Biodistribution of 111In- and 90Y-Labeled DOTA and

Dec 15, 1997 - Division of Diagnostic Radiology, City of Hope National Medical Center, Duarte, California 91010, City of Hope. Graduate Program in Bio...
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Bioconjugate Chem. 1998, 9, 87−93

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Biodistribution of 111In- and 90Y-Labeled DOTA and Maleimidocysteineamido-DOTA Conjugated to Chimeric Anticarcinoembryonic Antigen Antibody in Xenograft-Bearing Nude Mice: Comparison of Stable and Chemically Labile Linker Systems Lawrence E. Williams,*,† Michael R. Lewis,‡ Gregory G. Bebb,§ Kenneth G. Clarke,| Tamara L. Odom-Maryon,| John E. Shively,⊥ and Andrew A. Raubitschek# Division of Diagnostic Radiology, City of Hope National Medical Center, Duarte, California 91010, City of Hope Graduate Program in Biological Sciences, Duarte, California 91010, Division of Surgery and Department of Biostatistics, City of Hope National Medical Center, Duarte, California 91010, Division of Immunology, Beckman Research Institute of the City of Hope, Duarte, California 91010, and Department of Radioimmunotherapy, City of Hope National Medical Center, Duarte, California 91010. Received July 18, 1997; Revised Manuscript Received October 15, 1997X

Biodistributions of two radiometal chelate conjugates of the human/murine chimeric anticarcinoembryonic antigen monoclonal antibody cT84.66 were obtained in nude mice bearing LS174T human colorectal carcinoma xenografts. Derivatives of the macrocyclic chelating agent 1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetic acid (DOTA) were covalently attached to the antibody by a stable amide linkage and by a maleimidocysteineamido side chain (MC-DOTA) that has been shown to be chemically labile at physiological temperature and pH. Biodistributions of both 111In and 90Y labels were obtained in these studies. At common biodistribution time points, it was found that the 111In label had greater uptake in the liver than 90Y for both conjugates. No significant differences were found with respect to bone uptake of 90Y using either chelate. Blood curves were generally lower at comparable time points for MC-DOTA, indicative of faster clearance as compared to DOTA. Tumor uptake was high for both conjugates (57-68% ID/g at 48 h), with a longer tumor residence time in the case of the DOTA conjugate, probably a result of its longer blood circulation times. We conclude that bone uptake of 90Y would be minimal if either DOTA or MC-DOTA were used as the bifunctional chelator. This would imply preference for these macrocyclic ligands if radiation doses to the bone marrow would be considered to be dominated by skeletal uptakes. Alternatively, if bone marrow radiation dose is dominated by circulating antibody, the chemically labile linker system employed by the MC-DOTA conjugate offers the advantage of enhanced blood clearance.

INTRODUCTION

Radiolabeled monoclonal antibodies (mAbs) have applications for both imaging and therapy of malignant tumors (1). A large number of radionuclides, including radioiodines but primarily radiometals, are available for these applications. Generally, pure photon emitters and particulate emitters are favored for imaging and therapy, respectively. In the case of clinical radioimmunotherapy (RIT1), however, an imageable photon must be emitted along with the particulate radiation so the absorbed dose * Address correspondence to Lawrence E. Williams, Ph.D., Division of Diagnostic Radiology, City of Hope National Medical Center, 1500 E. Duarte Rd., Duarte, CA 91010. Phone: (626) 301-8252. Fax: (626) 930-5451. E-mail: lwilliams@smtplink. coh.org. † Division of Diagnostic Radiology, City of Hope National Medical Center. ‡ City of Hope Graduate Program in Biological Sciences. § Division of Surgery, City of Hope National Medical Center. | Department of Biostatistics, City of Hope National Medical Center. ⊥ Beckman Research Institute of the City of Hope. # Department of Radioimmunotherapy, City of Hope National Medical Center. X Abstract published in Advance ACS Abstracts, December 15, 1997.

can be estimated. If one utilizes only one radiolabel, such as a radioiodine, the associated photon yield may be of the wrong energy and/or too intense for effective quantitation. Human therapies require total injected activities approaching or exceeding 100 mCi (2). An example is the application of 131I-labeled anti-CD20 in the treatment of B-cell lymphomas (3), where injected activities have exceeded 500 mCi. Such levels lead to γ camera count rate limitations at early time points postinjection. This may limit the accuracy of patient iodine uptake measurements and consequently the estimation of the absorbed dose. Because of this ineffective or hazardous photon production, in many situations two radiolabels are used in RIT. One label must be suitable for the imaging or therapy planning situation, and the second, essentially of a particulate type, provided for the subsequent treatment(s). If the particulate emitter has no associated 1 Abbreviations: RIT, radioimmunotherapy; BCA, bifunctional chelating agent; DOTA, 1,4,7,10-tetraazacyclododecaneN,N′,N′′,N′′′-tetraacetic acid; MC-DOTA, maleimidocysteineamidoDOTA; DTPA, diethylenetriaminepentaacetic acid; CEA, carcinoembryonic antigen; cT84.66, human/murine chimeric anti-CEA mAb; cT84.66-DOTA, cT84.66 conjugated with the N-hydroxysulfosuccinimide ester of DOTA; cT84.66-MC-DOTA, cT84.66 conjugated with MC-DOTA.

S1043-1802(97)00137-7 CCC: $15.00 © 1998 American Chemical Society Published on Web 01/12/1998

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photon suitable for imaging, the provision of an imaging label becomes a necessity. From the earliest discussions of therapeutic efficacy, 90Y(III) has been described as a possible RIT radionuclide (4). Its pure, high-energy β(range, 1.1 cm) leads to the clinical advantage of crossfire capability in treating tumor cells distant from those to which the radioimmunoconjugate has localized. The corresponding γ emitter 111In(III) generally has been used for imaging and dosimetry applications in 90Y therapy. Both trivalent metals can be attached to mAbs using the same bifunctional chelating agent (BCA). 111In emits two γ photons at 171 and 247 keV, energies that are efficiently detected with commercial Anger cameras. Given that two labels must be used for 90Y-based RIT, it is incumbent upon the investigator to demonstrate that variation of the radiometal has minimal and predictable impact upon biodistributions. If this is so, then an imaging sequence can be used to plan and document patient RIT treatments. Biodistributions in an animal model of human cancer can be used to show the variation of organ uptake with a change in the radiometal or bifunctional chelate coupled to the mAb. An important consideration for the selection of the BCA is the formation of a physiologically stable radiometal complex, to minimize uptake in and radiation toxicity to normal tissues (5). In the case of 90Y, uptake in mice approaching 20% of the injected dose per gram (% ID/g) of bone has been seen for the ionic form (6). Since bone marrow is an organ at risk in cancer radiotherapy, the dissociation of 90Y in the bloodstream and other tissues as a function of time postinjection of the radiolabeled mAb must be evaluated. The stability of the conjugated radiometal complex will depend upon the type of BCA used. Macrocyclic ligands have been suggested as one possible methodology for reducing 90Y loss in vivo (7-10). Meares and co-workers (7-9) have demonstrated bone uptakes on the order of 2% ID/g in nude mice receiving 1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetic acid (DOTA) derivatives labeled with 90Y and conjugated to Lym-1 (7) and chimeric L6 (9) antibodies. In these studies, the radiometal was incorporated into the BCA prior to mAb conjugation. Similar bone uptake values have been demonstrated by Harrison et al. (10) using the anti-colon cancer antibody B72.3, which had been covalently linked to DOTA before radiolabeling. In these earlier works, however, little numerical comparison was made between 111In and 90Y biodistributions in animals. Deshpande et al. (7) utilized Lym-1 mAb, which targets to B lymphocytes within the bone and confounds analyses. Harrison and co-workers utilized only 90Y in their B72.3 studies (10). One recent report (9) included both radiometals, but did not analyze their differences statistically in vivo. The need to compare 111In- and 90Y-labeled antibody biodistributions for DOTA-based BCAs led to the studies described herein. The human/murine chimeric anticarcinoembryonic antigen (anti-CEA) mAb cT84.66 (11) has shown minimal cross-reactivity with normal human or murine tissues, and biodistributions of 111In- and 90Y-labeled cT84.66 were evaluated earlier using a bifunctional DTPA derivative (12). Athymic nude mice implanted with CEAexpressing LS174T human colorectal tumor xenografts were used in that study. In this work, we obtained the 111 In and 90Y biodistributions of cT84.66 conjugated to two new DOTA derivatives in the LS174T-bearing nude mouse model. When endogenous primary amino groups of the mAb were reacted with the N-hydroxysulfosuccinimide ester of DOTA, the radiometal-labeled conjugate

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displayed extremely high kinetic stability in vitro (13). In contrast, radiometals conjugated to reduced disulfide bonds in the mAb with maleimidocysteineamido-DOTA (MC-DOTA) were chemically labile at physiological temperature and pH. This BCA exhibited pH-dependent linker hydrolysis in vitro, which resulted in release of Y(III) in the chelated form (14). For both macrocycle-antibody conjugates, we compared the biodistributions obtained with 111In versus those obtained with 90Y. The likelihood of bone uptake of 90Y was evaluated with either macrocycle. In addition, blood clearance curves were analyzed for all four combinations of label and BCA. The latter two studies provided information that can be used to select an optimal BCA for reducing bone marrow absorbed dose during clinical trials. EXPERIMENTAL PROCEDURES

Conjugation of mAb and Radiolabeling. The preparation, 111In and 90Y labeling, and in vitro characterization of cT84.66-DOTA have been described previously (13). Reagents, synthesis of MC-DOTA, antibody conjugation, radiometal labeling, and in vitro evaluation of cT84.66-MC-DOTA were detailed in the preceding paper by Lewis and Shively (14). Briefly, the mAb conjugates were labeled with 111In by incubating the conjugate with 111InCl3 in 0.25 M ammonium acetate for 45 min at 43 °C. Then EDTA was added to a final concentration of 1 mM, and the mixture was incubated at 37 °C for 15 min. Labeling with 90Y was performed using a 1 h incubation at 43 °C, after which DTPA was added to a final concentration of 1 mM, and the mixture was incubated for an additional 15 min at 37 °C. The radiometal-labeled mAb conjugates were purified by size exclusion HPLC, using a TosoHaas TSKgel G2000 SW column (10 µm, 7.5 × 300 mm) and an isocratic mobile phase of normal saline at a flow rate of 0.5 mL/min. In each case, the radiolabeled mAb peak was collected in 0.5 mL fractions containing 1 drop of 25% (w/v) human serum albumin, and the fraction containing the highest activity of radiolabeled mAb was used to prepare biodistribution doses. The cT84.66-MC-DOTA conjugate used for the biodistribution experiments was not treated with iodoacetic acid, a procedure which was shown to afford a moderate improvement in serum stability (14). cT84.66-DOTA Biodistributions. In the case of cT84.66-DOTA, separate animal experiments were conducted with either the 111In or 90Y label. Female nu/nu BALB/c mice were injected subcutaneously with approximately 106 LS174T tumor cells in the hind flank, and tumors were allowed to grow for 10-14 days. Radiolabeled immunoconjugate (8 µCi of 111In per 3.79 µg of mAb or 30 µCi of 90Y per 3.49 µg of mAb) was injected into the tail vein of the animals when single tumors were palpable. Biodistributions were obtained at 0, 4, 18, 48, 72, and 96 h postinjection of the 111Inlabeled antibody. Corresponding values for the 90Y label were 0.75, 6, 24, 48, 72, and 96 h. At these times, animals were euthanized and tissues (drained of blood) were taken, weighed, and counted in a well counter. Tissues included blood, liver, spleen, kidney, lung, bowel, bone, tumor, and carcass for the 111In label. 90Y biodistributions included blood, liver, spleen, kidney, bone, tumor, and carcass sampling. For the 111In label, γ radiation was counted directly. 90Y samples were quantitated using a β- counter detecting Cerenkov radiation produced in a fixed volume (tissue plus saline) of 10 mL. Prior to Cerenkov counting, blood samples were clarified

DOTA and MC-DOTA Immunoconjugate Biodistributions

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by treatment with 10% sodium dodecyl sulfate at room temperature, bleach at 56 °C, and 30% hydrogen peroxide at room temperature. Other tissue samples were dissolved in perchloric acid at 56 °C, followed by treatment with 30% hydrogen peroxide at 56 °C and with bleach at room temperature, to make the emitting volume transparent to Cerenkov radiation. By comparison with a standard equal in activity to the injected dose per animal, the decay of either radiolabel was corrected so that normal organ and tumor uptake were calculated as the percent injected dose per gram of tissue. cT84.66-MC-DOTA Biodistributions. When the cT84.66-MC-DOTA biodistributions were evaluated, we had become aware that tumor sizes could be different in separate groups of animals used for different radiolabels. Thus, in the case of cT84.66-MC-DOTA, the two radiolabeled preparations were mixed (8 µCi of 111In per 10 µCi of 90Y per 2.22 µg of mAb) and co-injected into the same animal group. Biodistributions were performed at 0, 4, 12, 18, 48, 96, and 120 h postinjection. 111In and 90 Y counting were performed as described above, and count crossover was taken into account in these analyses. Because of the logistical difficulty of processing a large number of tissues simultaneously for both γ and Cerenkov counting, only blood, liver, bone, and tumor were evaluated for 90Y activity per gram. In addition to those tissues, 111In counts from spleen, lung, kidney, bowel, and carcass were also measured. Statistical Methods. To compare differences between 111In and 90Y for the DOTA chelate at comparable time points (48, 72, and 96 h) and for each organ separately, the independent two-sample t-test was used (15). Similarly, the t-test was used to compare differences between the cT84.66-DOTA and cT84.66-MC-DOTA conjugates for a given label at comparable time points (4, 18, 48, and 96 h) and for each organ separately. For the cT84.66-MC-DOTA biodistribution studies, the agent was labeled with both 111In and 90Y. Therefore, to evaluate the agreement between the organ biodistributions for the two labels, Lin’s concordance correlation coefficient (16) was estimated for each time point separately and all time points combined (referred to as pooled concordance coefficients). The concordance correlation coefficient evaluates the degree to which the pairs of data fall on the line of identity. All statistical testing was done at the p ) 0.05 level using the SAS/STAT software package (17). Blood Clearance. The rapidity of blood clearance was evaluated for all four combinations of BCA and radiometal. In these analyses, first and second biological half-lives were obtained by using the ADAPT II program of D’Argenio and Schumitzky (18). A biexponential function of time (t) having the form

UB(t) ) A1 exp(-k1t) + A2 exp(-k2t)

Figure 1. (Top) Percent injected dose per gram (% ID/g) of blood (b), liver (9), bone (O), and tumor (4) for 111In-labeled cT84.66-DOTA in LS174T tumor-bearing nude mice. (Bottom) Percent injected dose per gram (% ID/g) of spleen (2), kidney (0), lung ([), and bowel (3) for 111In-labeled cT84.66-DOTA in LS174T tumor-bearing nude mice. Standard errors of the mean (SE) are indicated; all data were corrected for radiodecay.

(1)

was fit to the blood data that had been corrected for radiodecay. In the equation, UB(t) represents the blood activity in percent injected dose per gram. Inverting the first and second rate constants allowed us to determine the two corresponding biological half-lives. These were intracompared among the radioimmunoconjugates studied here and also to the values of the antibody-chelate conjugates in the literature (9). It should be kept in mind that a double-exponential function may not be the best description of blood clearance. It is, however, a standard form that has been used in earlier analyses and can be applied, to the lowest order, in these data sets.

Figure 2. Percent injected dose per gram (% ID/g) of blood (b), liver (9), bone (O), tumor (4), spleen (2), and kidney (0) for 90Ylabeled cT84.66-DOTA in LS174T tumor-bearing nude mice. Standard errors of the mean (SE) are indicated; all data were corrected for radiodecay. RESULTS

cT84.66-DOTA Biodistributions. The results of separate biodistribution studies for 111In- and 90Y-labeled cT84.66-DOTA are given in Figures 1 and 2, respectively. For both radiometals, blood was the dominant normal tissue out to 48 h. High uptakes were seen in

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Figure 4. Percent injected dose per gram (% ID/g) of blood (b), liver (9), bone (O), and tumor (4) for 90Y-labeled cT84.66-MCDOTA in LS174T tumor-bearing nude mice. Standard errors of the mean (SE) are indicated; all data were corrected for radiodecay.

Figure 3. (Top) Percent injected dose per gram (% ID/g) of blood (b), liver (9), bone (O), and tumor (4) for 111In-labeled cT84.66-MC-DOTA in LS174T tumor-bearing nude mice. (Bottom) Percent injected dose per gram (% ID/g) of spleen (2), kidney (0), lung ([), and bowel (3) for 111In-labeled cT84.66MC-DOTA in LS174T tumor-bearing nude mice. Standard errors of the mean (SE) are indicated; all data were corrected for radiodecay.

the lung (18% ID/g) initially and in the liver as time exceeded 18 h. With the 111In label, hepatic accumulation (11-14% ID/g) eventually exceeded that of 90Y (4-7% ID/ g). The highest accumulation, however, was ultimately obtained in the tumor, where values approached 60% ID/g at time points as early as 18-24 h. This level of tumor uptake remained relatively constant out to 96 h for both radiometals. cT84.66-MC-DOTA Biodistributions. Organ and tissue uptakes for the simultaneous biodistribution of 111 In- and 90Y-labeled cT84.66-MC-DOTA are shown in Figures 3 and 4, respectively. As in the case of the DOTA conjugate, blood was the dominant normal tissue out to 48 h. At early time points, lung accumulation was the second highest value with the 111In label (9-17% ID/g). No lung data were obtained with 90Y. Hepatic uptakes for both labels approached 10% ID/g by the end of the experimental period. Tumor accumulation exceeded 60% ID/g for both radiometals at 48 h but began to decrease slowly at later time points. Comparison of Radiometals. Comparison of the 111In and 90Y uptakes resulting from the cT84.66-DOTA agent revealed that only the liver showed consistently different values at the three common time points (48, 72, and 96 h). At these intervals, hepatic uptake of 111In was consistently higher (p < 0.05) than that of 90Y. None of the other six tissues showed more than one point at which the uptakes differed significantly (p > 0.05).

Figure 5. Concordance correlation analysis of blood data for cT84.66-MC-DOTA. Uptakes are plotted along both axes, with the 111In-labeled cT84.66-MC-DOTA along the horizontal and the 90Y-labeled cT84.66-MC-DOTA along the vertical axis. All time points are considered. The reference line shown is the line of identity. 111In and 90Y uptakes from the cT84.66-MC-DOTA biodistributions were evaluated using the Lin concordance coefficient (16). For blood, bone, tumor, and liver, pooled concordance coefficients were 0.9942, 0.8698, 0.9042, and 0.6460, respectively. Graphical representations of the blood and liver data are shown in Figures 5 and 6. It was noteworthy that the reduced concordance of hepatic samples was a result of greater accumulation of 111In from the MC-DOTA conjugate in liver tissues. Reference lines shown on each of the two graphs are the lines of identity. Bifunctional Chelate Effects. The biodistribution properties of the cT84.66-DOTA and cT84.66-MCDOTA conjugates were compared for the 111In label. Five time points were common to the two experiments: 0, 4, 18, 48, and 96 h. Blood values of 111In were generally higher for cT84.66-DOTA than for cT84.66-MC-DOTA throughout, but statistically significant differences were found only at 18 and 48 h. The cT84.66-MC-DOTA conjugate showed significantly greater 111In uptake in the kidneys at two of five times (48 and 96 h). Corresponding tissue uptakes of 90Y from cT84.66DOTA and cT84.66-MC-DOTA were also compared. Four tissues were available at two common time points.

DOTA and MC-DOTA Immunoconjugate Biodistributions

Figure 6. Concordance correlation analysis of liver uptake for cT84.66-MC-DOTA. The 111In-labeled cT84.66-MC-DOTA is plotted along the horizontal and the 90Y-labeled cT84.66-MCDOTA along the vertical axis. All time points are considered. The reference line shown is the line of identity.

At both times, significantly lower 90Y blood levels were obtained with cT84.66-MC-DOTA. No significant differences were observed for hepatic or bone uptakes. 90Y Loss from Macrocycle and Bone Uptake. Bone uptake of 90Y was compared for the two macrocyclic BCAs using the t-statistic. Using data from Figures 2 and 4, no significant differences were seen in bone uptake at the 5% level of confidence. Initial values (t ) 0) were on the order of 2% ID/g and decreased to 1.07% ID/g by the end of the experiment (t ) 96 h) for 90Y-labeled cT84.66DOTA. In the case of cT84.66-MC-DOTA, the experiments were carried out to 120 h, at which time the bone uptake had been further reduced to 0.95% ID/g. Blood Curve Analyses. Results from the blood curve analyses are given in Table 1. Half-lives were determined from the rate constants by the relationship T1/2 ) ln(2)/k. The first exponential (k1) could be estimated, but its error or that of its associated half-life was indeterminate for either macrocycle-antibody conjugate. The primary reason for this difficulty appeared to be a slight inflection in the blood curves for cT84.66-DOTA and cT84.66-MC-DOTA, making eq 1 a more approximate representation for the early time points (18). For both radiometals, it was found that characteristic blood halftimes were comparable for cT84.66-DOTA and cT84.66MC-DOTA. Initial biological half-times were generally on the order of 0.2-0.4 h, while the second biological halftime was much larger, on the order of 40 h or longer. Figure 7 shows the 90Y blood clearance curves for cT84.66-DOTA and cT84.66-MC-DOTA. The curve for the MC-DOTA conjugate was below that of the corresponding DOTA conjugate throughout the time range; significant differences were observed at both common time points (48 and 96 h). It should be noted that 111Inlabeled cT84.66-MC-DOTA exhibited faster blood clearance throughout the experimental period, but this difference was only significant at two time points, as described above. DISCUSSION

Figures 1-4 show that both the amide-linked cT84.66DOTA conjugate and the cT84.66-MC-DOTA conjugate, with its cleavable imide linker, demonstrate high tumor targeting in the LS174T nude mouse model. These results were consistent for both radiometals. At the 48 h time point, tumor uptake of the DOTA conjugate was

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between 3- and 5-fold greater than that of the normal tissue having the next highest accumulation (typically blood or liver). The MC-DOTA conjugate exhibited an approximate 7-fold difference between tumor and the highest normal tissue uptake at 48 h. The absolute magnitude of tumor uptake was between 50 and 70% ID/g for both conjugates. These values were comparable to those observed previously in the LS174T nude mouse model by our group (12) for cT84.66 conjugated with a benzylisothiocyanate derivative of DTPA. Other xenograft data using the Lym-1 (7) and chimeric L6 (9) antibodies conjugated to DOTA derivatives have indicated maximum uptakes approaching only 17 and 18% ID/g, respectively. These lower values may be a result of differences in antibody-antigen affinity constants and/ or xenograft models. Tumor masses were not specified in those earlier analyses; our xenograft sizes ranged from approximately 0.1 to 0.2 g at the 48 h time point. Table 2 contains a summary of our biodistribution comparisons. It was seen that hepatic accumulation of 111In was significantly greater than that of 90Y when either BCA was coupled to cT84.66. Transchelation of 111In by transferrin is one possible mechanism for transport to the hepatocytes, but we found no evidence of this mechanism with either conjugate in vitro or in vivo. Kinetic stability studies of radiolabeled cT84.66-DOTA in human serum (13) showed that 90Y > 90Y MC-DOTA < DOTA DOTA < MC-DOTA MC-DOTA < DOTA 111In 111In

Significant when p < 0.05. b Significant difference was determined by concordance analysis.

son of blood clearance data. Since β--induced tissue radiation doses are proportional to the area under that tissue’s curve (AUC), it is appropriate to compare the ratios of such areas for the two BCAs studied here. For blood-associated radioactivity, the relevant ratio is AUC(tumor)/AUC(blood). In the case of cT84.66-MC-DOTA, this value was 4.06, while the comparable magnitude for cT84.66-DOTA was only 3.22. Thus, assuming that the blood activity curve was of primary toxicity significance as a result of the blood-induced marrow dose, the cT84.66-MC-DOTA conjugate appeared to be superior. This result could provide a reason for selecting this conjugate for use in clinical RIT trials. ACKNOWLEDGMENT Figure 7. Blood clearance curves (% ID/g) for 90Y-labeled cT84.66-DOTA (b) and cT84.66-MC-DOTA (O). All data have been corrected for radiodecay. Measured standard errors of the mean (SE) are indicated.

Blood clearance kinetic analyses were consistent (Table 1) with biphasic curves for all combinations of radiometal and BCA. The difference between the two half-times was almost 2 orders of magnitude, with the larger value being between 40 and 70 h. This T1/2,2 was somewhat smaller than the comparable quantity observed by DeNardo et al. (9). This difference may not be significant, as no errors were quoted for their T1/2,2 values. By comparing 90Y data for our DOTA and MC-DOTA conjugates, we have been able to show that the latter BCA results in a generally lower blood clearance curve (Figure 7). This difference may have important consequences in the bone marrow-absorbed dose in a clinical situation. The dominant source organ for the bone marrowabsorbed dose in patients cannot be predicted at this time. If we assume that murine data correspond to the human case, it can be anticipated that blood and bone will be contributors. Both BCAs studied here exhibited substantially reduced bone uptake compared to the benzylisothiocyanato-DTPA chelate studied earlier by our group (12). Thus, the use of a macrocycle can be generally justified for minimization of the marrow dose arising from bone as a source organ. As noted above, skeletal uptake of 90Y was comparable for both BCAs. If the bone marrow radiation dose in human subjects proves to be dominated by circulating 90Y-labeled antibody, selection of an optimal agent for RIT applications may be accomplished using a direct numerical compari-

This work was supported by funds from NIH Research Grant CA43904, Cancer Center Core Grant 33527, and Department of the Army Grant DAMD17-96-1-6047 (predoctoral fellowship to M.R.L.). The content of this article does not necessarily reflect the position or the policy of the United States government, and no official endorsement should be inferred. Supporting Information Available: Tables of mean percent injected dose per gram (% ID/g) with standard errors (SE) of 111In- and 90Y-labeled cT84.66-DOTA and cT84.66-MC-DOTA for all tissues and time points evaluated (4 pages). Ordering information is given on any current masthead page. LITERATURE CITED (1) Waldmann, T. A. (1991) Monoclonal Antibodies in Diagnosis and Therapy. Science 252, 1657-1662. (2) Williams, L. E., Beatty, B. G., Beatty, J. D., Wong, J. Y. C., Paxton, R. J., and Shively, J. E. (1990) Estimation of Monoclonal Antibody-associated Y-90 Activity Needed to Achieve Certain Tumor Radiation Doses in Colorectal Cancer Patients. Cancer Res. 50 (Suppl.), 1029s-1030s. (3) Kaminski, M. S., Zasadny, K. R., Francis, I. R., Milik, A. W., Ross, C. W., Moon, S. D., Crawford, S. M., Burgess, J. M., Petry, N. A., Butchko, G. M., Glenn, S. D., and Wahl, R. L. (1993) Radioimmunotherapy of B-Cell Lymphoma with [131I]Anti-B1 (Anti-CD20) Antibody. N. Engl. J. Med. 329, 459-465. (4) Wessels, B. W., and Rogus, R. D. (1984) Radionuclide selection and model absorbed dose calculations for radiolabeled tumor associated antibodies. Med. Phys. 11, 638-645. (5) Kozak, R. W., Raubitschek, A., Mirzadeh, S., Brechbiel, M. W., Junghaus, R., Gansow, O. A., and Waldmann, T. A. (1989) Nature of the Bifunctional Chelating Agent Used for Radio-

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