[CANCER RESEARCH 51. 3251-3256. June 15, 1991] Uptake and Distribution of Specific and Control Monoclonal Antibodies in Subcutaneous Xenografts following Intratumor Injection Gail RowHnson-Busza,1 Aristotelis Bamias, Thomas Kraus/, and Agamemnon A. Epenetos Imperial Cancer Research Fund Oncology Group, Department of Clinical Oncology ¡G.R-B., A. B., A. A. E.¡,and Histopathology Department ¡A.B., T. K.J. Royal Postgraduate Medical School, Hammersmith Hospital, DuCane Road, London W12 OHS, England ABSTRACT Nude mice bearing s.c. xenografts of the human colon adenocarcinoma HT29 were given intratumor injections of a mixture of I2sl-labeled specific antibody (AUA1) and "'¡-labeled control antibody (HMFG1), or with the labels reversed. After dissection at 1 and 4 h postadministration, both specific and control antibodies had 47-63% of the injected dose (% ID) in the tumor. By 24 h. the tumor contained 43 ±11% ID of AI Al which persisted at around this level for 5 days and remained at nearly 20% ID at 18 days. In contrast, the HMFG1 activity was 23 ±9% ID at 24 h, which continued to fall and was less than 5% ID by 7 days. Normal organ levels were less than 2% ID/g for both antibodies, with HMFG1 being higher than AU AI at all times, resulting in specificity indices greater than 20 by 5 days. Autoradiography of tumors removed 2 h postinjection of '"I-labeled M'AI or HMFG1 showed high levels of antibody at the injection site. At 48 h and 7 days postinjection, the specific antibody was bound to the surface of tumor cells in islands remote from the injection site, whereas the control antibody was found only in the strenna and blood vessels, or as diffuse nonspecific uptake. These data indicate that intratumor injection of radiolabeled monoclo nal antibodies may achieve high radiation doses in accessible tumors without systemic irradiation. INTRODUCTION Although radiolabeled monoclonal antibodies are being in vestigated for diagnosis and treatment of malignant disease (1, 2), antibodies administered i.v. for therapy have several draw backs which limit the radiation dose delivered to the tumor. These include the high systemic radiation dose due to circulat ing antibody, the low absolute uptake by the tumor, typically 0.001-0.01% of the injected dose/g (3), and the catabolism of the antibody in vivo by enzymes in the liver and other normal organs. In addition, patients may develop antibodies against the murine monoclonal antibodies used for therapy (4), pre cluding the repeated treatments which may be necessary to achieve a tumoricidal dose of radiation. Regional therapy is an alternative to i.v. injection in cases when the disease is confined to a body cavity, and may overcome some of these problems. In a xenograft model of i.p. tumors, we have shown the short term advantage of i.p. over i.v. admin istration of radiolabeled antibodies to be approximately 50-fold (5). Clinically, i.p. administration of antibodies has been used by Stewart et al. (6) for the treatment of ovarian cancer; Lash- ford et al. (7) have given antibody therapy intrathecally for brain tumors and observed objective responses; and malignant pleural and pericardial effusions have been successfully treated by intracavitary radiolabeled antibodies (8). For accessible tumors such as primary prostate and primary Received 11/14/90; accepted 4/5/91. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this faci. 1To whom requests for reprints should be addressed. 2 Financially supported by the Ministry of National Economy of Greece (Technical Assistance Department). breast cancer, which can be treated by radioactive wires and implants (9), direct intratumor injection of radiolabeled mono clonal antibodies may be advantageous. Intratumor injection of tumor necrosis factor for the therapy of human tumor xeno grafts implanted in nude mice has been shown to be far more effective than the same dose given i.p. (10). Clinically, glioblas- toma has been treated by intratumor injection of autologous lymphocytes plus human lymphoblastoid interferon, and mor phological studies showed that the injected lymphocytes re mained within the tumor (1 1). LAK1 cells treated with bispecific antibody have been shown to be effective when given locally after tumor debulking in patients with malignant glioma (12). Intratumor injections of unlabeled human cytotoxic IgM anti bodies, with or without complement supplement, have been used to treat patients with cutaneous metastatic melanoma, resulting in tumor regression in the majority of cases (13). The aims of the work presented here were to improve the tumor uptake of radiolabeled monoclonal antibody by direct intratumor injection, and to study the diffusion through the tumor at the cellular level. In addition, the uptake and distri bution of a specific and a control antibody were compared, to determine to what extent antibody specificity is important via this route of administration. To the best of our knowledge, this is the first study of intratumor injection of radiolabeled mono clonal antibodies. MATERIALS AND METHODS Tumors. HT29 was established in 1964 (14) from the primary tumor in a female patient with adenocarcinoma of the colon. The cell mor phology is epithelioid. Cells were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (Gibco, Paisley, Scotland) in 175-cm2 Falcon tissue culture flasks (Becton Dickinson, Lincoln Park, NJ). When confluent, cells were harvested by using 0.06% trypsin in 0.02% EDTA solution, washed with medium, and resuspended at a concentration of 5 x IO7cells/ml in tissue culture medium. Tumor xenografts were initiated in the right flank of male nude mice of mixed genetic background (ICRF Animal Breeding Unit, South Mimms, Hertfordshire, United Kingdom) by a s.c. injection of 5 x 10* cells in a volume of 100 n\. For some studies, bilateral tumors were implanted into groups of mice. Animals were used for experiment 4 to 5 weeks later when the tumor diameter was 7-10 mm. Antibodies. AUAI was raised against a human colon adenocarcinoma cell line (IS). It is an IgGl immunoglobulin directed against a M, 35,000 protein associated with the majority of human gastrointestinal, ovarian, and breast carcinomas, as well as normal proliferating epithe lial cells (16). It reacts strongly with a cell surface antigen on HT29 cells, both in vitro and in vivo. HMFG1, also IgG 1, was raised by Taylor-Papadimitriou ( 17) against delipidated human milk fat globule. It recognizes a carbohydrate deter minant on a high molecular weight glycoprotein (M, > 400,000) nor mally produced by lactating human mammary cells, but was also found on many carcinomas (18). It does not bind to HT29 cells and was used as the control antibody. Antibody Biodistribution. Antibodies were labeled with ''I or '"I (IMS30 and IBS30, respectively, Amersham International, Amersham, 'The abbreviations used are: LAK. lymphokine activated killer; ID, injected dose; AUC, area under the curve. 3251 Research. on November 28, 2021. © 1991 American Association for Cancer cancerres.aacrjournals.org Downloaded from