Effects of Radiolabeling Monoclonal Antibodies with a Residualizing Iodine Radiolabel on the Accretion of Radioisotope in Tumors1

Home , Isotopes of yttrium

[CANCER RESEARCH 55. 3132-3139, July 15. 19*51 Effects of Radiolabeling Monoclonal Antibodies with a Residualizing Iodine Radiolabel on the Accretion of Radioisotope in Tumors1

Rhona Stein,2 David M. Coldenberg, Suzanne R. Thorpe, Amartya Basu, and M. Jules Mattes

Garden Siale Cancer Center al the Center far Molecular Medicine and Immunology [R. S., D. M. G.. M. J. M.] and Department of Biochemistry and Molecular Biology [A. B.], Graduate School of BiomÃ©dicalScience. University of Medicine and Dentistry of New Jersey. Newark, New Jersey 07103; and Department of Chemistry, University of South Carolimi. Columbia. Smith Carolina 29208 [S. R. T.I

ABSTRACT Thus, only mAbs internalized via clathrin-dependent endocytosis (coated pits) will be internalized efficiently within 1-2 h. Most mAbs The effect of using a "residualizing" iodine radiolabel. dilactitol-iodo- are probably internalized by the non-clathrin-dependent pathway, u ramine, for radioimmunolocali/ation of antibodies to tumors was inves which is much slower (reviewed in Ref. 3). Similar results have been tigated. This tracer is designed to be lysosomally trapped after catabolism of the labeled antibody, m \lis RS7 and RSI 1 were used for m vivo and in obtained with carcinomas of various histolÃ³gica! types, astrocytomas, vitro studies on the uptake and retention of radioisotope into tumor cells. and melanomas (1, 2). Both are murine IgGl mAbs with pancarcinoma reactivity, which react Once the antibody is catabolized, which occurs within lyso- with integral membrane glycoproteins. mAb RS7 has been shown to be somes, the fate of the radiolabeled catabolic product becomes a key relatively rapidly catabolized by the antigen-bearing cell line Calu-3, factor. It is known that iodotyrosine rapidly exits from the lyso- whereas RSI I is catabolized more slowly in the same cells. An "'In- or ""Y-p-isothiocyanatobenzyl-diethylenetriamine pentaacetic acid conjugate some and the cell after its generation after catabolism of conven tionally iodinated proteins (4-6). However, other radiolabels are was also tested because these radiometals are known to be lysosomally trapped within lysosomes, essentially because of their inability to trapped, and iodination via chloramine T was used to provide a baseline. In vitro, a substantial increase in retention of the label by cells was cross the lysosomal membrane. Iodinated radiolabels have been observed when the dilactitol-tyramine DLT- or "'In-labeled mAbs were designed to be lysosomally trapped by taking advantage of their linkage to nonmetabolizable disaccharides (reviewed in Ret". 7). It used, and the improvement gained by the use of these residualizing labels was greater with the use of the rapidly catabolized mAb (RS7) than it was might be expected that such radiolabels would accumulate within with the more slowly catabolized mAb 1RS II). In biodistribution studies tumor cells to a greater extent than conventional iodine labels, and in nude mice bearing Calu-3 tumor xenografts, a dramatic improvement such results have been demonstrated in vitro (4, 8, 9). However, in in the tumor accretion of the radiolabel was seen with the use of the '"1-labcled DLT- or ""Y-labeled mAbs. For example, at day 7 the per vivo results with such residualizing labels have not, to date, been impressive, partly because of accretion in certain normal tissues. centage of injected dose/g in the tumor was 5.54 Â± 1.47% (SD), The cellobiose-tyramine label was tested by the methods of Ali et 38.06 Â±8.04%, and 43.18 Â± 19.50% for the conventionally iodinated, DLT- and ""Y-labeled RS7, respectively. Dosimetry calculations per al. (9, 10) using an anti-Thy-1.1 mAb and a mouse T-cell lym- formed on the biodistribution data predict increases of approximately 8- phoma, but only slightly increased tumor accretion was demon and 4-fold in the absorbed dose to tumor with the use of ' " l-laholt'il 1)11- strated. Demignot et al. (11), using radioiodine-labeled DLT mAb and '"'Y-labeled mAbs, respectively, compared to the conventional 'â€¢"!.In 79IT/36 and the human sarcoma 79IT, found increased tumor contrast to in vitro findings, these results were similar for both RS7 and accretion of the DLT label, relative to chloramine T, but also RSI 1, suggesting that the use of DLT may be advantageous for most of the increased accretion in many normal tissues, and there was not a mAbs binding to the cell surface, including antibodies that are catabolized relatively slowly. The advantage of >3ll-labeled DLT over *"Y is due to the large improvement in tumor/nontumor ratios. However, it seemed longer physical half-life of the '" I. worthwhile to perform additional experiments with the DLT label for several reasons. The only direct comparison of the two residu alizing labels suggested that DLT might be superior to cellobiose- INTRODUCTION tyramine (12). The DLT label was tested only with mAb 791T/36, which appears to localize within tumors primarily in the The delivery of radiation to tumor cells by radiolabeled mAbs3 stroma adjacent to the tumor, rather than on the tumor cell surface depends on both the ability of antibodies to penetrate solid tumors to (13). Clearly, the advantage of residualizing labels should best be reach antigens within them and the processing of the antibodies after seen with the use of mAbs that bind to the cell surface and are antigen binding. mAbs binding to the cell surface can potentially be catabolized. internalized and catabolized by the cells. Although it is frequently assumed that few mAbs are "rapidly" internalized, we have recently In previous studies of radioimmunodetection and radioimmuno therapy, we have utilized mAbs RS7 and RS11, which react with reported that all mAbs binding to the surface of human tumor cells in integral membrane proteins on the surface of lung carcinoma cells, vitro are gradually catabolized with a half-life of 1-2 days (1, 2). Such and other carcinomas (14-17). RS7 was shown to be catabolized a rate of catabolism will have a major effect on radioimmunotherapy with isotopes such as "'I and '"'Y, with decay half-lives of 3-8 days. relatively rapidly (18), and RS11, an antibody to EGP-2, is catabo lized more slowly at a rate similar to that for most mAbs binding to The apparent controversy regarding mAb internalization is readily the cell surface (2). Although we have not directly examined the mode explained in terms of the time point utilized in different experiments. of endocytosis involved, it is likely that RS11 internalization is via noncoated vesicles, as discussed previously (1). The faster catabolism Received 1/12/95; accepted 5/12/95. The costs of publication of Ihis article were defrayed in part by the payment of page of RS7 is compatible with either a coated or noncoated vesicle charges. This article must therefore be hereby marked advertisement in accordance with pathway (19). This experimental system seemed useful for investigat 18 U.S.C. Section 1734 solely to indicate this fact. ' Supported in part by USPHS Grant CA-60039 (R. S.) and DK-25373 (S. R. T.) from ing the advantage of residualizing radiolabels. We anticipated that the NIK. DLT would be most useful with the rapidly catabolized mAb and be - To whom requests for reprints should be addressed, at the Center for Molecular less of an advantage with the slowly catabolized mAb. However, our Medicine and Immunology, I Bruce Street. Newark. NJ 07103. *The abbreviations used are: DLT. dilactitol-tyramine; DTPA. diethylcnetriamine results demonstrated that the use of DLT provided a large advantage pentaacetic acid; ITC-Bz. /i-isothiocyanatobenzyl; %ID/g. percentage of injected dose/g. with both mAbs. 3132

MATERIALS AND METHODS animals. Details on the quantities of radioisotope injected are indicated in "Results" for each study. The animals were sacrificed at the times indicated, in Mis, Cell Lines, and Radiolabeling. The production and initial charac and the radioactivity in the tumor, liver, spleen, kidneys, lungs, small and large terization of RS7 and RS11 (also referred to as RS7-3G11 and RS11-51, intestines, muscle, bone (whole femur), and blood was determined after cor respectively) have been described previously (14, 18). Ag8 (American Type rection for physical decay in a "y-scintillation counter. Results are given as the Culture Collection, Rockville. MD), an irrelevant mouse myeloma IgGl des mean Â±SD of four to five animals/group. ignated P3 X 63 AgH, was used as a negative control antibody in this study. Radiation dose estimates were determined by first integrating the trapezoi The antibodies were purified from ascites fluid by passage through a protein dal regions (for tumors) or exponential regions (for normal tissues) defined by A-immunoadsorbent column. the time activity data (corrected for physical decay). Trapezoidal integration Calu-3, a human adcnocarcinoma of the lung cell line, was purchased from was also used for estimation of the bone and spleen dose in the yttrium study the American Type Culture Collection. The cells were grown as monolayers in because the correlation coefficient was <0.y for the exponential fit for these RPM1 1640 (JRH Biosciences, Lenexa, KS) supplemented with 5% fetal tissues. To generate conservative dose estimates that avoid ovcrestimation of bovine serum, 5% horse serum, 100 units/ml penicillin, 100 fig/ml streptomy the tumor cumulative dose and underestimation of the cumulative dose in the cin, and 2 HIML-glutamine. The cells were routinely passaged after detachment normal tissues calculated with the use of trapezoidal integration, a zero time with trypsin-0.2% EDTA. value of zero is assumed for the trapezoidal fit of the tumor; for the normal mAbs were radioiodinated with I31I or '-'I (New England Nuclear, North tissues, the zero time value is estimated by extrapolating the line described by Billerica, MA) by the chloramine-T method (20) or via dilactitol-tyramine by the first data pair. For the other tissues, the zero time point was extrapolated the method of Strobel et al. (12). Briefly, 5 nmol DLT in 25 (nl 0.5 M sodium according to the exponential curve. The resulting integral for each organ is phosphate buffer (pH 7.6) were added to 0.5 ml polypropylene vials (number converted to cGy/mCi with the use of S values appropriate for isotope and 72.730; Sarstedt, Pennsauken, NJ), which had been coated with 60 /j.g lodogen organ weight. These S values arc calculated for each isotope by assuming (Pierce Chemical Co., Rockford, IL) evaporated from 150 fÃÃdichloromethane. uniformly distributed activity in small unit-density spheres (25). Five mCi "'I, diluted to 50 /A!in the same buffer, were added and incubated 30 min at room temperature. After transfer to a clean vial, 4 units of galactose RESULTS oxidase (Sigma Chemical Co., St. Louis, MO) were added in 5 /Â¿Iofthe same buffer and incubated 45 min at 37Â°C.After addition of 0.25 mg mAb in 15-50 Retention and Processing of mAbs on Calu-3 Cells in Culture. H.1PBS [0.15 M NaCI-0.02 M sodium phosphate (pH 7.2)], 2.0 M sodium Calu-3, an adenocarcinoma of the lung cell line, was used as a model cyanoborohydride was added to a final concentration of 40 mM. After 3 h at 37Â°C,conjugated mAb was collected by passage over a PD-10 column (Phar system for evaluating the retention and cellular processing of radio- macia, Piscataway, NJ) with a column buffer of PBS-IO mM Nal-l() mM labeled mAbs by tumor target cells with the use of mAbs RS7 and NaN,-0.1% gelatin. The iodination efficiency with this method ranged from RS11. Comparisons were made between the binding and processing of 3-6%, resulting in a specific activity of 0.5-1.0 mCi/mg. the two mAbs radioiodinated by either the conventional chloramine-T '"In- and s*Y-labcled mAbs were prepared as described previously (21), procedure or with the use of the residualizing adduci dilactitol- tyramine. The mAbs were also labeled with '"in with the use of with the use of indium from New England Nuclear and Y from Los Alamos National Laboratory (Los Alamos. NM). The bifunctional chelate used in the ITC-Bz-DTPA. The results of the processing studies of the three preparation of these radioimmunoconjugates was the ITC-Bz derivative of radioconjugates of each of these mAbs are summarized in Table 1. DTPA, provided by Immunomedics, Inc., Morris Plains, NJ, and was synthe The data indicate a difference in the retention of the two mAbs sized following the method of Cummins et al. (22). The preparation and testing when radioiodinated by the conventional chloramine-T procedure. of the antibody conjugates were performed as described by Brechhiel et al. The processing of I2?l-labeled RS7 was faster than '-sl-labeled RSI 1, (23) and Meares et al. (24). Specific activities of 3â€”5mCi/mg and 0.1 mCi/mg were produced with '"In and SKY,respectively. consistent with the results of earlier studies on the relative rates of internalization of the two mAbs (2, 18). This was especially apparent Assessment of immunoreactivity after radiolabeling was performed with the use of a direct cell-binding assay (14). at 21 h when only 22.9 Â±0.8% (SD) of initially bound RS7 remained In Vitro Antibody Retention Experiments. As described previously in bound to the cells, whereas with RSI 1, 47.9 Â±2.4% remained. When detail (1), confluent cells in 96-well plates were incubated with 5 X IO5 cpm the residualizing labels were used, the percentage of radiolabel re of labeled antibody for 2 h at 37Â°C,then washed four times to remove unbound tained by the cells increased substantially. With I25l-labeled DLT and labeled antibody. Tissue culture medium (0.2 ml) was added and incubation '"in-labeled mAbs, approximately 60% of initially bound cpm re was continued for various times as indicated in "Results." Under these con mained at 21 h. Nonspecific binding averaged 4.8% (ranging from 2.4 ditions, the cells remain healthy and continue to divide for at least 3 days. At to 7.8%). Both I25l-labeled DLT and '"in labeling yielded approxi desired times, 0.1 ml of media was collected, and after additional washing, the mately equal retention of the labels with either mAb RS7 or RSI 1, cells were solubilized with 2.0 M NaOH. After determining the cpm in the indicating that the rate of antibody internalization and catabolism does culture medium, iodinated samples were precipitated with 5 ml cold 10% trichloroacetic acid, and the indium-labeled samples were precipitated with 5 not affect the cellular retention of the radiolabel when residualizing labels are used. ml methanol with the use of 1.0 mg of bovine IgG as a carrier protein, and the precipitate was collected by centrifugation. Nonspecific antibody binding was determined in each experiment by adding a large excess of unlabeled antibody Table I Pnti'i's.Ã¯inf!of radinconjitfzates h\ Culit-3 cells to control wells. The percent of originally hound mAhs retained by cells at the indicated times were In some experiments, the incubation with radiolabelcd mAbs was continued determined as described in "MalcriÃ¡is and Methods." Values represent the mean Â±SD of for 3 days, in a total volume of 0.2 ml, with other conditions being the same two independent experiments, each done in triplicate. The designation 1251(chloramine-T) represents labeling of the mAbs with the use of the conventional chloramine-T procedure. as described above. At various times, cells were washed 4 times, and the cell-bound cpm was determined. In these experiments, >90% of the binding cpm retained by cells was antigen specific, as shown by inhibition with a large excess of unlabeled mAb Label 4 h 21 h 45 h W h mAb, at 50 fig/ml, which was also present continuously. '-5I (chloramine-T) 79.28 + 2.69 22.91 Â±0.82 9.93 Â±0.98 4.70" RS7 In Vivo Biodistribution Studies. Tumors were propagated in female mi/nu I25l-lahcled DLT 74.37 Â±9.03 64.90 Â±6.00 52.35 + 0.79 45.39 Â±2.33 mice (HarÃanSprague-Dawley. Indianapolis, IN) at 6-8 weeks of age by s.c. "'In 77.(X)Â±11.35 60.11 Â±18.20 49.11 Â±12.16 42.72 Â±11.77 injection of 1-2 X IO7 washed Calu-3 cells, which had been propagated in RSI1125 '-I (chloramine-T) 85.38 Â±1.70 47.86 Â±2.39 33.27 Â±7.09 16.49" tissue culture. The mice were used for in vivo biodistribution studies approx '-'I-labcIed DLT 80.27 + 6.03 58.64 + 6.39 48.48 Â±5.22 41.65 Â±5.37 imately 3 weeks after the injection of cells when tumors reached a weight of "'in 82.51Â±7.90 67.03 Â±13.90 56.28 Â±8.84 51.30 + 8.08 approximately 0.2 g (generally in the range of 0.1-0.4 g). Radioiodinated " No SD is reported because this time point was not determined in the second antibodies were injected i.V.. via the lateral tail vein, into the tumor-bearing experiment. 3133

ments were performed in which the cells were incubated with the mAbs continuously for up to 3 days. This protocol can potentially result in the accumulation of large amounts of radiolabel/cell. At various times, wells were harvested to determine the accumulation of cell-bound cpm. A double label study was performed comparing the residualizing label, nlln, with conventionally conjugated I25I in the same wells. To ensure that uptake was via antigen-specific binding, and not due to fluid-phase uptake of mAb present in the medium, control wells contained a large excess of unlabeled mAb; this was effective at inhibiting mAb uptake by >90%. The results, shown in Fig. 1, demonstrate that accumulation of 125I continued to increase from 2 to 24 h but reached a plateau after 24 h. In contrast, 1uln continued to accumulate for 3 days, the duration of the experiment, with both mAbs. Note, however, that the advantage of the residual izing label, although clearly seen, is less pronounced in this experi ment than it was in the earlier in vitro experiments. This can be attributed to the fact that I25I uptake increases markedly from 2 to 24 h, probably due to cell multiplication, which makes the retention of "'In less prominent. The ratio of the percentage of specifically bound cpm of the two radioisotopes (%'"ln specifically bound/%125I specifically bound) is Fig. 1. Accumulation of radioisÃ³topos in Calu-3 tumor cells in vitro. Adherent tumor cells were incubated for up to 3 days in tissue culture medium containing a mixture of presented in Fig. 1C. This graph shows that the advantage of the mAhs labeled with either 125I(â€¢)or '"In (â€¢).The Ab was either RS11 (A) or RS7 (fl). residualizing label is considerably greater for RS7 than it is for RSI 1 At varying times, cells were washed and collected and the percentage cpm specifically because the ratio increases by 2.4-fold with RS7 and only 1.2-fold for bound was determined. Nonspecific binding was determined by including a large excess of unlabclcd mAb in control wells, and the specific binding was calculated by subtraction. RSI 1, from 2 to 72 h. Therefore, this experiment also suggests that SDs of triplicates were all S0.381* of the total cpm/well for RSI Land so. 11% for RS7. residualizing labels should produce more of an advantage with RS7 C, from the same experiment, the value plotted is (% "'In specifically bound)/(%125l specifically hound). The increase in this ratio with time indicates greater retention of than with RS11. indium in comparison with iodine. (A). RS7; (V), RSI 1. Similar results were obtained in In Vivo Studies. A comparison of the in vivo targeting of mAbs a second experiment. RS7 and RS11 conjugated with the conventional and residualizing radiolabels was performed in nude mice bearing xenografts of Calu-3. As reported previously (1), a relatively small fraction of bound An unreactive mAb, Ag8, was included as a control. These biodistri antibody was released intact from the cells. The majority of released bution studies were performed to assess whether the increases in cpm was found in a degraded form (trichloroacetic acid or methanol tumor cell retention observed in vitro when the residualizing labels (I3'l-labeled DLT and radiometal) were used translate into higher soluble fraction; data not shown). At 3 days, the percentage of bound antibody released in intact form (i.e., not catabolized) varied from ID accretion of radiolabel at the target site in vivo. In addition, the to 27% of the initially bound antibody, depending on the conjugate. In contribution of the rate of mAb internalization and catabolism to general, this value was reached within the first day, in contrast to the radioisotope accretion and tumor:nontumor ratios was investigated by release of degradation products, which continued to increase during comparing the results obtained with the use of the two mAbs. The the 3-day time course. results are summarized in Fig. 2. Marked improvements in the tumor in Vitro Experiments Using a Prolonged Antibody Incubation. accretion of radiolabel were seen with the use of the 1MI-labeled In vivo, an antibody remains at high levels in the circulation for DLT- or xxY-labeled mAbs in comparison to the conventional 131I- several days, although it gradually decreases due to clearance from the labeled antibodies. For example, at day 7 the %ID/g in tumor was blood. To more closely mimic the in vivo situation, in vitro experi 5.54 Â±1.47% (SD), 38.06 Â±8.04%, and 43.18 Â±19.50% for the

60 - 'I-DLT E a 50 - CD OJ o. 40 - CD Fig. 2. Accretion of radioactivity in Calu-3 tumors xe co nografts in nude mice. Mice with tumors were given injec â€¢oo 30 - tions i.v. on day 0 with 10 /iCi (10 /ig) of the conventional -o 1"l-labeled- and "'[-labeled DLT mAbs or 3 /Â¿Ci(30 /ig) of _o> the ""Y-lahelcd mAhs and sacrificed successively. â€¢,RS7; 20 - O, RS11; A, Ag8. .92.'e

10 - B--ZS a

100 200 300 O 100 200 300 O 100 200 300 400 Hours post injection 3134

Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1995 American Association for Cancer Research. RESIDUALIZING RADIOLABELS ON mAbs conventional '-"I-, ml-labeled DLT-, and 88Y-labeled RS7, respec 100 -. Liver Spleen Kidney tively. No significant differences in tumor accretion were observed between RS7 and RS11 with any of the labels. For certain data points, the SDs for tumor accretion of mAb were relatively high. This 10 -3 variation among tumors could not be fully explained by variation in tumor size or by any other characteristic that has been identified; thus, variation can be attributed only to unknown variation in the physio 1 logical properties of the tumors.

The results of these studies are shown in more detail in Figs. 3 and 100 Lungs Sm Int Lglnt 4, which show the percentage of the injected dose of radiolabel/g of --a normal tissue and the tumonnontumor ratios for these tissues for the CO RS7 studies over the 14-day time course. Similar results were ob DC 10 -. served with the radiolabeled RS11 (data not shown). As seen in Fig. 3, the % ID/g of radioisotopes were relatively low for all three labels in the normal tissues. This indicates that although the 131I-labeled 1 DLT and 88Y labels are accumulating in the tumor to levels approx imately 7-8 times greater than those observed with the use of con 100 Blood Bone Musei ventional 13ll-labeled mAbs (Fig. 2), the accretion in normal tissues is not changed as much by the use of the residualizing labels. The effect of increased tumor accretion without matching increases in normal 10 -. tissue accretion is the generation of increases in tumonnontumor ratios (Fig. 4). Tumonliver ratios at day 7 are 3.40 Â±0.69 (SD) with 13ll-(conventionally)labeled RS7 compared to 16.26 Â±4.12 with 131I-labeled DLT-RS7 and 15.49 Â±4.66 with the use of 88Y-labeled RS7. Maximal tumonnontumor ratios were seen in the muscle with 0 100 200 300 0 100 200 300 0 100 200 300 400 90.64 Â±25.49 and 84.00 Â±31.62 observed at day 7 after injection of Hours post injection 131I-labeled DLT-RS7 and 88Y-labeled RS7, respectively, compared Fig. 4. Tumonnontumor ratios for radiolabeled RS7 in nude mice bearing Calu-3 to 11.60 Â±2.42 with the use of conventional 131I-labeled RS7. At day tumors. Mice with tumors were injected i.V. on day 0 with conventional '31I-labeled-RS7 7, the lowest ratios were seen in the blood, at 0.87 Â± 0.19, (â€¢),l31I-DLT-labcled RS7 (O), or ""Y-labeled RS7 (A) and sacrificed successively as 8.84 Â± 1.86, and 8.05 Â±2.73, for the conventional 131I-labeled, described in Fig. 1. Tumornontumor ratios were calculated from the data presented in 131I-labeled DLT, and 88Y-labeled RS7, respectively. Figs. 2 and 3. 5m Int, small intestine; Lg Int. large intestine.

The increases over time in the tumonnontumor ratios were similar Liver Spleen Kidney for the I31l-labeled DLT and 88Y-labeled mAbs, the only exception 12 - being bone, where the tumonnontumor ratio decreases after day 3 with the 88Y label but not with the I3'l-labeled DLT-antibodies. This 8 - most likely results from the known bone-seeking properties of the

4 - yttrium, which has become dissociated from the chelator. The accre tion of yttrium by bone seen here is only significantly different from E that of the iodine labels at the latest time point (14 days after injection) CO Lungs Sm Int Lglnt and would not contribute a significant radiation dose to the bone when 90Y is used therapeutically, due to the short physical half-life (64 h) (D 12 - (A of 9"Y. O Q Residualizing labels will accumulate not only in tumor cells to T3 which the antibody binds but also in any normal tissues that are jg T5 4 - responsible for antibody clearance and catabolism. The organs most CD "E" active in IgG catabolism/g of tissue are the liver, spleen, and possibly the kidney (26). Although the kidney was noted to have increased Blood Bone Muscle accretion of IgG (or its degradation products), the results are difficult 12 - to interpret because the kidney may accumulate degradation products that are generated in other organs (26). Differences in the accretion of the radiolabels in various normal organs are addressed in Fig. 5, which shows the accumulation of 131I-labeled DLT-RS7 in various organs in 4 - the Calu-3-bearing nude mice, in comparison to RS7 labeled conven tionally with 131I. Elevated localization ratios, defined as (%ID/g 131I-labeled DLT mAb)/(%ID/g conventional 131I-labeled mAb), are 0 100 200 300 0 100 200 300 0 100 200 300 400 seen in the liver, spleen, and kidney, as expected (26). Although the Hours post injection liver, spleen, and kidney accrete more radioiodine with I31l-labeled DLT than with the conventional 13II label, the increases in the ratios Fig. 3. Biodistribution of radiolabeled RS7 in nude mice bearing Calu-3 tumors. Mice with tumors were given injections i.v. on day Ãœwithconventional "'Â¡-labeled RS7 (â€¢), are not as large as those observed in the Calu-3 tumor. Thus, the '-"1-labeled DLT-RS7 (O), or ""Y-labeled RS7 (A) and sacrificed successively as de tumonnontumor ratios are increased even for these organs. Fig. 5 scribed in Fig. 2. Tumor, organ, and blood radioactivity were counted. The percentage of suggests that 13ll-labeled DLT slowly accumulates to a small extent in the injected dose/g of tissue was calculated from this data. 5m Ini, small intestine; Lg Ini, large intestine. the bone because the localization ratio increased from day 7 to day 14. 3135

tumonliver ratio with RS7 was lower than that with RSll. This 10 - â€¢Calu-3 difference, which was also seen in other normal tissues, is due to â€¢Spleen faster normal blood clearance of 13I1-RS11 rather than increased D Kidney tumor accretion. Thus, the marked advantage obtained with residual- O Liver T Bone Â¡zinglabels is seen equally well with both mAbs. A Lungs Dosimetry. Cumulative absorbed radiation doses were calculated from the biodistribution data of the above studies (Table 2). X8Ywas O Small Int. g used in the biodistribution experiments in lieu of ""'Y, which would be â€¢S* ^ Large Int. Q V Muscle used therapeutically. This substitution was made because the y emis A Blood sions of S8Y allow counting of the tissues in the y counter. Mean cumulative absorbed doses were computed for '"'Y with the use of the 88Y data because the distribution of the two isotopes of yttrium can be O assumed to be the same. To compare the three studies, the absorbed 05 doses were normalized to a blood dose of 1500 cGy, a level which has O been observed to be approximately the maximal tolerated dose in this 1 - system. The data are summarized in Table 3. The administered doses of these conjugates necessary to achieve a 1500-cGy absorbed dose to co blood are 242 jnCi conventionally l31I-labeled-RS7, 368 /xCi 131I- OC labeled DLT-RS7, and 170 /Â¿Ci""Y-labeled RS7. Although the max-

Table 2 Mean cumulative absorbed dose in Â¡issues to tissue(cGy/mCi)131I-labeled

0 100 200 300 400 500 cr mAb456162089491559945186920574928561322421350577511772558226821814826591069DoseDLTmAb240514077821282957824821817348W22952498834378453344728382016974777992386''"Y-labeledDTPAmAb''3029388442141300510484708317573613423103286261002522893315118555584331Sill14753217 Hours post injection RS7Calu-3BloodBoneKidneyLarge Fig. 5. Relative accumulation of '"l-labeled DLT-RS7 in tumor and normal mouse tissues. The value plotted is the (% injected dose/g of tissue with 131l-labeledDLT- RS7)/(% injected dose/g of tissue with conventional ml-labeled-RS7), to show the preferential accumulation of DLT in certain tissues. For tissues that do not retain either intestineLiverLungsMuscleSmall label preferentially, the ratio will be 1.0.The ratios are derived from the data presented in Figs. 2 and 3. //(/., intestine.

intestineSpleenRSllCalu-3BloodBoneKidneyLarge Similar results were observed in the other experiments described here, and also in experiments utilizing normal BALB/c mice (data not shown). This accretion is likely to be due to the macrophage content of the bone marrow. The possible contribution of the rate of mAb internalization and catabolism to radioisotope accretion and tumonnontumor ratios was intestineLiverLungsMuscleSmall investigated by directly comparing the results obtained with the use of the two mAbs. Tumor:liver ratios of the mAbs are shown in Fig. 6. No significant differences in tumonnontumor ratios were observed be intestineSpleenml-labeled tween RS7 and RSll with the 13ll-labeled DLT or 8SY labels. With " CT. labeling performed with the use of the conventional chloramine-T procedure. the conventional l;"l-labeled mAbs, at day 14 after injection, the ''Calculated from 8NY-labclcdDTPA-RS1I biodistribution data.

crCO i_ CD Fig. 6. Comparison of tumor/liver ratios for radiolabeled RS7, RSll, and Ag8 in nude mice bearing Calu-3 tumor xenografts. Ratios are calculated with the use of data de rived from the animals presented in Fig. 2. â€¢.RS7; O, O RSll; A, Ag8. ,4" 1 _

100 200 300 O 100 200 300 O 100 200 300 400 Hours post injection 3136

Table 3 Mean cumulative absorbed dose normalized to 1500 cGy dose to blood icantly in the rate at which they turn over their membranes. In to tissue(cGy)ml-labeled potential clinical use, antibodies will also bind to certain normal cells that express the particular target antigen, so radioisotope accretion in DTPAmAb''(170 CTÂ°mAb(242 DLTmAb(368 these cells also must be considered. nu)''1,1021,500229377228452497119207319(297 RS7Calu-3BloodBoneKidneyLarge fiCi)8,8491,5003021,041213913669128347844(436M.CÃŒ)5,1381.500363510178799539125228526(150The relationship between radiometals and DLT must be considered because both labels increase the cellular retention of radioisotope. It was recently reported that radiometals bound to strong chelators such as ITC-Bz-DTPA are trapped within cells after catabolism of the intestineLiverLungsMuscleSmall antibodies to which they were conjugated (8) and that they are trapped within lysosomes (28). Although no radiolabel appears to be trapped permanently within lysosomes (7, 8), the retention of DLT and ITC- intestineSpleenRS11Calu-3BloodBoneKidneyLarge Bz-DTPA seemed to be very similar in a direct comparison (8). Thus, it could be predicted that these two labels would produce very similar u.Ci)1,2501,500223526166673647143195317Dose/Â¿Ci)10,9051.5003691,4593181,6677412083491,041"Â°Y-labeledu.Ci)4,2831,5003424%177832648120221481antibody localization results, and this is confirmed in the present study. It has been widely accepted that the difference in tumor localization between radiometals and a conventional iodine label is

intestineLiverLungsMuscleSmall primarily due to deiodination. However, as discussed previously in detail (8), experimental evidence points directly to an explanation based on differences in the processing of the catabolic products rather intestineSpleen'-""[-labeled than deiodination. Because chelated radiometals have been utilized in a large number " CT, labeling performed with the use of the conventional chloramine-T procedure. of investigations and sometimes have been compared with a conven * Calculated from stiY-labeled DTPA-RS11 biodistribution data. 'Numbers in parentheses, the administered dose necessary to achieve 1500 cGy tional iodine label, such results can potentially provide additional adsorbed dose to blood. insight into the advantage of residualizing labels. However, this analysis is complicated by two factors: (a) most earlier studies used relatively unstable chelators, mainly the cyclic anhydride or mixed imal tolerated doses using 131I-labeled DLT mAbs have not yet been anhydride of DTPA. These chelators release a substantial amount of determined, the levels calculated for the conventionally iodinated and free metal, which then binds to transferrin and is taken up by the liver 9"Y-labeled RS7 are comparable to previous experimentally deter (29). Work with these chelators did demonstrate increased accumu mined values for 13II- (27) and 90Y-labeled mAbs.4 Dosimetry cal lation in tumors, relative to an iodine label (30, 31), but in regard to culations performed on the biodistribution data show that increases of normal tissue uptake the results obtained with these unstable chelators approximately 8- and 4-fold in absorbed dose to tumor can be ex are difficult or impossible to compare with our experiments; and (b) a pected to be achieved with the use of 1MI-labeled DLT- and 9"Y- considerable variety of the newer, stronger metal chelators have been labeled mAbs, respectively, compared to the conventional 131I.These developed (29, 32, 33) with some significant differences between results were similar for both RS7 and RSI 1. Comparing ml-labeled them. Although all chelators may release very small amounts of free DLT with 90Y, an increase of about 2-fold in absorbed dose to tumor metal, they may differ in other characteristics, including lysosomal is observed with DLT, due primarily to the longer half-life of 131Iin retention. In view of these considerations, only a few comparisons of comparison to 9"Y. Increased doses to the normal tissues, which had strong metal chelators with iodine labels are clearly relevant to the increased accretion of the residualizing labels (i.e., liver, spleen, and present study. kidney) were also observed. However, the doses to these organs Sharkey et al. (34) compared ITC-Bz-DTPA with iodine using an remain below levels expected to cause toxicity. anticarcinoembryonic antigen mAb. Their results demonstrated in creased tumor accretion of the metal, but there was also increased DISCUSSION normal tissue accretion, and dosimetry calculations indicated that yttrium was probably inferior to iodine for purposes of radioimmu- We report here that a residualizing label such as DLT can provide notherapy. The most extensively characterized mAb tumor system has a great improvement in tumonnontumor localization ratios in com been mAb B72.3 and the LS174T colon carcinoma, in which a wide parison with a conventional iodine label. Improvement is marked not variety of benzyl-DTPA derivatives have been tested (35, 36). In this only for tissues such as lung, muscle, and intestinal, which are not case, it was reported that there was no significant difference in major sites of normal antibody catabolism, but also for the liver, biodistribution between a strong metal chelator and iodine. This result spleen, and kidney, where DLT tends to accumulate. This effect was can perhaps be attributed to the fact that the B72.3 antigen is not a cell observed with use of the lung adenocarcinoma cell line, Calu-3, but surface antigen but rather a secreted mucin, and that the mAb is, was less dramatic with a second carcinoma, LoVo.5 Results published therefore, not internalized and catabolized. This consideration may previously with DLT (11) were more similar to the LoVo results than also apply to the results with carcinoembryonic antigen antibodies were the Calu-3 results, in that relatively high accretion in liver, (34) because the carcinoembryonic antigen occurs in both secreted spleen, and kidney were observed. We cannot currently explain these and membrane-bound forms (37). In this context, we note that differ differences, and additional experimentation in this direction is in ent benzyl-DTPA derivatives may vary in their level of lysosomal progress. Clearly, the rate of mAb uptake in tumors and/or the rate of retention, but this of course can be seen only with mAbs that are mAb catabolism are significant variables that must be taken into efficiently internalized and degraded. Stein et al. (15) compared consideration. At present, we speculate that tumors may differ signif- iodine and indium using mAb RS7 on ME 180 cervical carcinoma tumors in a system essentially identical to that used here except for the 4 R. M. Sharkey. R. D. Blumenthal. G. Y. Wong, G. L. Griffiths, and D. M. Golden berg. Radioimmunothcrapy with cquitoxic doses of I3II-, WY-, and 188Re-labeled mono different target cell. In that study, indium produced higher absolute clonal antibody in human colonie tumor xenograft models, manuscript in preparation. tumor accretion and higher tumonnontumor ratios for some organs 5 M. J. Mattes and R. D. Blumenthal, unpublished data. but lower ratios for the liver and spleen. In summary, although the use 3137

Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1995 American Association for Cancer Research. RESIDUALIZINO RADTOLABELS ON mAbs of indium does generally result in increased tumor accretion, the ments but not for clinical studies. However, we have recently im dramatic difference in tumornontumor ratios described here has not proved the efficiency to approximately 33%, simply by increasing the been described in other experimental systems. Thus, the advantage of concentration of mAb used by a factor of 20. Additional improve residualizing labels will depend on the particular mAb and on the ments in efficiency are under investigation. particular tumor target. In summary, the prolonged retention of 13'l-labeled DLT mAbs in Although DLT and benzyl-DTPA seem to be very similar in terms tumor cells, the 8-day half-life of the I3'I, and the relatively low levels of retention within lysosomes, significant differences between these of accretion in normal tissues combine to make radioiodinated DLT labels remain. One difference is in the accretion of yttrium by bone. an adduci of great potential for radioimmunotherapy. The effective Such bone accretion can occur only after the metal escapes from the ness of this methodology appears not to be limited by whether a mAb chelator; therefore, it is relatively minor when strong chelators such as is rapidly catabolized by antigen-expressing cells and should be of benzyl-DTPA are used. Some release of yttrium does still occur, and general applicability. this material appears to be efficiently taken up by bone. Sharkey et al. (34) concluded that bone accretion was an obstacle to the use of ACKNOWLEDGMENTS benzyl-DTPA chelates of yttrium. This factor, therefore, represents an advantage of DLT over yttrium. The release of yttrium can be reduced The excellent technical assistance of Susan Chen, Rosarito Aninipot, Philip with the use of even stronger chelators, namely the macrocycles (32), Andrews, Mark Przybylowski, and Donald Varga is gratefully acknowledged. but such chelators are inconvenient to use because radiolabeling is less efficient. Our data indicate that bone accretion is probably not REFERENCES entirely due to uptake of free metal but also due to accumulation of 1. Kyriakos, R. J., Shih, L. B., Ong. G. L., Palei, K., Goldenberg, D. M., and Malles. residualizing labels in the bone. Such accumulation was seen with M. J. The fate of antibodies bound to the surface of tumor cells in vitro. Cancer Res., DLT, although at a level much less than in liver, spleen, and kidney. 52: 835-842, 1992. 2. Mattes, M. J., Griffiths, G. L., Diril, H, Goldenberg, D. M.. Ong, G. L., and Shih. This accumulation is unlikely to be due to uptake in the bone itself, L. B. Processing of antibody-radioisotope conjugates after binding to the surface of and we tentatively attribute this to uptake by bone marrow macro tumor cells. Cancer (Phila.). 73: 787-793, 1994. phages. It would be expected that benzyl-DTPA-yttrium would sim 3. van Deurs. B., Petersen, D. W.. Olsnes, S., and Sandvig. K. The ways of endocytosis. Int. Rev. Cytol., 117: 131-178, 1989. ilarly accumulate in bone. Thus, bone accretion can occur by two 4. Geissler. F., Anderson, S. K.. Venkatesan, P.. and Press, O. Intracellular catabolism of distinct mechanisms. However, it should be emphasized that the level radiolabeled anti-n antibodies by malignant B-cells. Cancer Res.. 52.- 2907-2915. 1992. 5. Vaes, G. Digestive capacity of lysosomes. In: H. G. Hers and F. Van Hoof (eds.), of bone marrow accretion of the residualizing labels is very low and, Lysosomes and Storage Diseases, pp. 43-77. New York: Academic Press, 1973. therefore, unlikely to represent a toxicity problem. 6. LaBadie, J. H.. Chapman. K. P., and Aronson, N. N., Jr. Glycoprotein catabolism in The major difference between yttrium and iodine, however, is seen rat liver. Lysosomal digestion of iodinated asialo-fetuin. Biochem. J., 152: 271-279, 1975. not in the tumor:nontumor ratios but only in the dosimetry and is due 7. Thorpe, S. R., Baynes, J. W.. and Chroneos, Z. C. The design and application of to the difference in decay half-life. Thus, an approximately 2-fold residualizing labels for studies on protein catabolism. FASEB J., 7: 399-405, difference in radiation delivered to the tumor is seen with estimated 1993. X. Shih, L. B.. Thorpe, S. R., Griffiths, G. L.. Diril, H., Ong, G. L., Hansen, H. J., equitoxic doses. It is apparent that, due to relatively slow uptake and Goldenberg. D. M., and Mattes, M. J. The processing and fate of antibodies and their accumulation of the radiolabel within the tumors, a 3-day radiation radiolabels bound to the surface of tumor cells in vitro: a comparison of nine half-life is not long enough to take full advantage of the retention of radiolabels. J. NucÃ.Med., 35: 899-908, 1994. 9. Ali, S. A., Warren, S. D., Richter. K. Y.. Badger, C. C., Eary, J. F., Press, O. W., the label at the tumor site. There are few if any other residualizing Krohn, K. A.. Bernstein, I. D., and Nelp. W. B. Improving the tumor retention of radiolabels with relatively long half-lives (1-2 weeks) that have been radioiodinated antibody: aryl carbohydrate adducts. Cancer Res.. 5i>(Suppl.).- utilized. One potential candidate is l77Lu (38). However, it cannot be 783s-788s, 1990. K). Ali, S. A., Eary. J. F., Warren, S. D., Badger, C. C., and Krohn, K. A. Synthesis and assumed that lutetium or other metals will behave identically to the radioiodinalion of tyramine cellobiose for labeling monoclonal antibodies. NucÃ. benzyl-DTPA chelate of indium or yttrium, and results may depend on Med. Biol., 15: 557-561, 1988. 11. Demignot, S.. Pimm, M. V.. Thorpe. S. R.. and Baldwin. R. W. Differences between the particular chelator used. the catabolism and tumor distribution of intact monoclonal antibody (791T/36) and its Although we expected that the advantage of a residualizing label Fab/c fragment in mice with tumor xenografts revealed by the use of a residualizing radiolabe] (dilactitol-I25I-tyramine) and autoradiography. Cancer Immunol. Immu- would be greater for a rapidly catabolized mAb, RS7, than it would be nother., 33: 359-366, 1991. for a slowly catabolized mAb, RSI 1, such a difference was not seen. 12. Strobel. J. L.. Baynes, J. W., and Thorpe, S. R. 125I-glycoconjugate labels for In retrospect, this is perhaps not surprising. The difference in the rate identifying sites of protein catabolism in vivo: effect of structure and chemistry of coupling to protein on label entrapment in cells after protein degradation. Arch. of catabolism of these two mAbs (seen in vitro) is only approximately Biochem. Biophys., 240: 635-645, 1985. 2-fold, which perhaps is a small difference compared to the other 13. Pimm, M. V., and Baldwin, R. W. Localization of an anti-tumor monoclonal antibody processes that are involved. Although we attempted to modify our in in human tumor xenografts: kinetic and quantitative studies with the 791T/36 anti body. In: R. W. Baldwin and V. S. Byers (eds.). Monoclonal Antibodies for Cancer vitro experiment to more closely simulate the in vivo situation, by Detection and Therapy, pp. 97-128. New York: Academic Press, 1985. keeping the labeled mAb in the medium for 2-3 days, these results did 14. Stein, R., Chen, S., Sharkey. R. M., and Goldenberg, D. M. Murine monoclonal antibodies raised against human non-small cell carcinoma of the lung: specificity and not appear to more closely parallel the in vivo data. This discrepancy tumor targeting. Cancer Res.. 50: 1330-1336, 1990. may be due to differences in the physiological state of the cells when 15. Stein. R., Blumenthal, R.. Sharkey R. M., and Goldenberg, D. M. Comparative grown in nude mice or in tissue culture. For example, differences in biodistribution and radioimmunotherapy of monoclonal antibody RS7 and its F(ab')Ã¯ in nude mice bearing human tumor xenografts. Cancer (Phila.), 73: 816-823, 1994. the rate of membrane turnover would have major effects on the rate 16. Stein. R., Basu. A.. Chen. S.. Shih, L. B.. and Goldenberg. D. M. Specificity and of antibody processing. Because RSI 1 has consistently been observed properties of mAb RS7-3GI1 and the antigen defined by this pancarcinoma mono to be processed in a manner similar to the majority of "typical" clonal antibody. Int. J. Cancer, 55: 1-9, 1993. membrane-reactive mAbs on a variety of cell lines (2), we expect that 17. Stein, R., Basu, A., Goldenberg, D. M., Lloyd, K. O.. and Mattes, M. J. Character ization of Cluster 13: the epithelial/carcinoma antigen recognized by MAb RS7. Int. most mAbs reacting with the surface of Calu-3 would be processed J. Cancer, 8 (Suppl.): 98-102. 1994. 18. De Leij, L.. Helfrich, W., Stein, R., and Mattes, M. J. SCLC-Cluster-2 antibodies similarly. detect the pancarcinoma/epithelial glycoprotein EGP-2. Int. J. Cancer. 8 (Suppl.). Additional use of DLT for radioimmunotherapy is complicated 60-63, 1994. partly by relatively low conjugation efficiency. The efficiency of 19. Huet. C., Ash, J. F., and Singer, S. J. The antibody-induced clustering and endocytosis protein iodination obtained in these studies, 3-6%, is sufficient for of HLA antigens on cultured human fibroblasts. Cell, 21: 429-438, 1980. 20. McConahey. P. J., and Dixon, F. J. A method of trace iodination of proteins for radioimmunodetection experiments in mice and for in vitro experi immunologie studies. Int. Arch. Allergy Appi. Immunol.. 2V: 185-189. 1966. 3138

21. Motta-Hennessy, C., Sharkey, R. M., and Goldenberg, D. M. Metabolism of indium- and kinetics of ' "In-labeled monoclonal antitumor antibodies in normal animals and 111-labelcd murine monoclonal antibody in tumor and normal tissue of the athymic nude mouse-human tumor models. Cancer Res., 43: 5347-5355, 1983. mouse. J. Nuc!. Med., 31: 1510-1519. 1990. 31. Khaw, B. A., Cooney, J., Edgington, T., and Strauss, H. W., Differences in experi 22. Cummins, C. H., Rutter, E. W., Jr., and Fordyce, W. A. A convenient synthesis of mental tumor localization of dual-labeled monoclonal antibody. J. NucÃ.Med., 27: bifunctional chelating agents based on diethylenetriamine pentaacetic acid and their 1293-1299, 1986. coordination chemistry with yttrium (III). Bioconjug. Chem., 2: 180-186, 1991. 32. Meares, C. F., Moi, M. K., Diril, H., Kukis, D. L.. McCall, M. J., Deshpande, S. V., 23. Brechbiel M. W., Gansow, O. A., Atcher, R. W., Schlom, R. W., Esteban, J., De Nardo, S. J., Snook, D., and Epenetos, A. A. Macrocyclic chelates of radiometals Simpson, D. E., and Colcher, D. Synthesis of l-(/>isothiocyanatobenzyl) derivatives for diagnosis and therapy. Br. J. Cancer, 62 (Suppl.): 21-26, 1990. of DTPA and EDTA; antibody labeling and tumor-imaging studies. Inorg. Chem., 25: 33. CamÃ©ra,L., Kinuya, S., Garmestane, K., Pai, L. H., Brechbiel, M. W. Gansow, O. A., 2772-2781, 1986. Paik, C. H., Pastan, I., and Carrasquillo, J. A. Evaluation of a new DTPA-derivative 24. Meares, C. F.. McCall, M. J., Reardan, D. T., Goodwin, D. A., Diamanti, C. I., and chelator: comparative biodistribution and imaging studies of m In-labeled B3 mono McTigue, M. Conjugation of antibodies with bifunctional chelating agents: isothio- clonal antibody in athymic mice bearing human epidermoid carcinoma xenografts. cyanate and bromoacetamide reagents, methods of analysis, and subsequent addition NucÃ.Med. Biol., 20: 955-962, 1993. of metal ions. Anal. Biochem., 142: 68-78, 1984. 34. Sharkey, R. M., Motta-Hennessy, C., Pawlyk, D., Siegal, J. A., and Goldenberg, 25. Siegel, J. A., and Stabin, M. G. Absorbed fractions for electrons and beta particles in D. M. Biodistribution and radiation dose estimates for yttrium- and iodine-labeled small spheres. J. NucÃ.Med., 29: 803, 1988. monoclonal antibody IgG and fragments in nude mice bearing human colonie tumor 26. Henderson, L. A., Baynes, J. W., and Thorpe, S. R. Identification of the sites of IgG xenografts. Cancer Res., 50: 2330-2336, 1990. catabolism in the rat. Arch. Biochem. Biophys., 2/5: 1-11, 1982. 35. Esteban, J. M., Schlom, J., Gansow, O. A., Atcher, R. W., Brechbiel, M. W., Simpson, 27. Blumenthal, R. D., Sharkey, R. M., Natale, A. M., Kashi, R., Wong, G., and D. E., and Colcher, D. New method for the chelation of indium-111 to monoclonal Goldenberg, D. M. Comparison of equitoxic radioimmunotherapy and chemother antibodies: biodistribution and imaging of athymic mice bearing human colon carci apy in the treatment of human colonie cancer xenografts. Cancer Res., 54: noma xenografts. J. NucÃ.Med., 28: 861-870, 1987. 142-151, 1994. 36. Carney, P. L., Rogers, P. E., and Johnson, D. K. Dual isotope study of iodine-125 and 28. Duncan, J. R.. and Welch. M. J. Intracellular metabolism of indium-111-DTPA- Â¡ndium-lll-labeled antibody in athymic mice. J. NucÃ.Med., JO: 374-384, 1989. labeled receptor targeted proteins. J. NucÃ.Med., 34: 1728-1738, 1993. 37. Kinugasa, T., Kuroki, M., Yamanaka, T., Matsuo, Y., Oikawa, S., Nakazato, H., and 29. Deshpande, S. V., Subramanian, R., McCall, M. J., De Nardo, S. Jâ€žDeNardo, G. L., Matsuoka, Y. Non-proteolytic release of carcmoembyonic antigen from normal human and Meares, C. F. Metabolism of indium chelates attached to monoclonal antibody: colonie epithelial cells cultured in collagen gel. Int. J. Cancer, 58: 102-107, 1994. minimal transchelation of indium from benzyl-EDTA chelate in vivo. J. NucÃ.Med., 38. Schlom, J., Siler, K., Milenic, D., Eggensperger, D., Colcher, D., Miller, L. S., 31: 218-224, 1990. Houchens, D., Cheng, R., Kaplan, D., and Goeckeler, W. Monoclonal antibody-based 30. Halpem. S. E.. Hagan, P. L., Carver, P. R., Koziol, J. A., Chen, A. W. N., Frincke, therapy of a human tumor xenograft with a '77Lutetium-labeled immunoconjugate. J. M., Bartholomew, R. M., David, G. S., and Adams, T. H. Stability, characterization. Cancer Res., 5/: 2889-2896, 1991.

3139

Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1995 American Association for Cancer Research. Effects of Radiolabeling Monoclonal Antibodies with a Residualizing Iodine Radiolabel on the Accretion of Radioisotope in Tumors

Rhona Stein, David M. Goldenberg, Suzanne R. Thorpe, et al.

Cancer Res 1995;55:3132-3139.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/55/14/3132

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/55/14/3132. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.