[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 BiomédicalScience. 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 histológica! 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 ± 1.47% (SD), The cellobiose-tyramine label was tested by the methods of Ali et 38.06 ±8.04%, and 43.18 ± 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 '•"!.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 Downloaded from cancerres.aacrjournals.org on October 3, 2021. © 1995 American Association for Cancer Research. RESIDUALIZING RADIOLABELS ON mAhs 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 ±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.