to 37 MBq of ⁹⁰Y-labeled DOTA-biotin. As a result, radiolabeled DOTA-biotin cleared rapidly. All of the normal tissues had <2.6% of the injected dose per gram, whereas tumor uptake reached 15% ID/g. The total tumor uptake of radioactivity remained similar for 96 h or longer. In the first study, the median survival of the control group was 8 days, whereas it increased to >163 days in the 37 MBq ⁹⁰Y group (P < 0.005). In a second therapy group, 7 of 10 mice receiving 37 MBq of ⁹⁰Y and B3-SA were cured, and remained healthy for >180 days after therapy, compared with control groups, with 29.2 days mean survival time (P < 0.001). Tumor pretargeting with B3-SA and radiolabeled DOTA-biotin has shown favorable, specific, and fast targeting that has resulted in good tumor responses and, thus, serves as a rationale for human studies with the B3-SA pretargeting approach.

	INTRODUCTION

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Clinical studies using radiolabeled MAbs² for radioimmunotherapy of lymphomas have shown promising results (1) . In contrast, studies performed with MAbs directed against epithelial tumors have rarely shown partial or complete remissions (1) . Various physiological factors contribute to the limited therapeutic response of this targeting approach using intact MAb (1, 2, 3) . Approaches are needed that result in faster delivery of higher concentrations of radiolabeled MAbs to tumor with relative sparing of normal tissues.

Several pretargeting methods that dissociate the targeting step from the radioactivity delivery step have been proposed (1 , 4, 5, 6) . Some of the methods are based on the high affinity binding of avidin or streptavidin to biotin (7) . A promising pretargeting approach consists of three steps (8 , 9) : (a) the SA conjugate is allowed to target and accumulate in tumor, thus carrying the SA receptor that will later bind the radiolabeled biotin; (b) the nontumor bound MAb-SA is cleared from the circulation to prevent it from binding the radiolabeled biotin; (c) the radiolabeled biotin is administered i.v. and because of its small size, the radiolabeled biotin extravasates out of the circulation quickly and can bind to the SA on the MAb-SA conjugate that has localized in tumor (8, 9, 10) .

B3 is an excellent antibody to consider for pretargeting applications (11) . Biodistribution and radioimmunotherapy of ¹¹¹In/⁹⁰Y-radiolabeled B3 antibody has shown good tumor localization, although at the maximal tolerated doses tumor responses were not observed (12) . Using the pretargeting approach, it is likely that higher doses of ⁹⁰Y could be delivered to tumor compared with normal tissues and thus increase the therapeutic index. In this report, we performed a biodistribution study using ¹¹¹In- and ⁸⁸Y-labeled DOTA-biotin after pretargeting with B3-SA, as well as a radioimmunotherapy trial with ⁹⁰Y-labeled DOTA-biotin.

	MATERIALS AND METHODS

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Pretargeting Reagents.
B3 is a murine IgG1k MAb directed at Lewis^y and polyfucosylated-Lewis^x (11) . B3 was conjugated to SA by NeoRx by incubating for 1 h with succinimidyl 4-(N-maleimido-methyl) cyclohexane-1-carboxylate (10) . To confirm that the conjugated SA to B3 was still functional, excess B3-SA was incubated with ¹¹¹In-labeled DOTA-biotin, and instant thin-layer chromatography, using paper chromatography developed with normal saline, was performed and showed a bound fraction >99%.

The sCA provided by NeoRx consists of a bifunctional moiety with multiple N-acetyl-galactosamine residues linked to biotin (M_r 8651; Ref. 13 ). When sCA was injected to mice (n = 5) that had received ¹²⁵I B3-SA, a mean drop of 90% of the circulating ¹²⁵I was observed.

Biotinidase-resistant DOTA-biotin (M_r 900) was prepared by NeoRx corporation as described previously and labeled with either ¹¹¹In, ⁸⁸Y, or ⁹⁰Y (14) .

Radiolabeling.
Unmodified B3 and B3-SA were labeled with 4-[¹²⁵I]iodobenzoate (15) at a specific activity of 37 kBq/µg for biodistribution studies. B3 conjugated with 2-(p-isothiocyanato benzyl)-cyclohexyl-diethylenetriaminepentaacetic acid (CHX-A''-referred to as CHX) was also labeled with ¹¹¹In at a specific activity of 37 kBq/µg for biodistribution study as described previously (16) . Biotinidase-resistant DOTA-biotin was labeled with ¹¹¹In or ⁸⁸Y at a specific activity of 0.37 to 1.11 MBq/µg for biodistribution and with ⁹⁰Y at a specific activity of 37 MBq/µg for therapy (10) . These radiolabeled DOTA-biotin reagents consistently bound >99% to B3-SA.

Immunoreactivity Assay.
A431, a human epidermoid carcinoma cell line that expresses the antigen recognized by B3, was used for immunoreactivity determination (16) . The immunoreactivity of ¹²⁵I B3-SA (68–73%) was in the range of that of ¹²⁵I B3 (70–77%). A competition assay performed using 10⁵ A431 cells incubated with ¹²⁵I B3 and increasing doses of unlabeled B3 or B3-SA showed overlapping curves (data not shown). The IC₅₀ was 89 nM and 95 nM for B3 and B3-SA, respectively (GraphPad Prism, San Diego, CA).

Tumor Model.
Female athymic mice (nu/nu) were inoculated s.c. with A431 cells. Animal experiments were performed under an NIH Animal Care and Use Committee approved protocol. Biodistribution or therapy studies were performed when xenografted tumors typically reached a maximum diameter of 0.5 cm (0.11–0.31 g). Mice were killed when tumor size reached >2 cm in the longest diameter, tumor was ulcerated, or excessive weight loss (>25%) was noted, according to the protocol guidelines. Seven days before pretargeting, the mice were given a biotin-free diet to reduce their endogenous biotin level (Biotin Deficient Purina Diet 5836C; Purina Mills, Richmond, IN). One week of this diet in BALB/c mice (n = 3 per group) caused their biotin level in serum to drop from a baseline of 15.0 ± 5.2 ng/ml to 4.77 ± 1.12 ng/ml (Vitamin Diagnostics, Inc., Cliffwood Beach, NJ).

Optimization of B3-SA Dose.
To evaluate the effect of MAb mass on tumor targeting and thus determine the optimal MAb dose for pretargeting, biodistribution studies were performed with ¹¹¹In-CHX-B3. B3 was used rather than B3-SA because studies in tumor bearing mice showed a similar biodistribution in most tissues of ¹¹¹In-B3 and B3-SA (data not shown), and because the quantity of B3-SA was limited. Tumor-bearing mice were injected i.v. with 0.185 MBq of ¹¹¹In-CHX-B3. The amount of B3 was adjusted by adding unlabeled, unconjugated B3 in doses of 5, 50, 100, 200, 400, and 800 µg. At each dose level 5 mice were sacrificed 48 h after injection. Two additional groups were sacrificed at 24 and 72 h after receiving 400 µg to better characterize the kinetics of the dose finally selected for the therapy trial.

To evaluate the effect of dose on gross heterogeneity of distribution within the tumor, autoradiography was performed after injection of the ¹¹¹In-CHX-B3. The autoradiography was performed using a BAS-1500 Fujifilm Bio-imaging analyzer (Fuji Medical Systems, Inc.). The slides were exposed to the imaging plate, and developed and analyzed according to the manufacturer’s procedure. The autoradiograms were inspected visually, and areas of interest were drawn around the individual tumor autoradiogram, and the mean and SD of the counts were determined in the manufacture’s PSLs.

¹¹¹In-and/or ⁸⁸Y-labeled DOTA-Biotin Biodistribution.
Tumor-bearing mice were injected i.v. with 400 µg (1.91 nmol) of B3-SA for pretargeting. After 24 h were allowed for distribution, 100 µg (11.56 nmol) of sCA was injected i.v. to clear circulating B3-SA from the blood. Four h after injection of the sCA, 1 µg (1.11 nmol) of ¹¹¹In-labeled DOTA-biotin was injected i.v. Groups of 5 mice were sacrificed at 2, 24, 48, 96, and 168 h after injection. In a separate experiment, dual-isotope injection of the ¹¹¹In- and ⁸⁸Y-labeled DOTA-biotin was given to mice to allow direct comparison of both reagents at 2 and 96 h.

Dosimetry.
The mean %ID/g of ¹¹¹In in tumor and tissues at the various time points up to 7 days was used to obtain the area under the curve of the exposure for ⁹⁰Y using trapezoidal integration. From that point on, the activity in tumor was extrapolated, assuming it cleared with the estimated terminal half-life of the clearance curve (GraphPad Prism). ⁹⁰Y radiation dosimetry was then calculated based on biodistribution data using the medical internal radiation dose method (17) and adjusted for organ size using the "Nodule Module" of MIRDOSE 3 (18) . The data from our previous study of ¹¹¹In B3 (16) was analyzed in the same way and compared with that of ⁹⁰Y-labeled DOTA-biotin.

Therapy.
Radioimmunotherapy was performed in tumor-bearing mice by pretargeting ⁹⁰Y-labeled DOTA-biotin after allowing for localization of B3-SA and clearing of the blood as described above. Groups of mice (n = 5) were treated in a dose escalation trial with 9.25, 18.5, and 37 MBq of ⁹⁰Y-labeled DOTA-biotin and compared with a no-treatment control group. In an additional experiment, a group of 10 mice was treated with 37 MBq ⁹⁰Y-labeled DOTA-biotin after the same pretargeting approach with B3-SA and compared with 3 control groups: (a) no treatment; (b) radioactivity-free group receiving B3-SA, clearing agent, and nonradioactive DOTA-biotin; and (c) a nonspecific MAb consisting of HAT-SA, directed against the interleukin 2 receptor, which is not expressed on A431 cells and was pretargeted followed by 100 µg sCA and 37 MBq ⁹⁰Y-labeled DOTA-biotin (19) .

Response to Therapy and Toxicity Assessment.
Tumor growth was monitored twice a week for 3 weeks after treatment and then once per week. A digital caliper was used to measure the tumor in two orthogonal dimensions. The volume was calculated using the formula .

To determine hematological toxicity, 20 µl of blood was collected by bleeding the tail veins using an EDTA-coated capillary tube and diluted to 250 µl with PBS (n = 5/group). WBC, lymphocytes, RBC, and platelet count were performed before therapy and at weekly intervals thereafter. Serum was obtained at the time of sacrifice, and liver (total bilirubin, alk phosphatase, and albumin) and kidney (blood urea nitrogen and creatinine) function test were done.

Histological analysis was performed in 3 mice in each of the groups (control, 9.2 MBq, 18.5 MBq, and 37 MBq of the ⁹⁰Y-labeled DOTA-biotin-B3-SA, and the 37 MBq ⁹⁰Y-labeled DOTA-biotin-HAT-SA group). Tissues analyzed included the kidney and liver, and in some animals bone and bone marrow were also fixed in 10% buffered formalin for pathological studies by a veterinary pathologist.

Statistics.
Statistical analysis was performed using ANOVA for multiple groups and Student’s t test for unpaired data between two groups. Survival analysis was based on the Kaplan-Meier product limit, and groups were compared using the log-rank test (GraphPad Prism).

	RESULTS

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Biodistribution Study.
The concentration of ¹¹¹In-CHX-B3 and ¹¹¹In-B3-SA in tumor was similar, with concentrations of 13.89 ± 3.16 and 10.33 ± 2.20%ID/g at 24 h, respectively (P = 0.077). The fraction of administered ¹¹¹In-CHX-B3 (%ID/g) in tumor remained similar, with doses ranging from 5 to 400 µg (Fig. 1A) . Although the %ID/g of radioactivity dropped with the 800 µg dose, the absolute tumor-bound B3 still increased. The tumor uptake of 400 µg was near maximal at 24 h (21.8 ± 2.1%ID/g) and remained grossly unchanged for 24 h through 72 h after injection (P = 0.74; Fig. 1B ). On autoradiography no gross differences in tracer heterogeneity were noted visually in the tumors. The mean activity value and SD obtained from the region of interest analyses over the autoradiographs of tumors, which are a measure of the heterogeneity of the intensity per pixel, were 120 ± 23 PSL units, 164 ± 41 PSL units, 165 ± 62 PSL units, 140 ± 36 PSL units, 116 ± 19 PSL units, and 158 ± 37 PSL units for the 5, 50, 100, 200, 400, and 800 µg antibody doses, respectively (ANOVA, P = 0.197).

View larger version (38K): [in this window] [in a new window]   Fig. 1. The biodistribution of 111In-CHX-B3 in groups of mice (n = 5) bearing A431 tumor xenografts was evaluated. A, the effect of various doses of B3 ranging from 5 to 800 µg on its 24 h biodistribution expressed as the mean %ID/g; bars, ±SD. B, the biodistribution at various times after i.v. injection of 400 µg of 111In-CHX-B3 in A431 tumor-bearing mice. The data are expressed as mean %ID/g; bars, ±SD.

View larger version (24K): [in this window] [in a new window]   Fig. 2. Biodistribution of pretargeted 111In-labeled DOTA-biotin in mice bearing A431 tumor xenografts that received 400 µg of B3-SA followed by 100 µg sCA clearing agent. Values represent the mean %ID/g; bars, ±SD. The insert compares the mean %ID/g tumor and the mean total %ID; bars, ±SD (%ID = %ID/g * g tumor weight).

Radiation Dosimetry.
On the basis of ¹¹¹In-CHX-B3 and ¹¹¹In-labeled DOTA-biotin biodistribution data ⁹⁰Y-CHX-B3 and pretargeted ⁹⁰Y-labeled DOTA-biotin radiation dosimetry for tumor was calculated as 196 Gy/37MBq and 72 Gy/37MBq, respectively, assuming a 1-g tumor that does not increase in size during the therapy and correcting for absorption fraction. The radiation dose to tumor was higher for directly labeled B3. Nevertheless, the expected maximum tolerated dose for ⁹⁰Y B3 (nonpretargeted) is likely to be in the range of 7.4–11.1 MBq based on prior studies (10 , 20) . Whereas 37 MBq ⁹⁰Y could generally be delivered with the pretargeting. The tumor:blood ratios were 2.4 for ¹¹¹In B3 and 9.3 for pretargeted ¹¹¹In-labeled DOTA-biotin, respectively.

Therapy Study.
Effective and specific therapeutic responses were observed in tumor-bearing mice treated with ⁹⁰Y-labeled DOTA-biotin after pretargeting with B3-SA. A431 tumors in the no-treatment control group grew rapidly, from 0.2 cm³ to 2 cm³ in <2 weeks (Fig. 3 , Group 1), and these animals were sacrificed according to our animal protocol. Escalating doses of 9.25, 18.5, and 37 MBq ⁹⁰Y-labeled DOTA-biotin with B3-SA pretargeting showed a dose-related response in tumor size (Fig. 3 , Group 2, 3, 4, respectively). The median duration of survival of the control group was 7 days; it increased to 18, 28, and >180 days in the groups receiving 9.25, 18.5, and 37 MBq of ⁹⁰Y-labeled DOTA-biotin, respectively. Four of the 5 mice in group 4 and 1 in group 3 were cured and remained healthy for >180 days after therapy, at which time they were sacrificed electively for histology, CBC, liver, and kidney chemistries.

View larger version (23K): [in this window] [in a new window]   Fig. 3. Response to radioimmunotherapy. Tumor volume changes over time in control mice or mice receiving escalating doses of pretargeted 90Y-labeled DOTA-biotin after receiving 400 µg B3-SA followed by sCA (100 µg). Group 1 is control without any treatment; Groups 2, 3, and 4 received 9.25, 18.5, and 37 MBq of 90Y-labeled DOTA-biotin, respectively. Each symbol represents a single mouse in the group (n = 5 mice/group).

View larger version (25K): [in this window] [in a new window]   Fig. 4. Response to radioimmunotherapy in mice (n = 10) with A431 tumor xenografts. Four groups were studied: a no-treatment control; a group receiving B3-SA pretargeting (400 µg), sCA (100 µg), and DOTA-biotin but no 90Y; a group receiving 37 MBq of pretargeted 90Y-labeled DOTA-biotin after pretargeting steps with B3-SA and sCA clearing of blood; a group receiving 37 MBq of pretargeted 90Y-labeled DOTA-biotin after all three pretargeting steps with the nonspecific HAT-SA and sCA. Top panel, the mean tumor volumes of all surviving mice are shown. Note that follow-up is shorter for the three control groups because of the need to kill the mice when their tumors grew too large. If mice died or were killed early, their last measured tumor volume was used from that point forward and contributed to the mean. Bottom panel, the Kaplan-Meier survival curve of mice treated in this second therapy experiment is shown. Note that the control group (no treatment) and the B3-SA group without radioactivity survival curves are almost indistinguishable.

Although only a few animals (n = 3) had CBC at each therapy dose, comparison of hematology results in the therapy groups showed that there were no significant differences in platelet and RBC counts between the different groups (P > 0.12), with the nadirs occurring at 2 weeks. Nevertheless, a significant decrease in WBC and lymphocyte counts was observed 1–2 weeks after therapy in the 37 MBq-treated groups (P < 0.007), with the nadir occurring at 2 weeks and recovery by 3 weeks. This represented decreases of 79% and 94% for WBC and lymphocyte count, respectively. Liver and kidney function chemistry performed at the time of sacrifice was no different between nonradioactivity receiving controls or ⁹⁰Y pretargeted groups.

Histological analysis of the liver and kidney of mice receiving the 37 MBq of ⁹⁰Y-labeled DOTA-biotin-B3-SA showed similar finding as those of the control group that did not receive radioactivity. In addition, the bone and bone marrow of the mice receiving 37 MBq of ⁹⁰Y-labeled DOTA-biotin-B3-SA were histologically normal (performed 180 days after therapy). The mice receiving 37 MBq of ⁹⁰Y-labeled DOTA-biotin-HAT-SA showed similar findings as the control animals and did not have any histological findings in the kidney or liver to explain their toxicity.

	DISCUSSION

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The pretargeting approach based on SA-biotin depends on the successful delivery and function of three reagents. In this study we showed sufficient concentration of B3-SA to serve as an effective binding receptor for the ¹¹¹In/⁹⁰Y-labeled DOTA-biotin. In this study, the %ID/g of the MAb in tumor was shown to be similar for doses of 5–400 µg, whereas the total amount delivered continued to increase. Autoradiography showed that heterogeneity in tumor distribution was not grossly decreased at the higher antibody dose levels. There was some evidence of partial saturation of uptake at the 800 µg dose, as evidenced by the lack of uptake linearity at this higher dose. Our decision to use 400 µg was based on: (a) some evidence of saturation at the higher doses that resulted in lower tumor:normal organ uptake ratios; (b) very large doses of B3-SA that may be more difficult to clear from the blood and may result in higher tissue background; and (c) the results of these experiments, which indicated that the 400 µg dose level resulted in good tumor targeting.

The clearing step and administration of radiolabeled biotin at 24 h after giving B3-SA was chosen because near-peak antibody tumor levels were achieved by this time (Fig. 1B) . In addition, studies reported previously indicated that despite the low biotin diet, endogenous biotin fills additional binding sites of MAb-SA conjugate at 48 h as compared with 24 h, which decreases the binding sites for the subsequently administered radiolabeled biotin. The doses of sCA and DOTA-biotin we used were effective, as gauged by the high tumor concentration of radiolabeled DOTA-biotin and its fast clearance from the blood (Figs. 2) . The dose of these two reagents was based on that shown previously to be effective in other studies (10) . These two reagents are "universal" or independent of what MAb-SA conjugate is used, and the doses required for therapy are unlikely to be greatly dependent on what tumor antibody system is used.

Our studies showed that ¹¹¹In- or ⁸⁸Y-labeled DOTA-biotin was delivered in similar concentrations to tumor and normal organs. Thus, this similarity allowed us to make dosimetry estimates for ⁹⁰Y based on ¹¹¹In biodistribution. The pretargeting approach resulted in rapid delivery of high doses of radiation to the tumor. The calculated dose delivered to the tumor is likely to be an underestimate, because in our pretargeted animals, the tumor size increased, resulting in decreasing %ID/g. If these animals had received therapy, their tumors would not have continued to grow and the radiation dose would have been much higher. ⁹⁰Y B3 dosimetry extrapolated from previous biodistribution studies suggested that high radiation doses to tumor could be delivered and that much higher doses to normal tissues on an equivalent MBq basis would also be delivered compared with the pretargeting approach. Although the dose-limiting toxicity from ⁹⁰Y B3 (not pretargeted) was not evaluated in this study, works by others suggest that the maximal tolerated dose by nude mice was in the range of 7.4–11.1 MBq (10 , 20) . These lower tolerated doses would significantly limit the maximal radiation dose to tumor as compared with the pretargeted approach, where higher pretargeted amounts of ⁹⁰Y were possible. Our dosimetry estimates for ⁹⁰Y B3 on a per MBq basis were in the same range reported by Axworthy et al. (10) for nonpretargeted MAb NR-LU-10. This is because of similar peak concentration and kinetics in tumor for their MAb (29.9%ID/g) and ours (26.3%ID/g) at 24 h. In contrast, the peak dose observed in this study from pretargeted radiolabeled DOTA-biotin was lower than the 34.9%ID/g observed previously with NR-LU-10-SA antibody (10) . Given the fast clearance of the DOTA-biotin through the kidneys, the ratios of tumor:blood and tumor:bone marrow are more favorable than when ⁹⁰Y B3 is given without pretargeting. The average tumor:blood area under the curve ratio in our pretargeting studies was 9.3, which compares favorably with the 2.4 ratio seen with ⁹⁰Y directly conjugated to B3.

Significant decreases of WBC and lymphocyte counts were observed 1–2 weeks after therapy. The platelet counts in the treated and control groups failed to show any significant changes. These results are in line with those reported previously with NR-LU-10-SA, where rapid targeting was observed and where dose-limiting toxicity was not observed at doses of 29.6 MBq (800 µCi). The limited toxicity to the marrow was most likely attributable to the rapid clearing of the unbound ⁹⁰Y-labeled DOTA-biotin through the kidney, such that with the long physical half-life of ⁹⁰Y this would not have given a large dose to the whole body, bone marrow, or kidney. Evidence of toxicity was observed when pretargeted ⁹⁰Y-labeled DOTA-biotin was used with HAT-SA where the mice died or were sacrificed because of tumor ulceration or significant body weight loss within 41 days. Additional studies will be required to determine the cause of toxicity in this control group.

Whereas preclinical animal studies are useful and necessary to help optimize delivery strategies and show proof of principle, use in humans remains the final goal. Given the favorable targeting seen previously in vivo with ¹¹¹In-CHX-B3, the findings in this pretargeting study encourage us to pursue this system in humans (12) .

In summary, B3-SA using a three-step pretargeting approach allows rapid and specific delivery of radionuclides to tumor xenografts. High tumor uptake with fast clearance from the vasculature and low nontumor uptake resulted in high tumor doses and good radioimmunotherapeutic responses. These findings support the use of this reagent for a clinical trial in patients.

	ACKNOWLEDGMENTS

 
We thank Debby Drumm-Guisseppe for expert technical assistance in animal experiments and assays.

	FOOTNOTES

 
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.

¹ To whom requests for reprints should be addressed, at Nuclear Medicine Department, NIH, Building 10, Room 1C-496, 10 Center Drive, MSC 1180, Bethesda, MD 20892.

² The abbreviations used are: MAb, monoclonal antibody; %ID/g, injected dose per gram; SA, streptavidin; sCA, synthetic clearing agent; DOTA, 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid; PSL, photoluminescence stimulated unit; HAT-SA, humanized anti-Tac monoclonal antibody-streptavidin; CBC, complete blood count.

Received 3/20/02. Accepted 8/15/02.

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