445
WORK IN PROGRESS
Vol. 116
• Tumor Localization of 1311-Labeled Antibodies by Radionuclide Imaging 1
Work In Progress
•
Tarun Ghose, M.B., B.S., Ph.D., M.R.C.Path.(U.K.), Joseph Tai, M.Sc., Joe Aquino, M.D., F.R.C.P.(C), Alev Guclu, M.D., Ph.D., Steve Norvell, M.D., F.R.C.P.(C), and Alan MacDonald, M.D., F.R.C.P.(C) Intravenous injections of 13' I-Iabeled anti-EL4 lymphoma antibodies showed progressive localization of rad ioactivity in EL4 transplants but not in B 16 melanoma in mice carrying both tumors. Normal rabbit globulin labeled with 1311did not localize in either tumor and cleared more slowly from the internal organs. Metasta tic loc alization of intravenous ' 311-labeled anti-tumor antibodies was also observed in 2 cancer pat ients. TERMS : Antigens and Antibodies. Lymphoma. Melanoma. Neoplasms, experimental. Radiobiology, cell and tissue studies
INDE X
Radiology 116:445-448, August 1975
•
•
Pressman and Keighley (1) reported that radioiodinated anti-tissue antibodies injected intravenously localize in target tissue, and Day et al. (2) demonstrated that antisera against Murphy rat lymphosarcoma had higher " localization titers " in tumors than in normal tissues. Although the antibody used by Day et al. later proved to be directed against fibrinogen alone (3), significantly higher localization of radio-antibodies against antigens on the surface of tumor cells has now been confirmed in several experimental tumor models (4-8) and in 2 of 5 glioma pat ients (9). We now wish to describe the detection of tumor-specific localization of radio-antibodies by radionuelide imaging in a syngeneic mouse tumor and in 2 of 4 cancer patients, illustrating the potential usefulness of radiolabeled antibodies in the detection of tumors and metastases. MATERIALS AND METHODS EL4 lymphomas induced by dimethylbenzanthracene in C57BL/6 mice and B16 melanomas of spontaneous origin were maintained by serial transplantation in C57BLl6J mice.F Specific anti-EL4 serum was produced by injecting adult white New Zealand rabbits first with 4 X 10 8 viable tumor cells suspended in 4 ml of phosphate-buffered saline (PBS) (0.1 M, pH 7.1) mixed with an equal volume of Freund 's complete adjuvants followed one week later by injections of 4 X 108 tumor cells without the adjuvant (10, 11). Each rabbit received 10 more injections of 10 8 tumor cells tw ice a week and was bled 10 days after the last injection. Rabbits showing titers of 2: Y64 of specific anti-EL4 activity by immunofluorescence after appropriate absorption as described below were bled terminally. The resulting sera were inactivated at 56°C for 30 minutes and absorbed repeatedly with washed homogenates of liver , lung , kidney , and spleen from normal adult C57BL mice until the serum reacted with EL4 cells but not with cryostat sections of normal C57BLl6 mouse tissues , suspensions or smears of C57BL lymphoid cells from lymph nodes, spleen , and thymus, or B 16 melanoma cells. Antisera against four histologically proved human malignant tumors, including 1 renal adenocarcinoma, 1 squamous-cell carcinoma of the lung , and 2 malignant melanomas, were produced in a similar manner by injecting rabbits and goats with dissociated tumor cells and tumor homogenate from fresh tumor specimens.
Fig. 1. Autoradiograph of an EL4 tumor cell after incubation with n ' I_la_ beled rabb it anti-EL4 globulin in vitro for 30 minutes, showing localization of radioactivity on the surface of the tumor cell. (X 800)
An ti-human tumor sera were inactivated and absorbed first with human AB red cells and then repeatedly with homogenates of pooled normal human tissues, l.e., liver, lungs, kidney, spleen, and heart, and were then tested by immunofluorescence for the presence of specific anti-tumor antibodies. After appropriate absorptions, the anti-human tumor sera reacted only with the immunizing tumor and not with any normal adult human tissue, including the patient's own sk in and peripheral blood lymphocytes. Globulin fractions obtained from the antitumor serum and normal rabbit sera (NRG) by fractionation with 33 % saturated ammonium sulfate were freed of ammonium sulfate by repeated dialysis against PBS and bound to varying amounts of carrier- and reductant-free 131 1 4 by the chloramine T method (12). Prior to radioiodination , the anti-EL4 globulin was purified further by adsorption on and elution from formalinized EL4 cells according to the batch method (13). Binding of all radiolabeled anti-tumor antibodies to their respective tumor cells was confirmed by immunofluorescence as well as by autoradiography using Eastman Kodak NTB3 emulsion (Fig. 1). Mice and patients were scanned with a Nuclear Chicago Pho/Dot rectilinear scanner fitted with a 61-hole coll imator, and 131 1 activity was counted in a well-type gamma counter." Immunofluorescence was performed with proper controls (10-12) by the sandwich method, using fluorescein isothiocyanate-Iabeled goat antirabbit globulin 6 on 5Ji, cryostat sections of tissues or suspensions of viable tumor cells. Prior to scanning, 2 X 10 7 EL4 cells and 2 X 106 B 16 melanoma cells were SUbcutaneously inoculated into the right and left legs of adult C57BLl6 mice, respectively. Five days after tumor inoculation, one group of 3 mice received 70 Ji,Ci of 131 1 alone via the tail vein, a second group each received 70 Ji,Ci of 13 11 bound to 70 Ji,Q of NRG, and a third group was each given 70 Ji,Ci of 13 11 bound to 120 Ji,g of anti-EL4 globulin. All mice were scanned two hours after injection and every 24 hours thereafter. RESULTS All tumor-bearing mice died 6 days after injection, l.e., 11
446
WORK IN PROGRESS
3
Table I: Distribution of 13 '1 Activity in Mice with EL4 Lymphoma and B16 Mel anoma Transplants at Death 6 Days after Injection of l3l1-Labele d Anti-EL4 or Normal Rabbit Globulin *
/•
•
August 1975
.7
Fig. 2. Scan of a mouse with EL41ymphoma and 816 melanoma transplants 120 hours after intravenous injection of 1311_labeled normal rabbit globulin. There is virtually no activity in either tumor . Fig. 3. Scan of another mouse with EL4 Ivmphoma and 816 melanoma transplants 120 hours after intravenous injection of 131 1_ labeled anti-EL4 globulin. Localization of 13'1 activity can be seen in the EL4 transplant but not in the 816 melanoma . Compared to the mouse injected with 13'1 normal rabbit globulin, there is less activity in the thoracic and abdominal cavities and more in the bladder area . days after tumor inoculation. No preferential tissue localization was detected at any time in the mice injected with 131 1 alone. There was considerable reduction of activity in the whole body after 48 hours, and no activity above the background level could be detected in the whole body or in any individual organ 144 hours later. Most of the radioactivity detected 2 hours after the injection of 1311_NRG was localized in the thoracic and abdominal cavities ; at 24 hours, there was also considerable activity in
Heart Lung Liver Kidney Spleen Stomach Pancreas Femur Brain EL4 lymphoma B16 melanoma
13'1/Anti-EL4 Antibody
' 3'1/Normal Rabbit Globulin
1.44 ± 0.32 1.44 ± 1.01 6. 20 ± 1.35 2.51±0.73 1.55 ± 0.52 1.50±0.93 1.30 ± 0.87 1.44 ± 0.36 0. 24 ± 0.09 2.97 ± 0.37 1.77 ± 0.94
4.96 ± 0.34 4.21 ± 0.47 3.83 ± 0.21 4.63 ±0.75 3.07 ± 1.32 2.86 ± 0.72 4.27± 1.54 4.21 ± 0.57 0.53 ± 0.16 2_14 ± 0.20 2.03 ± 0.24
* 131' activity expressed as 10- 9 Ci/0.1 g of tissue as detected 144 hours after injection of radiolab eled globulin. The figures represent the mean and standard deviation from groups con taining 3 mice each.
the bladder. Localization in both tumors was low, and there was no consistent difference in activity between the EL4 lymphoma and the 816 melanoma. The same scanning pattern could be seen in all m ice until their death, with considerable retention of activity in the thoracic and abdominal cavities (Fig. 2). At 2 hours, the pattern seen in the mice injected with radioiodinated anti-EL4 globulin did not differ from that of the mice injected with 1311_NRG, i.e., most activity was localized in the thoracic and abdominal cavities with equally poor localization in both tumors. At 24 hours, one mouse showed definite preferential localization of activity in the EL4 lymphoma compared to the 816 melanoma, although localization of activity in the thoracic and abdominal cavities was still quite high in all 3 mice. At 48 hours, all 3 mice showed considerably more activity in the EL4 lymphoma than in the 816 melano-
Fig. 4 . Radiograph showing an osteolytic lesion (arrow) on the right side of the fifth lumbar vertebra in a patient with renal adenocarcinoma . Fig. 5. Scan of the same patient 24 hours after intravenous injection of radioiodinated anti-tumor globulin. Localization of radioactivity can be seen in the liver, spleen, lungs, bladder (urine), and metastasis (arrow).
447
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Vol. 116
ma, and the same pattern persisted at 72 and 96 hours. At 120 hours, there was a considerable reduction of radioactivity in the thorax in all 3 mice, though activity was still quite high in the liver and bladder and there was further improvement in the preferential localization of 1311_IRG in at least one EL4 tumor (Fig. 3). No change in the pattern of localization could be detected at 144 hours. TABLE I gives the postmortem 131 1activity in 12 tissues, including both tumors. The brain had the lowest 131 1activity in all cases. In the 1311-NRG-injected mice, both tumors had the lowest activity apart from the brain, and activity was approximately equal. In the 13111anti-EL4 globulin-injected mice, the highest activity was found in the liver, followed by the subcutaneous EL4 transplants. The EL4 transplants contained about twice the amount of 131 1 activity shown by the 816 transplants. Comparison of the tissue distribution of 131 1activity between the two groups of mice injected with 1311_NRG and 13111anti-EL4 globulin revealed that apart from the liver and the EL4 transplants, all tissues had lower activity in the 13111anti-EL4 globulin-injected group, thus supporting the scanning evidence of faster clearance of the radiolabeled antibody from these tissues. Histological examination of the EL4-bearing mice revealed invasion of subcutaneous tissue by lymphoma cells around the visible transplant nodules and all internal organs except the brain. The liver had by far the largest number of EL4 tumor cells, followed by the lungs and kidneys. In one patient, unequivocal localization of 131 1activity could be seen in the right pedicle of the fifth lumbar vertebra in a solitary metastasis from a histologically proved renal adenocarcinoma follOWing the injection of 4.2 mCi of 131 1 bound to 100 mg of goat antitumor globulin produced against the patient's own tumor (Figs. 4 and 5); in another patient, who had undergone wedge resection for advanced squamous-cell carcinoma of the right lower bronchus, 131 1activity was detected in a residual mediastinal tumor following intravenous injection of 5.6 mCi of 131 1bound to 120 mg of antitumor globulin produced against his own tumor. In both patients, localization of 131 1 activity in the tumor, liver, and spleen could be seen 24 hours after injection of 1311/anti-tumor globulins; it began to decrease in the liver and spleen after 48 hours but gradually increased in the tumor tissue until 96 hours. In one of the 2 patients with melanoma, tumor dissemination was too wide and massive to definitely correlate the scanning findings with the distribution of tumor tissue; the other patient, who had metastases in the skin and the right lung, had been given previous injections of antimelanoma globulin and had a mild anaphylactic reaction Iollowlnq intravenous injection of 3.48 mCi of 131 1bound to 120 mg of antimelanoma globulin. No localization of radioactivity could be detected in his lung or skin metastases. DISCUSSION Previous studies of in vivo localization of radiolabeled antitumor antibodies by radionuclide imaging involved either (a) a xenogenic tumor, e.g., human tumor growing in the cheek pouch of hamsters (6, 7) with or without conditioning of the hamsters with cortisone, or (b) an allogeneic hamster melanoma (8). A syngeneic mouse tumor model is much closer to what might be expected clinically in human tumors. It is interesting to note that in spite of the widespread invasion of the
Work In Progress
internal organs of the tumor-bearing mice by EL4 cells and the presence of EL4 cells in the circulation (11), specific localization of 1311/anti-EL4 globulin could still be visualized in the subcutaneous EL4 transplants but not in the 816 melanoma in the opposite limb; moreover, the time sequence of the pattern of localization of 13111anti-EL4 globulin and 1311_NRG was similar to that observed in mice with more localized tumors (6~8). Localization of considerable amounts of radioactivity in the liver in mice injected with 13111anti-EL4 globulin might be the result of large numbers of EL4 cells in the liver and uptake of the xenogeneic globulin by Kupffer cells. The faster clearance of injected anti-tumor antibodies from the internal organs and more prolonged localization in the target tumor compared to nonselective radioglobulins, i.e., 1311_NRG, add to the potentiality of the diagnostic and therapeutic uses of radiolabeled anti-tumor antibodies and might also offer at least a partial explanation of our observation of more effective tumor inhibition by 131 1_ or chlorambucil-bound tumor antibodies compared to antibodies, 131 1, or chlorambucil alone (10-12, 14). The anti-human renal carcinoma globulin cross-reacted with renal carcinoma cells (but not with normal kidney) from 3 of the 4 patients tested, while the anti-lung cancer globulin cross-reacted with pulmonary squamous-cell carcinoma cells from 2 other patients. Such cross-reactivity of xenogeneic anti-human tumor sera with histologically similar human tumors might extend the diagnostic and therapeutic uses of radiolabeled antibodies even to patients who do not have xenogeneic antibodies raised against their own tumor. Since our results on the localization of radiolabeled antitumor globulins in human cancer are of a very preliminary nature, widespread application of this technique is not indicated until the problems associated with in vivo localization of antitumor xenoglobulins are better understood.
REFERENCES 1. Pressman D, Keighley G: The zone of activity of antibodies as determined by the use of radioactive tracers; the zone of activity of nephritoxic antikidney serum. J Immunol 59: 141~ 146, Jun 1948 2. Day ED, Planinsek J, Korngold L, et al: Tumor-localizing antibodies purified from antisera against Murphy rat lymphosarcoma. J Natl Cancer Inst 17:517-532, Oct 19563. Day ED: Immunological distribution analysis. [In] Kwapinski JBG, ed: Research in Immunochemistry and Immunobiology. Baltimore, University Park Press, 1973, Vol 3, pp 41-90 4. Izzo MJ, Buchsbaum DJ, Bale WF: Localization of an 1251_ labeled rat transplantation antibody in tumors carrying the corresponding antigen. Proc Soc Exp Bioi Med 139:1185-1188, Apr 1972 5. Kellen JA, Lo JS: Localization of 1125 labelled antibodies against tumour-associated proteins from experimental rat mammary neoplasms. Res Commun Chem Pathol Pharmacol 5:411-420, Mar 1973 6. Hoffer PB, Lathrop K, Bekerman C, et al: Use of 1311_CEA antibody as a tumor scanning agent. J Nucl Med 15:323-327, May 1974 7. Goldenberg DM, Preston DF, Primus FJ, et al: Photoscan localization of GW-39 tumors in hamsters using radiolabeled antlcarcinoembryonic antigen immunoglobulin G. Cancer Res 34: 1-9, Jan 1974 8. Smith HJ, G6kcen M: Tumor localizing antibodies directed against the malignant melanoma of hamsters. Res Commun Chem Pathol Pharmacol 7:725-743, Apr 1974
448
9.
WORK IN PROGRESS
Day ED:
Myelin as a locus for radioantibody absorption in
vivo in brain and brain tumors. Cancer Res 28: 1335-1343, Jul 1968 10. Ghose T, Norvell ST, Guclu A, et al: Immunochemotherapy of cancer with chlorambucil-carrying antibody. Br Med J 3:495-499, 26 Aug 1972 11. Ghose T, Guclu A: Cure of a mouse lymphoma with radioiodinated antibody. Eur J Cancer 10:787-792, Dec 1974 12. Ghose T, Cerini M, Carter M, et al: Immunoradioactive agent against cancer. Br Med J 1:90-93, 14 Jan 1967 13. Yamana S, Read RSD, Davies OJ, et al: Antilymphocyte antibody purified by immunoabsorption and elution. Clin Exp Immunol 16:367-374, Mar 1974 14. Ghose T, Nigam SP: Antibody as carrier of chlorambucil. Cancer 29:1398-1400, May 1972
August 1975
1 From the Departments of Pathology and Microbiology (T. G., J. T., A. G.), Radiotherapy and Nuclear Medicine (J. A.), and Surgery (S. N., A. M.), Dalhousie University and Victoria General Hospital, Halifax, N.S., Canada. (Reprint requests to T. G., Dept. of Pathology, Dalhousie University, Halifax, N.S.) Accepted for publication in April 1975. This study was supported by a grant from the National Cancer Institute of Canada and by the Dean's funds for the development of cancer research, Faculty of Medicine, Dalhousie University. 2 Jackson Memorial Laboratories, Bar Harbor, Maine. 3 Difco Laboratories, Detroit, Mich. 4 NEZ 035A, New England Nuclear, Boston, Mass. 5 Picker Medicals, Arlington, Va. 6 Hyland, Los Angeles, Calif. sjh
WORK IN PROGRESS
Vol. 116
• Tumor Localization of 1311-Labeled Antibodies by Radionuclide Imaging 1
Work In Progress
•
Tarun Ghose, M.B., B.S., Ph.D., M.R.C.Path.(U.K.), Joseph Tai, M.Sc., Joe Aquino, M.D., F.R.C.P.(C), Alev Guclu, M.D., Ph.D., Steve Norvell, M.D., F.R.C.P.(C), and Alan MacDonald, M.D., F.R.C.P.(C) Intravenous injections of 13' I-Iabeled anti-EL4 lymphoma antibodies showed progressive localization of rad ioactivity in EL4 transplants but not in B 16 melanoma in mice carrying both tumors. Normal rabbit globulin labeled with 1311did not localize in either tumor and cleared more slowly from the internal organs. Metasta tic loc alization of intravenous ' 311-labeled anti-tumor antibodies was also observed in 2 cancer pat ients. TERMS : Antigens and Antibodies. Lymphoma. Melanoma. Neoplasms, experimental. Radiobiology, cell and tissue studies
INDE X
Radiology 116:445-448, August 1975
•
•
Pressman and Keighley (1) reported that radioiodinated anti-tissue antibodies injected intravenously localize in target tissue, and Day et al. (2) demonstrated that antisera against Murphy rat lymphosarcoma had higher " localization titers " in tumors than in normal tissues. Although the antibody used by Day et al. later proved to be directed against fibrinogen alone (3), significantly higher localization of radio-antibodies against antigens on the surface of tumor cells has now been confirmed in several experimental tumor models (4-8) and in 2 of 5 glioma pat ients (9). We now wish to describe the detection of tumor-specific localization of radio-antibodies by radionuelide imaging in a syngeneic mouse tumor and in 2 of 4 cancer patients, illustrating the potential usefulness of radiolabeled antibodies in the detection of tumors and metastases. MATERIALS AND METHODS EL4 lymphomas induced by dimethylbenzanthracene in C57BL/6 mice and B16 melanomas of spontaneous origin were maintained by serial transplantation in C57BLl6J mice.F Specific anti-EL4 serum was produced by injecting adult white New Zealand rabbits first with 4 X 10 8 viable tumor cells suspended in 4 ml of phosphate-buffered saline (PBS) (0.1 M, pH 7.1) mixed with an equal volume of Freund 's complete adjuvants followed one week later by injections of 4 X 108 tumor cells without the adjuvant (10, 11). Each rabbit received 10 more injections of 10 8 tumor cells tw ice a week and was bled 10 days after the last injection. Rabbits showing titers of 2: Y64 of specific anti-EL4 activity by immunofluorescence after appropriate absorption as described below were bled terminally. The resulting sera were inactivated at 56°C for 30 minutes and absorbed repeatedly with washed homogenates of liver , lung , kidney , and spleen from normal adult C57BL mice until the serum reacted with EL4 cells but not with cryostat sections of normal C57BLl6 mouse tissues , suspensions or smears of C57BL lymphoid cells from lymph nodes, spleen , and thymus, or B 16 melanoma cells. Antisera against four histologically proved human malignant tumors, including 1 renal adenocarcinoma, 1 squamous-cell carcinoma of the lung , and 2 malignant melanomas, were produced in a similar manner by injecting rabbits and goats with dissociated tumor cells and tumor homogenate from fresh tumor specimens.
Fig. 1. Autoradiograph of an EL4 tumor cell after incubation with n ' I_la_ beled rabb it anti-EL4 globulin in vitro for 30 minutes, showing localization of radioactivity on the surface of the tumor cell. (X 800)
An ti-human tumor sera were inactivated and absorbed first with human AB red cells and then repeatedly with homogenates of pooled normal human tissues, l.e., liver, lungs, kidney, spleen, and heart, and were then tested by immunofluorescence for the presence of specific anti-tumor antibodies. After appropriate absorptions, the anti-human tumor sera reacted only with the immunizing tumor and not with any normal adult human tissue, including the patient's own sk in and peripheral blood lymphocytes. Globulin fractions obtained from the antitumor serum and normal rabbit sera (NRG) by fractionation with 33 % saturated ammonium sulfate were freed of ammonium sulfate by repeated dialysis against PBS and bound to varying amounts of carrier- and reductant-free 131 1 4 by the chloramine T method (12). Prior to radioiodination , the anti-EL4 globulin was purified further by adsorption on and elution from formalinized EL4 cells according to the batch method (13). Binding of all radiolabeled anti-tumor antibodies to their respective tumor cells was confirmed by immunofluorescence as well as by autoradiography using Eastman Kodak NTB3 emulsion (Fig. 1). Mice and patients were scanned with a Nuclear Chicago Pho/Dot rectilinear scanner fitted with a 61-hole coll imator, and 131 1 activity was counted in a well-type gamma counter." Immunofluorescence was performed with proper controls (10-12) by the sandwich method, using fluorescein isothiocyanate-Iabeled goat antirabbit globulin 6 on 5Ji, cryostat sections of tissues or suspensions of viable tumor cells. Prior to scanning, 2 X 10 7 EL4 cells and 2 X 106 B 16 melanoma cells were SUbcutaneously inoculated into the right and left legs of adult C57BLl6 mice, respectively. Five days after tumor inoculation, one group of 3 mice received 70 Ji,Ci of 131 1 alone via the tail vein, a second group each received 70 Ji,Ci of 13 11 bound to 70 Ji,Q of NRG, and a third group was each given 70 Ji,Ci of 13 11 bound to 120 Ji,g of anti-EL4 globulin. All mice were scanned two hours after injection and every 24 hours thereafter. RESULTS All tumor-bearing mice died 6 days after injection, l.e., 11
446
WORK IN PROGRESS
3
Table I: Distribution of 13 '1 Activity in Mice with EL4 Lymphoma and B16 Mel anoma Transplants at Death 6 Days after Injection of l3l1-Labele d Anti-EL4 or Normal Rabbit Globulin *
/•
•
August 1975
.7
Fig. 2. Scan of a mouse with EL41ymphoma and 816 melanoma transplants 120 hours after intravenous injection of 1311_labeled normal rabbit globulin. There is virtually no activity in either tumor . Fig. 3. Scan of another mouse with EL4 Ivmphoma and 816 melanoma transplants 120 hours after intravenous injection of 131 1_ labeled anti-EL4 globulin. Localization of 13'1 activity can be seen in the EL4 transplant but not in the 816 melanoma . Compared to the mouse injected with 13'1 normal rabbit globulin, there is less activity in the thoracic and abdominal cavities and more in the bladder area . days after tumor inoculation. No preferential tissue localization was detected at any time in the mice injected with 131 1 alone. There was considerable reduction of activity in the whole body after 48 hours, and no activity above the background level could be detected in the whole body or in any individual organ 144 hours later. Most of the radioactivity detected 2 hours after the injection of 1311_NRG was localized in the thoracic and abdominal cavities ; at 24 hours, there was also considerable activity in
Heart Lung Liver Kidney Spleen Stomach Pancreas Femur Brain EL4 lymphoma B16 melanoma
13'1/Anti-EL4 Antibody
' 3'1/Normal Rabbit Globulin
1.44 ± 0.32 1.44 ± 1.01 6. 20 ± 1.35 2.51±0.73 1.55 ± 0.52 1.50±0.93 1.30 ± 0.87 1.44 ± 0.36 0. 24 ± 0.09 2.97 ± 0.37 1.77 ± 0.94
4.96 ± 0.34 4.21 ± 0.47 3.83 ± 0.21 4.63 ±0.75 3.07 ± 1.32 2.86 ± 0.72 4.27± 1.54 4.21 ± 0.57 0.53 ± 0.16 2_14 ± 0.20 2.03 ± 0.24
* 131' activity expressed as 10- 9 Ci/0.1 g of tissue as detected 144 hours after injection of radiolab eled globulin. The figures represent the mean and standard deviation from groups con taining 3 mice each.
the bladder. Localization in both tumors was low, and there was no consistent difference in activity between the EL4 lymphoma and the 816 melanoma. The same scanning pattern could be seen in all m ice until their death, with considerable retention of activity in the thoracic and abdominal cavities (Fig. 2). At 2 hours, the pattern seen in the mice injected with radioiodinated anti-EL4 globulin did not differ from that of the mice injected with 1311_NRG, i.e., most activity was localized in the thoracic and abdominal cavities with equally poor localization in both tumors. At 24 hours, one mouse showed definite preferential localization of activity in the EL4 lymphoma compared to the 816 melanoma, although localization of activity in the thoracic and abdominal cavities was still quite high in all 3 mice. At 48 hours, all 3 mice showed considerably more activity in the EL4 lymphoma than in the 816 melano-
Fig. 4 . Radiograph showing an osteolytic lesion (arrow) on the right side of the fifth lumbar vertebra in a patient with renal adenocarcinoma . Fig. 5. Scan of the same patient 24 hours after intravenous injection of radioiodinated anti-tumor globulin. Localization of radioactivity can be seen in the liver, spleen, lungs, bladder (urine), and metastasis (arrow).
447
WORK IN PROGRESS
Vol. 116
ma, and the same pattern persisted at 72 and 96 hours. At 120 hours, there was a considerable reduction of radioactivity in the thorax in all 3 mice, though activity was still quite high in the liver and bladder and there was further improvement in the preferential localization of 1311_IRG in at least one EL4 tumor (Fig. 3). No change in the pattern of localization could be detected at 144 hours. TABLE I gives the postmortem 131 1activity in 12 tissues, including both tumors. The brain had the lowest 131 1activity in all cases. In the 1311-NRG-injected mice, both tumors had the lowest activity apart from the brain, and activity was approximately equal. In the 13111anti-EL4 globulin-injected mice, the highest activity was found in the liver, followed by the subcutaneous EL4 transplants. The EL4 transplants contained about twice the amount of 131 1 activity shown by the 816 transplants. Comparison of the tissue distribution of 131 1activity between the two groups of mice injected with 1311_NRG and 13111anti-EL4 globulin revealed that apart from the liver and the EL4 transplants, all tissues had lower activity in the 13111anti-EL4 globulin-injected group, thus supporting the scanning evidence of faster clearance of the radiolabeled antibody from these tissues. Histological examination of the EL4-bearing mice revealed invasion of subcutaneous tissue by lymphoma cells around the visible transplant nodules and all internal organs except the brain. The liver had by far the largest number of EL4 tumor cells, followed by the lungs and kidneys. In one patient, unequivocal localization of 131 1activity could be seen in the right pedicle of the fifth lumbar vertebra in a solitary metastasis from a histologically proved renal adenocarcinoma follOWing the injection of 4.2 mCi of 131 1 bound to 100 mg of goat antitumor globulin produced against the patient's own tumor (Figs. 4 and 5); in another patient, who had undergone wedge resection for advanced squamous-cell carcinoma of the right lower bronchus, 131 1activity was detected in a residual mediastinal tumor following intravenous injection of 5.6 mCi of 131 1bound to 120 mg of antitumor globulin produced against his own tumor. In both patients, localization of 131 1 activity in the tumor, liver, and spleen could be seen 24 hours after injection of 1311/anti-tumor globulins; it began to decrease in the liver and spleen after 48 hours but gradually increased in the tumor tissue until 96 hours. In one of the 2 patients with melanoma, tumor dissemination was too wide and massive to definitely correlate the scanning findings with the distribution of tumor tissue; the other patient, who had metastases in the skin and the right lung, had been given previous injections of antimelanoma globulin and had a mild anaphylactic reaction Iollowlnq intravenous injection of 3.48 mCi of 131 1bound to 120 mg of antimelanoma globulin. No localization of radioactivity could be detected in his lung or skin metastases. DISCUSSION Previous studies of in vivo localization of radiolabeled antitumor antibodies by radionuclide imaging involved either (a) a xenogenic tumor, e.g., human tumor growing in the cheek pouch of hamsters (6, 7) with or without conditioning of the hamsters with cortisone, or (b) an allogeneic hamster melanoma (8). A syngeneic mouse tumor model is much closer to what might be expected clinically in human tumors. It is interesting to note that in spite of the widespread invasion of the
Work In Progress
internal organs of the tumor-bearing mice by EL4 cells and the presence of EL4 cells in the circulation (11), specific localization of 1311/anti-EL4 globulin could still be visualized in the subcutaneous EL4 transplants but not in the 816 melanoma in the opposite limb; moreover, the time sequence of the pattern of localization of 13111anti-EL4 globulin and 1311_NRG was similar to that observed in mice with more localized tumors (6~8). Localization of considerable amounts of radioactivity in the liver in mice injected with 13111anti-EL4 globulin might be the result of large numbers of EL4 cells in the liver and uptake of the xenogeneic globulin by Kupffer cells. The faster clearance of injected anti-tumor antibodies from the internal organs and more prolonged localization in the target tumor compared to nonselective radioglobulins, i.e., 1311_NRG, add to the potentiality of the diagnostic and therapeutic uses of radiolabeled anti-tumor antibodies and might also offer at least a partial explanation of our observation of more effective tumor inhibition by 131 1_ or chlorambucil-bound tumor antibodies compared to antibodies, 131 1, or chlorambucil alone (10-12, 14). The anti-human renal carcinoma globulin cross-reacted with renal carcinoma cells (but not with normal kidney) from 3 of the 4 patients tested, while the anti-lung cancer globulin cross-reacted with pulmonary squamous-cell carcinoma cells from 2 other patients. Such cross-reactivity of xenogeneic anti-human tumor sera with histologically similar human tumors might extend the diagnostic and therapeutic uses of radiolabeled antibodies even to patients who do not have xenogeneic antibodies raised against their own tumor. Since our results on the localization of radiolabeled antitumor globulins in human cancer are of a very preliminary nature, widespread application of this technique is not indicated until the problems associated with in vivo localization of antitumor xenoglobulins are better understood.
REFERENCES 1. Pressman D, Keighley G: The zone of activity of antibodies as determined by the use of radioactive tracers; the zone of activity of nephritoxic antikidney serum. J Immunol 59: 141~ 146, Jun 1948 2. Day ED, Planinsek J, Korngold L, et al: Tumor-localizing antibodies purified from antisera against Murphy rat lymphosarcoma. J Natl Cancer Inst 17:517-532, Oct 19563. Day ED: Immunological distribution analysis. [In] Kwapinski JBG, ed: Research in Immunochemistry and Immunobiology. Baltimore, University Park Press, 1973, Vol 3, pp 41-90 4. Izzo MJ, Buchsbaum DJ, Bale WF: Localization of an 1251_ labeled rat transplantation antibody in tumors carrying the corresponding antigen. Proc Soc Exp Bioi Med 139:1185-1188, Apr 1972 5. Kellen JA, Lo JS: Localization of 1125 labelled antibodies against tumour-associated proteins from experimental rat mammary neoplasms. Res Commun Chem Pathol Pharmacol 5:411-420, Mar 1973 6. Hoffer PB, Lathrop K, Bekerman C, et al: Use of 1311_CEA antibody as a tumor scanning agent. J Nucl Med 15:323-327, May 1974 7. Goldenberg DM, Preston DF, Primus FJ, et al: Photoscan localization of GW-39 tumors in hamsters using radiolabeled antlcarcinoembryonic antigen immunoglobulin G. Cancer Res 34: 1-9, Jan 1974 8. Smith HJ, G6kcen M: Tumor localizing antibodies directed against the malignant melanoma of hamsters. Res Commun Chem Pathol Pharmacol 7:725-743, Apr 1974
448
9.
WORK IN PROGRESS
Day ED:
Myelin as a locus for radioantibody absorption in
vivo in brain and brain tumors. Cancer Res 28: 1335-1343, Jul 1968 10. Ghose T, Norvell ST, Guclu A, et al: Immunochemotherapy of cancer with chlorambucil-carrying antibody. Br Med J 3:495-499, 26 Aug 1972 11. Ghose T, Guclu A: Cure of a mouse lymphoma with radioiodinated antibody. Eur J Cancer 10:787-792, Dec 1974 12. Ghose T, Cerini M, Carter M, et al: Immunoradioactive agent against cancer. Br Med J 1:90-93, 14 Jan 1967 13. Yamana S, Read RSD, Davies OJ, et al: Antilymphocyte antibody purified by immunoabsorption and elution. Clin Exp Immunol 16:367-374, Mar 1974 14. Ghose T, Nigam SP: Antibody as carrier of chlorambucil. Cancer 29:1398-1400, May 1972
August 1975
1 From the Departments of Pathology and Microbiology (T. G., J. T., A. G.), Radiotherapy and Nuclear Medicine (J. A.), and Surgery (S. N., A. M.), Dalhousie University and Victoria General Hospital, Halifax, N.S., Canada. (Reprint requests to T. G., Dept. of Pathology, Dalhousie University, Halifax, N.S.) Accepted for publication in April 1975. This study was supported by a grant from the National Cancer Institute of Canada and by the Dean's funds for the development of cancer research, Faculty of Medicine, Dalhousie University. 2 Jackson Memorial Laboratories, Bar Harbor, Maine. 3 Difco Laboratories, Detroit, Mich. 4 NEZ 035A, New England Nuclear, Boston, Mass. 5 Picker Medicals, Arlington, Va. 6 Hyland, Los Angeles, Calif. sjh
