Radioimmunotherapy of Small Volume Disease of Colorectal Cancer Metastatic to the Liver: Preclinical Evaluation in Comparison to Standard Chemotherapy and Initial Results of a Phase I

At the time of surgery, occult metastases (micrometastases) are present in more than 50% of colorectal cancer patients, and the liver is the most frequent site of apparent metastatic disease. Frequently, adjuvant chemotherapy is unable to prevent tumor recurrence. Thus, novel therapeutic strategies are warranted. The aim of this study was to establish a model of human colon cancer metastatic to the liver of nude mice, to assess, in this setting, the therapeutic efficacy of radioimmunotherapy (RAIT) compared to standard chemotherapy and to evaluate, in a Phase I/II trial, the toxicity and therapeutic efficacy of RAIT in colorectal cancer patients with small volume disease metastatic to the liver. Multiple liver metastases of the human colon cancer cell line GW-39 were induced by intrasplenic injection of a 10% tumor cell suspension. Whereas controls were left untreated, therapy was initiated on day 10 or 20 after tumor inoculation with the 131I-labeled, low affinity anticarcinoembryonic antigen (anti-CEA) monoclonal antibody (MAb), F023C5 ( K a = 1 0 7 liters/mol), or the high-affinity anti-CEA MAb, MN-14 (K a = 109 liters/mol), or chemotherapy (5-fluorouracil/leucovorin (folinic acid) versus irinotecan) at their respective maximum tolerated doses (MTDs). Twelve colorectal cancer patients with small volume disease metastatic to the liver (all lesions --<2.5 cm) were entered into a mCi/m 2based Phase I dose escalation study with 131I-labeled humanized version of MN-14, hMN-14. The patients were given single injections, starting at 50 mCi/m 2 and escalating in 10-mCi/m 2 increments. The MTD was defined as the dose level at which -<1 of 6 patients develop grade 4 myelotoxicity. In the mice, untreated controls died from rapidly progressing hepatic metastases at 6 8 weeks after tumor inoculation. The life span of mice treated with 5-fluorouracil/ leucovorin was prolonged for only 1-3 weeks, whereas irinotecan led to a 5-8-week prolongation. In contrast, at their respective MTDs, the 131I-labeled low-affinity antiCEA MAb, F023C5, led to a 20% permanent cure rate, and the high affinity MAb, MN-14, led to an 80% permanent cure rate, when therapy was initiated at 10 days after tumor inoculation. In the 20-day-old tumor stage, al though it prolonged life, 13~I-F023C5 was unable to achieve cures, whereas ~31I-MN-14 was still successful in 20%. Histologically, no remaining viable tumor cells could be demonstrated in these animals surviving > 6 months. In patients, the MTD was reached at 60 mCi/m 2 of hMN-14 (at 70 mCi/m 2, two of three grade 4 myelotoxicities). Of 11 assessable patients, 2 had partial remissions (corresponding to an objective response rate of 18%), and 5 (45 %) had minor/mixed responses or experienced stabilization of previously rapidly progressing disease. These data suggest that in small volume disease, RAIT may be superior to conventional chemotherapy. Antibodies of higher affinity seem to be clearly superior. The clinical response rates in patients with small volume disease are encouraging, being comparable to the response rates of conventional chemotherapeutic regimens but with fewer side effects. Ongoing studies will show whether t rea tment at the MTD will fur ther improve therapeutic results. 1 Presented at the "Seventh Conference on Radioimmunodetection and Radioimmunotherapy of Cancer," October 15-17, 1998, Princeton, NJ. Supported by Deutsche Forschungsgemeinschaft Grant DFG Be 1689/ 4-1 (to T. M. B.) and by Outstanding Investigator Grant CA39841 (to D. M. G.) and Program Project Grant CA 54425 (to David V. Gold) from the National Cancer Institute, NIH. 2 To whom requests for reprints should be addressed, at the Department of Nuclear Medicine, Georg-August-University of G6ttingen, RobertKoch-Strasse 40, D-37075G6ttingen, Germany. Phone: 49-551-398512; Fax: 49-551-39-8526; E-mail [email protected]. I n t r o d u c t i o n Colorectal cancer, making up 15% of all malignancies, is one of the most frequent cancer types in both sexes (1). To date, surgery is the only potentially curative therapeutic modality (1). However, despite the introduction of adjuvant chemotherapeutic regimens, which have been shown to reduce the relapse rate by approximately 30% (1, 2), the disease will still recur in approximately one-half of the patients. In unresectable cases, the 5-year survival is close to zero (3, 4), despite several promising Research. on October 16, 2017. © 1999 American Association for Cancer clincancerres.aacrjournals.org Downloaded from Clinical Cancer Research 3233s new chemotherapeutic developments, such as the introduction of the semisynthetic camptothecin derivative irinotecan as a topoisomerase I inhibitor (1, 5, 6). Immunotherapy with the murine MAb3/CO17-1A, which is directed against a 41-kDa cell surface glycoprotein of many epithelial cells, including colorectal malignancies, has shown, in an adjuvant setting, results comparable to those obtained with adjuvant chemotherapy (7, 8). This is in contrast to its lack of significant antitumor effects in established metastatic disease (9, 10). In this context, RAIT appears as an attractive therapeutic concept, aiming to deliver tumoricidal radiation doses to tumors that may be too large for being susceptible to a purely immunological approach (11). Indeed, in radiosensitive tumors, such as non-Hodgkin's lymphoma, RAIT has led to long-term remissions or even cures in a high percentage of treated patients (12, 13). In solid tumors, however, success is still limited, probably due to the low specific accretion of the radiolabeled antibody in the tumor target as compared to the normal tissues (11). However, as we have shown earlier, the tumor uptake, and thus the radiation dose to the tumor, increases exponentially with decreasing tumor size (11, 14, 15). Therefore, RAIT may be a viable option, especially in small volume and minimal residual disease (11, 14, 15). In colorectal cancer, the liver is the most frequent site of apparent metastatic disease (1). The aim of the present study was, therefore, to establish a model of human colon cancer metastatic to the liver of nude mice, to compare the therapeutic efficacy of radiolabeled MAbs ver sus equitoxic "standard" chemotherapy with 5-FU/folinic acid (leucovorin), or irinotecan, in this xenograft model, and to evaluate, in a pilot Phase I clinical trial in colorectal cancer patients, the toxicity and efficacy of RAIT in small volume disease metastatic to the liver. Patients and Methods Antibodies and Radiolabeling. The murine MAb antiCEA, clone F023C5, was obtained from Sorin Biomedica (Saluggia, Italy). It belongs to the IgGl isotype and has an affinity constant of approximately 0.5 • 1 0 7 liters/mol (16), whereas MN-14 is a high affinity MN-14 ( K a = 1 0 9 liters/ mol) IgG1 subtype murine anti-CEA MAb (obtained from Immunomedics, Inc., Morris Plains, NJ; Ref. 17). Preclinical and clinical experiences with these antibodies have been described previously (16-18). Both are directed against a similar class III epitope of the CEA molecule, according to the classification of Primus et al. (19). Recently, a humanized, CDR-grafted, form of MN-14, called hMN-14, has been developed and introduced in clinical trials (20). The anti-CD3 antibody, OKT3 (CILAG, Sulzbach/Taunus, Germany), was used as a nonspecific, antibody-isotype (IgGza)-matched control antibody (21). Iodine-131 was purchased as sodium iodide in 0.1 M NaOH from NEN DuPont (Bad Homburg, Germany). Radioiodination was performed using the iodogen method, as described previously (21). Briefly, the antibody in PBS buffer was transferred into an iodogen-coated glass vial (500 Ixg of iodogen coating the inner surface of a 10-ml vial) with a magnetic stirbar placed inside. Sodium phosphate buffer, 0.5 M, pH 7.4, was added in a volume that was twice as much as the volume of the radioiodine to be used. The specific activity used was 15-20 mCi/mg. The vial was placed on a magnetic stirrer, and the activity was added in 2.5 ml of 0.04 M sodium phosphate, pH 7.4. After a stilxing time of 10-15 min, Dowex 1 • 8-100 anion exchange resin (C1 form, Sigma, Deisenhofen, Germany) was added, and the incubation time was prolonged for another 5 min. Subsequently, the radioiodinated antibody was filtered through a sterile Millex-GV T M filter (pore size, 0.22 ~m; Millipore, Molsheim, France). The quality of each preparation was tested by instant TLC and size-exclusion HPLC. The amount of unbound radioiodine was less than 2% in each preparation. Animal Models. Female nude mice, 19-23 g and 4-5 weeks old, were purchased from Charles River (Sulzfeld, Germany). The human colon carcinoma cell line GW-39 was serially propagated as described in detail earlier (21, 22). Briefly, tumors were minced through a 40-mesh screen and rinsing with sterile HBSS (ICN Biomedicals, Eschwege, Germany) to yield a 20% cell suspension. A 200-~1 dose of this suspension was injected s.c. For the liver metastasis model, mice were anesthetized by i.p. injection of a mixture of 0.35 mg of 5,6-dihydro-2-(2,6xylidino-)-4H-1,3-thiazine hydrochloride (Rompun | Bayer, Leverkusen, Germany) and 1.5 mg of ketamin hydrochloride (Ketanest | Parke-Davis, Berlin, Germany). A small left subcostal incision was made ( c f Fig. 1), and the spleen was exposed and isolated between strips of sterile gauze soaked with alcohol. A 10% GW-39 cell suspension in HBSS was slowly injected into the spleen. Two min later, the splenic vessels were ligated, the spleen was removed, and the peritoneal cavity and abdomen were closed with staples. Multiple (as many as over 250) microscopic tumor colonies develop in the livers of such animals, reaching a size of approximately 250-500 Ixm at 10 days and a size of 1-2.5 mm at 20 days after tumor cell inoculation. With high reproducibility, the animals begin to lose weight by 4 6 weeks, and they eventually die at 6-9 weeks after tumor inoculation. Experimental RAIT and Chemotherapy. Animals were either left untreated (controls) or injected with a single dose of naked (200 ~g) or radiolabeled antibody, at their respective MTDs of radioactivity (260 t~Ci of 131I-MN-14 IgG or 600 [xCi of ~3~I_F023C5 IgG). 4 Eight to 20 (in most cases, 10) mice were studied in each treatment group. For chemotherapy, the mice received an i.v. injection of 1.8 mg of leucovorin, followed by 0.6 mg of 5-FU 1 h later (both 3 The abbreviations used are: MAb, monoclonal antibody; 5-FU, 5-fluorouracil; MTD, maximum tolerated dose; RAIT, radioimmunotherapy; CEA, carcinoembryonic antigen. 4 T. M. Behr, M. L6hr, M., Bdh6, A. L. Salib, and W. Becket, unpublished results. Research. on October 16, 2017. © 1999 American Association for Cancer clincancerres.aacrjournals.org Downloaded from 3234s Radioimmunotherapy of Colorectal Cancer Fig. 1 Surgical procedure of the induction of liver metastases of the human colonic cancer cell line, GW-39, in anesthetized nude mice. a, subcostal incision (top panel), exposure of the spleen on an alcohol gauze (middle panel), and intrasplenic injection of 100 txl of a 10% GW-39 tumor cell suspension (bottom panel), b, ligation of the splenic vessels (top panel), splenectomy (middle panel), and stapling of the wound (bottom panel). were obtained from Sigma), for five consecut ive days each in 200 OA of saline. This is the MTD in mice determined by Blumenthal et al. (23) and conf i rmed in our exper ience as well. 4 For ir inotecan (CPT-1 l) chemotherapy, the mice were given a single i.v. inject ion of 2 mg of i r inotecan hydrochloride in 100 txl of saline (Camptosar TM, Pharmacia & Upjohn, Kalamazoo, MI), which has been shown to represent the single administrat ion MTD. 4 Body weight was recorded weekly, and survival was monitored. The MTD was defined as the highest possible activity under the respective conditions that did not result in any animal deaths, with the next higher dose level resulting in at least 10% of the animals dying (24, 25). Total and differential leukocyte and thrombocyte counts, blood urea nitrogen, as well as glutamate oxaloactetate transammase and alkaline phosphatase were determined on the day of therapy and at weekly intervals thereafter, as described previously (24, 25). Initial Clinical Phase I/II RAIT in Patients with Small Volume Disease. Twelve colorectal cancer patients with small volume metastatic disease (all lesions --<2.5 cm) were entered in a mCi/m2-based dose escalation study with the 131I-labeled humanized anti-CEA MAb, hMN-14 (human IgG1 subtype; Ref. 20). All had histologically proven, CEA-expressing colorectal cancer. None of the patients had major surgery or external beam radiation within the last 4 weeks before the study, and they had a performance status of 60 or greater on the Karnofsky scale. After informed consent had been obtained, all patients were premedica ted with 200 mg of potassium iodide daily, initiated 24 h before the ant ibody administrat ion to decrease thyroid and gastric uptake. This medica t ion was cont inued until the patients were removed from radiat ion restrictions. The radiolabeled antibody was infused during a 10 -30-min period in a volume between 10 and 30 ml of sterile 0.9% NaC1 containing 1 .0-2 .5% human serum albumin. The patients were given single injections, starting at 50 mCi /m 2 and escalating in 10 mCi /m 2 increments . Rout ine blood chemistry parameters, b lood cell counts, and differential blood counts were obtained weekly until 8 weeks post therapy and then monthly thereafter. Red mar row and organ toxicity was graded according to standard toxicity criteria (Common Toxicity Criteria of the National Cancer Institute; Ref. 26). Three patients were studied on each dose level, six if one or more developed a toxicity higher than grade 3. The MTD was defined in this trial as the very dose level at which --< 1 of 6 patients develop a myelotoxic i ty higher than grade 3. Scanning was performed with a Picker Prism 2000 TM double-headed gamma camera equipped with high-energy colimaResearch. on October 16, 2017. © 1999 American Association for Cancer clincancerres.aacrjournals.org Downloaded from Clinical Cancer Research 3235s tors (Picker, Cleveland, OH). Anterior and posterior whole-body scans were obtained for 131I daily from 4 h to 240 h postinjection. MAb blood clearance rates were determined by counting samples at various times after the end of the infusion. Totalbody clearance rates were determined from whole-body scans and hand-held rate meter measurements. For organ and tumor dosimetry, regions of interest of the whole-body, organs, and visible tumors were generated from the anterior and posterior planar views. All calculations were performed using personal computer software developed for this purpose (27). The blood time-activity concentration data were fit by an exponential function to obtain the cumulated activity in the blood. The red marrow cumulated activity was calculated from the blood data by multiplying this concentration by 1500, as the assumed weight in grams of the marrow in an average adult. Time-activity curves were generated and integrated, and cumulated activities, as well as residence times, were derived (27). These data were entered into the MIRDOSE3 program (28), which yields the organ, red marrow, tumor, and wholebody dosimetry according to the MIRD scheme, based on derived residence times. All patients underwent computed tomography of the chest, abdomen, and pelvis on the day before therapy, as well as 4 and 12 weeks later. Afterward, patients were evaluated at 3-month intervals, including clinical follow-up, sonography of the abdomen, whole-body computed tomography, routine blood chemistry, and tumor marker evaluations. Therapeutic responses were graded according to oncological standard criteria (29), as follows. (a) Complete remission: absence of clinically detectable disease, with complete normalization of serum tumor marker (CEA) levels for at least 1 month. (b) Partial remission: at least 50% reduction in the sum of the diameter of measurable disease without appearance of new lesions and without increase in size of any lesion, for at least 1 month. (c) Minor/mixed response: less than 50% reduction in sum of the diameters of measurable lesions without increase in size of any lesion. (a t ) Stabilization of disease: no increase in the size of measurable lesions and no appearance of new lesions for at least 3 months in patients with rapidly progressing disease before therapy. (e) Progression: greater than 25% increase in measurable disease and/or any new metastasis on treatment.