The Japanese Journal of Surgery (1992) 22:155-158

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© Springer-Verlag 1992

Biodistribution of Radiolabeled Monoclonal Antibody after Intraperitoneal Administration in Nude Mice with Hepatic Metastasis from Human Colon Cancer KAZUHIKO YOSHIDA, TOURU FUJIKAWA, GEORGEYOSHIZAWA, AKIHIROTANABEA, and KENJI SAKURAI First Department of Surgery, The Jikei UniversitySchoolof Medicine, Tokyo, Japan

Abstract: The utility of the intraperitoneal (IP) administration of radiolabeled monoclonal antibody (mAb) for hepatic metastasis from human colon cancer was evaluated in congenital athymic mice. The intrasplenic injection of HT-29 LMM metastatic human colon cancer cell line reproducibly results in hepatic metastasis formation in nude mice. HT-2915, a murine mAb of IgG1 class reactive with the HT-29 LMM cell line was labeled with iodine 125. One lag/2 gCi of labeled HT-29-15 was injected intraperitoneally into mice with hepatic metastases, and additionally IP administration of the same dose of 1-125 labeled HT-29-15 with increased volume and intravenous (IV) administration of dose quantity of HT-29-15 were performed. Blood samples were obtained at 1, 3, 5 hours, and the animals were sacrificed on days 1, 3, and 5. The per cent of injected dose per gram (%ID/g) of blood after IP administration of 1-125 labeled HT-29-15 reached the same level of %ID/g after IV administration by 5 hours. The transfer from the peritoneal cavity to blood was delayed by increasing the volume injected. From day 1 to day 3, there was a progressive increase for hepatic metastasis/ blood ratios of 1-125 labeled HT-29-15 in each group. There was no difference in the hepatic metastasis/blood ratios among the three groups. IP administration of specific mAb, therefore, provides the same level of tumor uptake in hepatic metastasis from colorectal cancer, and would be advantageous in patients with both hepatic metastasis and peritoneal implants in which radioimmunodetection and radioimmunotherapy are appropriate. Key Words: Monoclonal antibody, nude mice, hepatic metastasis, colon cancer, HT-29LMM

Introduction The prevalence of colon cancer continues to increase in Japan. Liver metastasis has been found to represent the primary cause of therapeutic failure; in patients dying of colorectal cancer 48 per cent will have future liver involvement, a Both the detection and therapy of hepatic metastasis of colorectal cancer remain important goals of clinical oncology. Since the initial success reported by Goldenberg et al. for the clinical localization of primary and secondary colorectal cancer using polyclonal antibodies for carcinoembryonic antigen, 2 the use of radiolabeled antibodies has shown promise as a clinically useful modality for the diagnosis and treatment of colorectal cancer. 3 There is little data on absolute hepatic metastasis and normal tissue uptake of radiolabeled m A b uptake because there are considerable limitations in obtaining suitable hepatic metastasis samples and normal tissue in humans. Recently, we have reported a murine model with hepatic metastasis from h u m a n colon cancer, which appears to be useful for the evaluation of localization of radiolabeled m A b s to hepatic metastasis. 4 Using this model we are able to determine the utility of intraperitoneal (IP) administration of radiolabeled m A b for hepatic metastasis from human colon cancer. Materials and Methods

Establishment of Hepatic Metastasis in Nude Mice

Reprint requests to: Kazuhiko Yoshida, MD, First Department of Surgery, The Jikei University School of Medicine, Nishishinnbashi 3-25-8, Minato-ku, Tokyo, 105, Japan (Received for publication on Feb. 1, 1991)

Specific pathogen-free athymic B A L B / c female mice 3 to 4 weeks of age were cared for at the animal facility. The mice were kept under sterile conditions in a laminar flow r o o m in cages with filter bonnets and were fed a sterilized mouse diet and sterilized water. The h u m a n colon cancer cell line HT-29 L M M , a metastatic variant of the HT-29 cell line, ~ was gener-

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K. Yoshida et al.: Biodistribution of Radiolabeled Monoclonal Antibody

Fig. 1. Macroscopic findings of hepatic metastases an a splenic xenograft. The hepatic metastases appeared as multiple irregular gray-white nodules of varying size and were evenly distributed in both liver lobes. ×2 ously provided by Dr. I.J. Fidler (M.D. Anderson Hospital and Tumor Institute, Houston, TX, U.S.A.). The cell line was grown under humidified conditions in the presence of 5 per cent carbon dioxide in Dulbecco's modified minimum essential medium supplemented with 10 per cent fetal bovine serum (GIBCO, U.S.A.). Tumor cells grown in tissue culture were harvested by 5-minute treatment with 0.05 per cent trypsin and 0.02 per cent E D T A and suspended in phosphatebuffered saline (PBS). The mice were anesthetized with intraperitoneal pentobarbital (12~tg/gram body weight), and a small left subcostal incision was made to expose the spleen. 2 x 106 HT-29 LMM cells in 50 gl of PBS were injected beneath the splenic capsule using a 30-gauge needle. Approximately 4 weeks after intrasplenic injection, most of the mice with intrasplenic injection developed hepatic metastasis (Figs. 1, 2).

Radiolabeling of Monoclonal Antibody (mAb) HT-29-15, which was developed by Dr. J. Sakamoto at Memorial Sloan-Kettering Cancer Center in New York, 6 is a murine IgG1 mAb reacting with a neuraminidase-sensitive cell-surface antigenic determinant (200Kd) present on the HT-29 human colon cancer cell line. It reacts with more than 60 per cent of primary and metastatic colorectal cancers in immunohistopathlogy. A study on colorectal cancer patients with hepatic metastasis demonstrated that radiolabeled HT-29-15 localized to tumor tissue. 7 HT-29-15 mAb was labeled with iodine-125 sodium iodine by a modified chloramine-T method, s The radiolabeled mAb was purified by exclusion chromatography on a Sephadex G-25 column (Pharmacia, U.S.A.). Per cent labeling was 80 per cent. The frac-

Fig. 2. The hepatic metastasis were demonstrated to form distinct nodules and to displace hepatocytes. However, areas of necrosis were present in the center of the nodules. ×40 tion corresponding to the radiolabeled mAb typically had more than 95 per cent of radioactive iodine bound to protein, as determined by trichloroacetic acid precipitation. Immunoreactivity was tested by a modification of the method of Lindmo et al. 9 and was 55 per cent for HT-29-15 mAb.

Biod&tribution One ~tg/2~tCi of labeled HT-29-15 in 0.5ml was injected intraperineally in 11 mice with hepatic metastasis, and additionally an IP administration of the same dose of 1-125 labeled HT-29-15 in 2.0ml was given to 12 mice. An intravenous (IV) administration of dose quantity of HT-29-15 was also performed in 9 mice. Blood samples were obtained from the tail vein at 1, 3, 5 hours. Bleeding was obtained by nicking the tail vein with a scalpel, and 3-5 drops were collected into preweighed tubes. Data were expressed in terms of per cent injected dose per gram of olood (cpm/g of blood)/ (total injected dose). Three or 4 animals were sacrificed at days 1, 3, and 5. Normal tissue (liver, spleen, heart, ling, kidney, large intestine, thyroid, muscle, and brain), blood, splenic tumor, and all hepatic metastasis were removed, weighed and their radioactivity counted in a gamma well counter. Per cent injected dose per gram of tissue; (cpm/g of tissue)/(total injected dose) and tissue/blood ratios were calculated. A stastical analysis was performed by the Student's t-test. Results

Efflux from Peritoneal Cavity to Blood As shown in Fig. 3, with an injection volume of 0.5 ml, mAb was transferred from the peritoneal cavity to the

K. Yoshida et al. : Biodistribution of Radiolabeled Monoclonal Antibody

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Fig. 3. Transfer of 1-125 labeled HT29-15 from peritoneal cavity to blood. The per cent injected dose per gram of blood was determined at various times after injection.*

blood with 5 hours. We then attempted to decrease the efflux rate by injecting larger volumes. An injection of 2.0ml resulted in a relatively small but consistent decrease in the efflux rate (Fig. 3). The rate of transfer of 1-125 labeled HT29-15 from the peritoneal cavity into the blood did not differ significantly between injections of 0.5 and 2.0ml (p > 0.05). The maximum %id/g was approximately 20 per cent.

HT-29-15 mAb Uptake in Hepatic Metastasis Specific mAb uptake by tumor after both IP and IV administration progressively increased from day 1 through day 5 (Fig. 4). However, there was no difference (p > 0.05) in the hepatic metastasis/blood and normal liver/blood ratios between the IP group and IV group. On the other hand, the specific mAb uptake by normal tissues after both IP and IV administration progressively decreased from day 1 through day 5 as observed in our previous study 4 (data not shown).

Discussion

A theoretical analysis of intraperitoneal chemotherapy has assumed that the peritoneal cavity is lined by mesothelial cells and that any transfer from the peritoneal cavity to blood across the mesothelium is carried out transferred by diffusion. 1° This model may be appropriate for small molecules that are cleared rapidly from the blood, but it is not applicable to proteins. The efflux of large molecules, colloids, and erythrocytes from the peritoneal cavity occurs within hours via lymphatics of the lower surface of the diaphragm. The

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Fig. 4. 1-125 labeled HT-29-15 uptake to hepatic metastasis

and the normal liver. At days 1, 3, and 5 following both IV and IP injection,.tissue was removed and radioactivity was counted. There was no difference (P > 0.05) found in the hepatic metastasis/blood ratios between the IP group and IV group, o - o , IV (0.2ml); © .... ©, IP (0.5ml); A .... A, IP (2.0ml) *o-o, IV (0.2ml); ©----©, IP (0.5ml); A .... A, IP (2.0ml)

lack of a substantial barrier to efflux is due to the presence of a specialized, very permeable mesothelium and lymphatics at this location. 11 Our experiments showed that proteins injected intraperitoneally transferred almost completely to the blood by 5 hours in mice. Injecting large volumes decreases the efflux rate, but this effect was relatively small. Only about 20 per cent of the IP administrated mAb was found in plasm. Mattes suggested that in large animals, including rats, cats, and human, the efflux rate is inherently lower, and the effect increasing the volume injected may be greater and essentially all of the injected sample will be transformed to the blood.12 This rapid transfer from the peritoneal cavity to the blood may be useful in the clinical setting. Colcher et al. demonstrated the advantage of mAb B72.3 to target peritoneal implants from colorectal cancer when administering IP. 13 This is an advantage of IP administration to metastasis to colorectal cancer patients who often not only have hepatic metastases but peritoneal implants as well. The studies reported herein have also shown that only a minor component of the IP administered radiolabeled mAb is found in the plasm (Fig. 3), an important point to consider if one wishes to minimize plasma borne radiolabeled mAb as a potential source of marrow toxicity. In conclusion, IP administration of specific mAb in nude mice bearing hepatic metastasis from human colorectal cancer gives the same level of tumor uptake in hepatic metastasis as IV administration. We believe

158

K. Yoshida et al.: Biodistribution of Radiolabeled Monoclonal Antibody

that this study has important implications for future clinical studies involving radioimmunodetection and radioimmunotherapy for patients with both hepatic metastasis and peritoneal implants of colorectal cancer.

6.

Acknowledgments. The

authors wish to thank Drs. C. Divgi and E. Sigurdson (Memorial $1oan-Kettering Cancer Center, New York, U . S . A . ) for providing the HT-29-15 m A b .

7.

References 1. Cedermak B J, Schults SS, Baskshi S, Parthasarathy KL, Mittleman A, Evans JT (1977) The value of liver scan in the follow-up study of patients with adenocarcinoma of colon and rectum. Surg Gynecol Obstet 144:745-748 2. Goldenberg DM, DeLand F, Kimm E, Bennett S., Primus F, van Nagell JR, Estes N, DeSimone P, Rayburn P (1978) Use of radiolabeled antibodies to carcinoembryonic antigen for the detection and localization of diverse cancers by external photoscanning. N Eng J Med 298:1384-1388 3. Larson SM (1987) Lymphoma, melanoma, colon cancer: Diagnosis and treatment with radiolabeled monoclonal antibodies. The 1986 Eugene P. Pendergrass New Horizon Lecture. Radiology 165:297-301 4. Yoshida K, Rivoire M, Divgi C, Welt S, Cohen AH, Sigurdson ER (1990) Radiolocalization of monoclonal antibodies in hepatic metastases from human colon cancer in congenitally athymic mice. Cancer Res 50:862s-865s 5. Irimura T, Carlson DA, Price J, Yamori T, Giavazzi R, Ota DM, Cleary KR (1988) Differential expression of a sialoglycoprotein

8. 9.

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with an approximate molecular weight of 900,000 on metastatic human colon carcinoma cells growing in culture and in tumor tissues. Cancer Res 48:2353-2360 Sakamoto J, Cordon-Cardo C, Friedman E, Finstad CL, Enker WE, Melamed MR, Shiku H, Lloyd KO, Oettgen HF, Old LJ (1983) Antigens of norma~ and neoplastic human colonic mucosal cells defined by monoclonal antibodies. Proceedings of AACR, p 225 Cohn KH, Welt S, Banner WP, Harrington M, Yeh S, Sakamoto J, Cardon-Cardo C, Daly J, Kemeny N, Cohen A, Lloyd K, DeCosse J, Oettegen H, Old L (1987) Localization of radiolabeled monoclonal antibody in colorectal cancer. Arch Surg 122:1425-1429 Johnstone A, Thorpe R. In: Immunochemistry in practice, Blackwell, Boston, p 107 Lindmo T, Boven E, Cuttitta F, Fedorko J, Bunn PA Jr (1984) Detection of the immnoreactive fraction of radiolabeled monoclonal antibodies by linear extrapolation to binding at infinite antigen excess. J Immunol Methods pp 77-89 Markman M (1984) Medical principle of intraperitoneal and intrapleural chemotherapy. In: Howell SB (ed) Intra-arterial and intra-cavitary cancer chemotherapy. Nijihoff, Boston, pp 1-69 Raybuck, HE, Allen L, Harms W (1950) Absorption of serum from the peritoneal cavity. Am J physiol 199:1021-1024 Mattes MJ (1987) Biodistribution of antibodies after intraperitoneal or intravenous injection and effect of carbohydrate modifications. JNCI 79:855-863 Colcher D. Esteban J, Carrasquillo JA, Sugarbaker P, Reynolds JC, Bryant G, Larson SM, Schlom J (1987) Complementation of intracavitary and intravenous administration of a monoclonal antibody (B72.3) in patient with carcinoma. Cancer Res 47: 4218-4224

Biodistribution of radiolabeled monoclonal antibody after intraperitoneal administration in nude mice with hepatic metastasis from human colon cancer.

The utility of the intraperitoneal (IP) administration of radiolabeled monoclonal antibody (mAb) for hepatic metastasis from human colon cancer was ev...
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