Scintigraphy and biodistribution of monoclonal adrenocortical antibody in mice grafted with human adrenocortical carcinoma
Ulf Öhrvall, MD, Karin Backlin, BSc, Claes Juhlin, MD, Jan-Erik Westlin, MD, Henrik Arnberg, MD, Gunnel Bjerneroth, MD, Sten Nilsson, MD, Göran Åkerström, MD, and Jonas Rastad, MD, Uppsala, Sweden
Background. A murine monoclonal antibody recognizing normal and neoplastic human adrenocortical cells has been evaluated for scintigraphic localization and biodistribution in 53 nude mice grafted subcutaneously with human adrenocortical cell lines SW-13 and T-CAR 1.
Methods. The immunoglobulin GI antibody and its Fab’ 2 fragment were purified and labeled with 125 I. The tumor grafts exhibited diameters of 5 to 15 mm at 4 to 6 weeks after transplantation, when mice received a single subcutaneous or intraperitoneal injection of 50 µg iodinated intact or fragmented antibody, respectively.
Results. Examination up to 8 days after immunoglobulin G administration showed mean radioactivity ratios less than 1.0 for tumor to blood and corresponding ratios in tumor to lung, liver, spleen, and kidney from 0.6 to 5.3 at the time of peak tumor to blood ratio. A high background activity was noted on scintigraphic tumor visualization with the iodinated immunoglobulin G. In contrast, the radiolabeled Fab’ 2 fragment displayed gradually rising tumor to blood ratios, which, 4 days after injection, averaged 10.5 for T-CAR 1 and 5.3 for SW-13. Tumor transplants were scintigraphically visualized without substantial background activity 3 days after Fab’ 2 injection, when the ratio of radioactivity in the tumor to the investigated murine organs was 0.5 to 7.3.
Conclusions. The findings substantiate that immunoscintigraphy with the Fab’ 2 fragment of the antiadrenocortical Ac5 antibody may become a tool to localize human adrenocortical carcinoma. (SURGERY 1995;118:893-900.)
From the Departments of Surgery and Oncology, University Hospital, Uppsala, Sweden
ADRENOCORTICAL. CARCINOMA CONSTITUTES a rare tumor with dismal prognosis and 5-year survival rates generally ranging between 16% and 35%.1,2 This neoplasm rarely responds to chemotherapy, although transient regres- sions have been reported, notably with mitotane alone or in combination regimens.3-6 Because cure of adreno- cortical carcinoma is mainly related to the possibility of macroscopically radical operation, accurate procedures of tumor localization comprise important requisites for successful primary and reoperation in these pa- tients.7-9 Currently available methodologies, however, exhibit meager specificity for the adrenocortical tissue and are hampered by a limited ability to visualize local
Supported by the Swedish Cancer Society.
Accepted for publication Feb. 21, 1995.
Reprint requests: U. Öhrvall, MD, Department of Surgery, Uppsala University Hospital, S-751 85 Uppsala, Sweden.
Copyright @ 1995 by Mosby-Year Book, Inc.
0039-6060/95/$5.00 + 0 11/56/65407
recurrences and metastases of adrenocortical can- cer.10, 11
Monoclonal antibodies recently have been generated by immunization of mice with human endocrine cells to explore possibilities for improved diagnosis and ther- apy of patients harboring hormone-producing neo- plasms. 12-15 The concept of using human endocrine cells to obtain monoclonal antibodies arose from the circumstance that neoplasms originating from these tis- sues commonly comprise differentiated lesions with maintained secretory function. Such antibodies have been used in the treatment and localization of endo- crine tumors in vivo and to improve the histopathologic diagnosis and understanding of pathophysiologically important derangements of these lesions.13-16 This analysis examines a murine immunoglobulin G anti- body, denoted Ac5,17 for scintigraphic localization and biodistribution in immunoincompetent mice bearing subcutaneous grafts of human adrenocortical carci- noma.
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% Injected dose/gm
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MATERIAL AND METHODS
Tumor cell lines and animals. Two cell lines of human adrenocortical carcinoma were expanded and
maintained in a monolayer culture at 37° C in a humid- ified atmosphere containing 5% CO2. SW-13 (American Type Culture Collection, Rockville, Md.18) was grown in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum and T-CAR 119 in RPMI contain- ing 10% human A+ serum (Flow Lab, Irvine, Scot- land). Confluent cells were harvested after 5-minute exposures to 0.5% trypsin (Sigma Chemical Com- pany, St Louis, Mo.) in phosphate-buffered saline solution (PBS; Flow). Viability of the resulting single and small groups of cells routinely exceeded 95% on Trypan blue exclusion.
Female, 4- to 8-week-old nu/nu-Balb/c A Bom mice (Bomholtgaard Ltd, Ry, Denmark) were used for trans- plantation and tumor propagation. Animals primarily were injected subcutaneously into both proximal thighs with 5 to 25 x 106 cells in 300 ul medium, whereby mac- roscopic tumors were visible 3 weeks later in approxi- mately 90% of the thighs. The tumors of single mice bearing SW-13 and T-CAR 1 were removed and cut into cubes of about 1 mm3, whereupon two to three pieces immediately were reinserted subcutaneously into the proximal thighs of other mice. Altogether 25 mice har- boring SW-13 and 28 with T-CAR 1 tumors subsequently were analyzed for scintigraphy and biodistribution. An- tibody administrations were performed 4 to 6 weeks af- ter transplantation, when grafts exhibited palpable diameters of 5 to 15 mm. During tumor propagation both donors and recipients were anesthetized intra- peritoneally with chloral hydrate (0.36 mg/gm), which was also used during scintigraphy. Animals were killed by cervical dislocation.
Monoclonal antibody and Fab’2 fragment. Produc- tion and characterization of the monoclonal Ac5 an- tibody has been briefly described elsewhere.17 The antibody was generated by immunization of mice with dispersed cells from a human adrenocortical
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%ID/gm
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SW-13
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adenoma causing cortisol excess and application of a conventional hybridoma technique.20-22 This immu- noglobulin Gl antibody exhibits intense immun- ostaining encompassing the interior and surface of normal human adrenocortical cells and reacts with cryosectioned parenchyma of all 21 hitherto investi- gated adrenocortical adenomas and 17 cortical carci- nomas associated with different patterns of steroid overproduction. Cytospins of SW-13 and T-CAR 1 also displayed unequivocal reactivity with Ac5 (15 ug/ml) on examination with a peroxidase-anti- peroxidase technique.16 Other human tissues such as placental syncytiotrophoblasts, ovarian epithelium, and testicular Leydig cells, as well as the intestinal mucosa, have shown variably intense and extensive immunoreactivity with the antibody. In contrast, the adrenal medulla and pheochromocytomas, as well as a variety of other normal tissues including liver, pan- creas, spleen, lymph nodes, and thyroid gland, have failed to react with the antibody.
The Ac5 antibody was purified by affinity chroma-
tography on protein A-Sepharose columns (Kabi- Pharmacia, Uppsala, Sweden) and dialyzed overnight against 0.1 mmol/L NaHCO3 (pH 9.0). The antibody was biotinylated during exposure for 4 hours and at room temperature to N-hydroxysuccinimidebiotin es- ter (1 mg/ml; Sigma Chemical Company) in dimeth- ylsulfoxide22 followed by dialysis against PBS. Cryosec- tions of 6 um from excised SW-13 and T-CAR 1 tumors were stained with hematoxylin-eosin for rou- tine analysis and with the biotinylated antibody (5 ug/ml) by using an avidin-biotin-peroxidase complex (Vectastain ABC kit; Vector Lab, Burlingame, Calif.). Omission of the primary antibody or substitution with a subclass matched antiparathyroid antibody de- noted E11 (10 µg/ml) showed no immunostaining of the tumors. This control antibody previously has shown no immunoreactivity with human adrenocorti- cal tissue.21
A Fab’2 fragment was obtained by adding concen- trated acetic acid (to pH 4.0) to the antibody in PBS, which was followed by pepsin to an antibody/pepsin
Tumor/blood ratio
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SW-13
T-CAR 1
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Time (days)
Tumor/blood ratio
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molar ratio of 100:1.23 This digestion was performed at 37° C and terminated after 12 hours by adding 1.0 mol/L PBS (pH 7.4). The solution was separated into 1 ml fractions on a 1 x 60 cm Sephadex G-100 column (Kabi-Pharmacia). Peak fractions containing the Fab’2 fragment were pooled, kept at 4 ℃, and exhibited more than 95% purity on sodium dodecylsulfate polyacryl- amide gel electrophoresis. Chloramine T24 was used to label 0.5 mg of the intact and fragmented antibody with 37 MBq 1251 (Amersham Int., Amersham, U.K.). Free iodine was removed on a 1 x 5 cm Sephadex G-25 col- umn (PD-10; Kabi-Pharmacia), which yielded an activ- ity of approximately 50 MBq per µg protein on exam- ination of pooled fractions. All mice were injected within 2 hours after completion of the iodination pro- cedure.
Biodistribution and scintigraphy. Altogether 13 mice with SW-13 and 10 with T-CAR 1 tumors each received a single subcutaneous 0.2 ml injection of 50 ug radiolabeled Ac5 antibody in PBS, and the same amount of its Fab’2 fragment was administered intra- peritoneally into 9 and 15 mice, respectively. Thyroid
nuclide uptake was reduced by adding 10 mmol/L KI into the drinking water of 30 mice during the 24 hours preceding these injections. Up to four mice were killed at predetermined intervals 1 to 8 days after injection of the radiolabeled immunoglobulin G and Fab’2 fragment. Tumor grafts, both kidneys, spleen, and samples from lung, liver, and small intes- tine (without contents) were blotted dry and weighed wet together with transcardially aspirated blood. Ra- dioactivity of tissue samples was determined by scin- tillation counting using uninjected mice (n=4) as controls. These values in blood were expressed as percentages of the injected dose (ID), and the pro- portion of ID per gram tissue (% ID/gm) was used to show uptake in murine organs and to calculate tumor to blood and tumor to organ ratios.
Scintigraphy was performed repeatedly up to 8 days after injection of the radiolabeled antibody compounds using a computerized gamma camera (Nuclear Diag- nostics, Hägersten, Sweden) equipped with a low- energy, general-purpose collimator. Intraperitoneal ad- ministration of the antibody fragment prevented tem- porary nuclide signaling from the injection site, which was noticed on scintigraphy 1 and 2 days after subcuta- neous Fab’2 injection. Single mice harboring SW-13 and T-CAR 1 tumors received 50 µg of a subclass-matched control antibody denoted HMFG 125 (gift from Dr. W. D. Allen, Unilever, Bedford, U.K.). This antibody was radiolabeled as described above, administered subcuta- neously, and failed to immunostain human adrenocor- tical carcinoma including SW-13 and T-CAR 1 (not shown).
RESULTS
Routine investigation of cryosections from the extir- pated SW-13 and T-CAR 1 tumors showed solid neo- plasms with only traces of necroses and mostly delicate fibrous stroma. Examinations with the biotinylated Ac5 antibody were consistent with maintained antibody re- activity of transplants, because vast numbers of these cells displayed intense immunostaining of the cyto- plasm and cell surface (Fig. 1).
At the time of primary examination, radioactivity val- ues in blood averaged 15% ID/gm and 1.5% ID/gm for the iodinated Ac5 antibody and its Fab’ fragment, re- spectively (Fig. 2). Clearance from the circulation essentially was accomplished 4 to 5 days later. Compar- ison at individual points of measurement showed vari- able blood levels between animals, especially for the fragmented immunoglobulin, whereas no consistent discrepancies were discernible in this respect between mice harboring SW-13 and T-CAR 1. Standardized radioactivity values were the highest in SW-13 tumors (mean, 4.8% ID/gm) 24 hours after administration of
ratio
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intact Ac5, and a similar level (mean, 4.6% ID/gm) oc- curred 4 to 5 days later in T-CAR 1 (Fig. 3, A). The Fab’2 fragment was accompanied by less dynamic levels of tu- mor radioactivity (Fig. 3, B), which averaged 2.9 to 3.0% ID/gm in T-CAR 1 and 0.9 to 1.1% ID/gm in SW-13 1 to 3 days after injection. Calculation of tumor to blood ratios showed values approaching 1.0 for the intact an- tibody in SW-13, whereas considerably lower levels with less temporal variation occurred in T-CAR 1 (Fig. 4, A). In contrast, the Fab’2 fragment displayed gradu- ally rising tumor to blood ratios, which reached 10.5 in T-CAR 1 and 5.3 in SW 13 on the last day of analysis (Fig. 4, B).
Solid murine tissues like lung, liver, spleen, and kid- ney displayed considerable accumulation of radioactiv- ity, particularly after injection of intact Ac5 (Fig. 3). An early peak (mean, 8.7% ID/gm) was noticed in the kid- ney for the Fab’2 fragment. Radioactivity clearance, however, was swifter in these tissues as compared with both SW-13 and T-CAR 1 tumors; this difference was greatest for the antibody fragment. At the time of
recorded peak tumor to blood ratio for the intact anti- body, the ratio of SW-13 to lung, liver, spleen, kidney, and gut averaged 0.6 to 2.7, whereas the correspond- ing values for T-CAR 1 ranged between 1.1 and 5.3 (Fig. 5, A). At 3 days after administration of the radiolabeled fragment these ratios were 0.5 to 2.3 and 1.7 to 7.3 for T-CAR 1 and SW-13, respectively, with the lowest values for the ratio toward the spleen and kidney (Fig. 5, B).
The tumors became unequivocally detectable by scintigraphy 5 days after injection of the intact anti- body (Fig. 6, A and B). Activity in mainly the liver, spleen, and blood, however, was associated with con- siderable background disturbances, although this time involved the highest tumor to blood ratio. Moreover, the background disturbances remained es- sentially unchanged during the subsequent 3 days. The radiolabeled fragment resulted in less scinti- graphic background activity. Both tumor types were clearly visualized on the third day after Fab’2 injec- tion (Fig. 6, C and D), with an essentially unchanged
A
B
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pattern of activity distribution the next day. The tu- mors of mice injected with the HMFG 1 control antibody merely displayed background activity.
DISCUSSION
The monoclonal Ac5 antibody was generated by im- munization of mice with dispersed cells from an adreno- cortical adenoma associated with Cushing’s syndrome.17 This antibody has been found to recognize an 80 kd an- tigen of unknown function located on the surface and in the interior of normal human adrenocortical cells. Ac5 has also been shown to intensely stain panels of adrenocortical adenomas and carcinomas, causing highly discrepant patterns of peripheral steroid excess. The antibody was found to react with vast majorities of SW-13 and T-CAR 1 cells, both of which comprise cell lines derived from human adrenocortical carcino- ma. 18, 19 Immunohistochemical examination substanti- ated that these cells maintained an essentially unaltered antibody reactivity after 6 weeks of subcutaneous trans- plantation. Moreover, the tumors of both cell lines rap- idly proliferated in the nude mice and essentially failed to exhibit signs of regressive changes on routine mi- croscopy. The ability to proliferate among these cells was substantial in comparison with tissue implants of freshly excised metastases of human parathyroid carci- noma14 and midgut carcinoid tumors (unpublished data).
The values of standardized radioactivity were similar
for the two tumor types after injection of the iodinated antibody, whereas the Fab’2 fragment accumulated more efficiently in T-CAR 1 than in SW-13. The reason for this discrepancy is unclear and is perhaps related to different bioavailability of the recognized antigen in vivo. The groups of mice harboring these grafts thus ex- hibited similar levels of radioactivity in blood and solid tissues. In addition, consistent differences between the tumor types were absent on antibody staining before and after transplantation and on routine microscopy of the excised grafts. Dynamics of radioactivity uptake were also similar in the two grafted tissues, although the tem- poral variation was greater for the intact than the frag- mented antibody. These findings support that explora- tion of the Ac5 antibody for targeting in a clinical set- ting may be accompanied by considerable differences in tracer accumulation between individual tumors, al- though immunohistochemical staining of tissue sam- ples might display similarly intense and extensive anti- body reactivity.
The intact Ac5 antibody showed unsatisfactory tar- geting to the adrenocortical carcinoma grafts with aver- age ratios of radioactivity in tumor to blood approach- ing 1.0 for SW-13 and 0.4 for T-CAR 1. Moreover, the radiolabeled antibody showed rapid accumulation in lung, liver, spleen, and kidney, although investigation of human specimens has substantiated limited immuno- histochemical reactivity in these tissues.17 Clearance of radioactivity was rapid in all investigated murine organs,
Surgery Volume 118, Number 5
however, whereby improved ratios of radioactivity were attained with time on analysis, especially of SW-13. Although scintigraphic tumor visualization was attained with the intact antibody, the background activity was disturbing. These circumstances suggest limited in vivo applicability of the intact Ac5 antibody in human beings with adrenocortical carcinoma.
The iodinated Fab’2 fragment displayed consider- able tumor uptake, especially in T-CAR 1 grafts, where tumor to blood ratios rose gradually up to 10.5, with meager accumulation particularly in the lungs and gut. These characteristics allowed excellent scinti- graphic visualization of the transplants. Fab’2 frag- ments, however, display higher uptake in reticuloen- dothelial system organs such as the liver and spleen and are excreted through the kidneys. Although this cir- cumstance posed no problem in this scintigraphic eval- uation but rather provided landmarks facilitating tumor identification, it may interfere with the recognition of mainly adrenocortical cancer metastasis in vivo. This analysis, however, relied on planar image acquisitions. Biodistribution of the antibody fragment in mainly nor- mal organs suggested that single photon emission tomography might provide better means of visualiza- tion. Moreover, 125I was used as a conventional label for primary antibody evaluation,26 whereby findings could be improved by use of radioligands exhibiting more op- timal decay characteristics and less decomposition in vivo.27-29
Clinical in vivo applicability of scintigraphy with the Fab’2 fragment of the Ac5 antibody can only be speculated on. Tentatively, it could participate in dif- ferentiating lesions in the suprarenal area, because this antibody invariably has been found to recognize adrenocortical adenoma and carcinoma and similarly consistently has failed to immunoreact with normal or neoplastic adrenal medulla and adrenal metastases of renal cell cancer.17 Furthermore, there currently ex- ist sensitive biochemical markers for primary and recur- rent adrenocortical carcinoma,30 whereas available means of tumor localization exhibit limited specificity and sensitivity for the adrenocortical tissue.10, 11 Immu- noscintigraphy thus may become an adjunct in the treatment of this disease, because operation generally is considered also on relapse.1,2, 7-10
The skillful technical assistance of Carina Nordlund is gratefully acknowledged.
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