Imaging and Therapy of Small Cell Carcinoma Xenografts Using 13II-labeled Monoclonal Antibody SWA111

The IgG2a monoclonal antibody SU All has been evaluated as a radioimmunotherapeutic agent for use in the treatment of small cell cancer of the lung. This antibody was initially selected for in vivo localization studies in a nude mouse model system because of its high ultimi) for the SU 2 small cell cancer cell line in vitro. Following i.v. injection of '"I labeled antibody into nude mice bearing SVY2xenografts good selective accumulation was observed with 10.5% of injected material/g of tumor. The level remained constant from day 2 to day 4 following injection. At day 4 the tumor: blood ratio was 7:1 and munirli UT. tumorkidney, and tumor:lung ratios were 17:1, 24:1, and 12:1, respectively. Initial radioimmunotherapeutic studies performed on established small cell cancer of the lung xenografts have shown reduction in tumor burden following a single injection of 300 u.C\ of '"I labeled SUAI 1 with no evidence of regrowth up to day 34 postinjection. Histological evaluation of treated tumors revealed large areas of necrosis and extensive fibrosis. A few residual cells of tumor origin could be observed and these displayed atypical morphology. The clonogenic potential of such cells remains to be determined by long term observation. Introduction The use of MAbs1 for diagnosis, imaging, and now RIT of malignant disease is an increasingly realistic objective (1-3). Clearly, in terms of RIT the nature of the disease chosen for attack must significantly affect the likelihood of success. SCC may be a particularly appropriate clinical objective for this therapeutic approach. SCC is a highly chemoand radiosensi tive condition and yet the prognosis for patients is very poor. Because of the metastatic nature of the disease a strong reliance is found on the use of chemotherapy as the first treatment modality. Although initially highly sensitive to this approach SCC is characterized by posttreatment emergence of chemoresistance. The residual form of the disease following first treat ment, which is ultimately responsible for relapse, is widely dispersed, is radiosensitive, and exists as small cellular foci. Such characteristics encourage the application of RIT and when taking into consideration the short remission time for most patients the overall picture is one in which RIT may be advan tageous and in which data relating to the efficacy of treatment can be quickly obtained. We describe here studies on the M Ab IgG2a SWA11, which appears (by cross-competition experiments) to belong to Cluster w4 as defined at the International Workshop on SCC, London, 1987 (4). This antibody has been chosen for in vivo evaluation as a potential agent for RIT because of its strong reactivity with SCC lines in vitro (5). SWA11 is characterized by a very high capacity for in vivo localization, long biological half-life at the 1Presented at the "Second Conference on Radioimmunodetection and Radioimmunothcrapy of Cancer," September 8-10. 1988. Princeton. NJ. Supported in part by the Swiss Cancer League (FOR.302.85) and the Stiftung fÃ1⁄4r wissen schaftliche Forschung an der UniversitätZÃ1⁄4rich. 2To whom requests for reprints should be addressed, at Division of Oncology. University Hospital, CH-8091 /urich. Switzerland. 'The abbreviations used are: M Ab, monoclonal antibody; RIT. radioimmunotherapy; SCC. small cell carcinoma of the lung: CEA, carcinoembryonic antigen; PBS, 0.05 Mphosphate buffer-0.1 Msodium chloride; "Ã-ID/g.percentage of injected dose per g of tissue. tumor, and relatively low background levels in nontarget or gans. These characteristics suggest SWA 11 to be a suitable candidate for use in the establishment of an in vivo model for the radioimmunotherapy of SCC tumors and possibly as a clinical reagent for both imaging and therapy. The requirement for new agents such as MAbs to be used as therapeutic tools for use against SCC is indicated by the high mortality of the disease (6, 7). Materials and Methods Cell Lines. The SCC cell line SW2 was established in the laboratory of Dr. S. D. Bernal, Dana Farber Institute. It was routinely grown in RPMI supplemented with l mM glutamine and 10% fetal calf serum. Monoclonal Antibodies. Our procedure for antibody generation has been described previously (8). Antibody SWA11 was purified as follows. A 30-55% ammonium sulfate fraction was taken from culture super natant and adsorbed onto a protein A column in PBS. The adsorbed IgG was eluted with 100 mM citrate buffer (pH 4.5) and then dialyzed against 10 m\i phosphate buffer (pH 6.8) containing 0.01 HIMCaCI2. The antibody was then applied to a hydroxylapatite column (Bio-Gel HPHT; Bio-Rad, Richmond, CA) and then eluted with a linear gradient to 350 niM phosphate. Antibody Labeling Techniques. lodo-Gen (0.1 mg; Pierce) was dis solved in 0.2 ml chloroform and added to a 1-ml vial. The solvent was evaporated with a gentle stream of nitrogen and then 0.5 mg of antibody in 0.25 ml of water was added. "'I or I25I(0.3 mCi in 0.03 ml) was added and the reaction continued for 15 min at 10°Cwith stirring. The reaction mixture was applied to a prepacked Sephadex G-50 column which had been equilibrated with PBS. The solution was sterilized by passage through a 0.22 urn filter (Millex GV). 0.04 ml human serum albumin (25%) was added as a protein carrier, and then radiochemical purity was assessed by thin layer chromatography using a CEL300 polygram (Machet)' Nagel) and methanol (85%). Radiochemical purity was generally in excess of 95%. In Vitro Immunoreactivity. To determine the biological activity of radiolabeled SWA11 SW2 cells were washed 3 times in PBS (with 5% nonfat milk-0.05% azide) and varying cell numbers were then incubated for 2 h at 4°Cwith a fixed amount of radiolabeled antibody (690.000 cpm). After a washing, the activity in the cell pellet was counted. The number of counts remaining unbound were plotted against the recip rocal of the cell number (9). Resistance to Radiolabeling. To assess the maximal specific activity which could be achieved without impairment of biological activity of SWA 11 the antibody was labeled over the range of 2.5 to 25 mCi/mg. Labeled SWA 11 at a concentration determined to give half-maximal binding was then evaluated in a fixed cell radioimmunoassay and the number of counts bound was plotted against specific activity. SW2 Xenograft Model. Female NMRI-nw/nu mice were bred within the Biologisches Zentrallabor, Universitaetsspital, Zurich. Pathogen free food and acidified drinking water were given ad libitum. Xenograft passage was performed by s.c. transplantation of 2-3-mm1 pieces of SW2 tumor into 4-6-week-old animals. Within approximately 3 weeks tumors were ready for use in antibody localization studies having reached a size of approximately 1 cm'. In Vivo Localization Studies. The in vivo distribution of S WA11 was determined by simultaneous i.v. injection of 20 jjg (10 ^Ci) of '"I labeled SWA11 and the same amount and activity of a I25Ilabeled antiCEA. Both antibodies were of the IgG2a subclass. Thyroid blocking was achieved by the administration of 2-3 drops of Lugol's solution