Cetuximab Preclinical Antitumor Activity

104 Cetuximab preclinical antitumor activity (monotherapy and combination based) is not predicted by relative total or activated epidermal growth factor receptor tumor expression levels Robert Wild, Krista Fager, Christine Flefleh, the cetuximab resistant/weakly responsive HT29, A549, David Kan, Ivan Inigo, Stephen Castaneda, and WiDr models. We conclude that preclinical activity Feng (Roger) Luo, Amy Camuso, associated with cetuximab monotherapy does not corre- Kelly McGlinchey, and William C. Rose late directly with relative basal levels of total or activated (pY1068) EGFR in a tumor. Moreover, robust single-agent Bristol-Myers Squibb Co., Pharmaceutical Research Institute, activity by cetuximab may be the best predictor for this Oncology Drug Discovery, Princeton, New Jersey agent to potentiate chemotherapy-mediated antitumor activities. [Mol Cancer Ther 2006;5(1):104–13] Abstract Although Erbitux (cetuximab) has proven therapeutic Introduction benefit in the clinical setting, the molecular determinants Erbitux (cetuximab) is a chimeric mouse/human monoclo- predicting responsiveness to this agent are still not very nal antibody of the IgG1 subclass that targets the human well understood. Here, we assessed the relationship epidermal growth factor receptor (EGFR; ref. 1). EGFR is between basal total and activated (pY1068) epidermal overexpressed in about one third of all human cancers and growth factor receptor (EGFR) levels in a tumor and the has been directly implicated in tumor growth and responsiveness to cetuximab monotherapy or combina- progression (1). In particular, elevated expression levels tion-based treatment using human xenograft models. of EGFR in colon carcinomas have been linked to more Cetuximab treatment alone (0.25–1 mg/mouse/injection, aggressive disease and poor prognosis (2). Hence, strate- q3d, i.p.) effectively delayed the growth of GEO and gies aimed at disrupting the function of the EGFR have L2987 tumors by a minimum of 10 days corresponding to been investigated as exciting new approaches for the log cell kill values of z1.0. Borderline activity was seen in treatment of cancer and include small molecule inhibitors the A549 and WiDr xenografts. However, cetuximab as well as various biologics approaches (3). In the case of failed to show any significant antitumor activity in the the monoclonal antibody cetuximab, clinical studies have HT29, HCT116, LOVO, Colo205, LX-1, HCC70, and N87 shown that this agent is capable of significantly inhibiting models. All of the studied tumors had detectable yet tumor growth (4). In particular, about 11% to 23% of variable levels of EGFR. For combination regimens, irinotecan (CPT-11) refractory, EGFR-positive colon cancer cetuximab (1 mg/mouse/injection, q3dx5, i.p.) and cis- patients dramatically benefited from this antibody therapy platin (4.5 mg/kg/injection, q3dx5, i.v.) proved to be when administered as a single agent or in combination significantly more efficacious than individual monothera- with irinotecan (5). Thus, in February 2004, cetuximab was pies in the cisplatin-refractory yet cetuximab-responsive approved in the United States for the treatment of GEO tumor model (P < 0.001). However, no therapeutic advanced colon cancer in combination with irinotecan. enhancement was observed in the cisplatin and cetuximab Nevertheless, several questions remain unanswered about weakly responsive A549 xenograft. Similarly, combina- this therapeutic. Specifically, it is still not very well tions of CPT-11 (48 mg/kg/injection, q3dx5, i.v.) with understood why only a fraction of patients with EGFR- cetuximab (1 mg/mouse/injection, q3dx5, i.p.) failed to positive tumors respond to cetuximab treatment (i.e., which show any improvements over individual monotherapies in exact patient population will most likely benefit from this antibody therapy). Preclinically, many studies have shown that treatment with cetuximab results in inhibition of tumor growth in nude mice bearing xenografts of human cancer cell lines Received 7/19/05; revised 10/11/05; accepted 11/15/05. (6–14). Moreover, treatment with the antibody in combi- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked nation with chemotherapeutic drugs or radiation has been advertisement in accordance with 18 U.S.C. Section 1734 solely to reported to be more efficacious than individual mono- indicate this fact. therapies in the same or similar xenograft model systems Requests for reprints: Robert Wild, Bristol-Myers Squibb Co., Oncology (6–11, 13–20). Although published preclinical reports Drug Discovery, P.O. Box 4000, Mailstop K23-03, Princeton, NJ 08543- 4000. Phone: 609-252-5808; Fax: 609-252-7340. suggest that EGFR expression may be needed for cetux- E-mail: [email protected] imab activity (21, 22), recent clinical evidence indicates that Copyright C 2006 American Association for Cancer Research. response to cetuximab treatment is independent of the doi:10.1158/1535-7163.MCT-05-0259 relative degree of EGFR expression levels in tumors (23). Mol Cancer Ther 2006;5(1). January 2006 Downloaded from mct.aacrjournals.org on September 27, 2021. © 2006 American Association for Cancer Research. Molecular Cancer Therapeutics 105 The objective of the following studies was to further Tu m o r L in e s expand on our knowledge of cetuximab monotherapy All human carcinoma xenografts were propagated in activities in a broad panel of EGFR-positive human nude mice as s.c. transplants using tumor fragments carcinoma xenografts. In particular, we wanted to assess obtained from donor mice. The models employed included whether any correlation existed between basal total EGFR the colon carcinomas: GEO, HT29, HCT116, LOVO, WiDr, or basal activated (pY1068) EGFR levels in a tumor and the and Colo205; the lung carcinomas: L2987, A549, and LX-1; responsiveness to cetuximab treatment. To this effect, we the breast carcinoma, HCC70; and the gastric carcinoma, employed tumor models from several different histologies N87. Tumor passage occurred approximately every 2 to 4 (colon, lung, breast, and gastric cancer), all known to weeks depending on the model. All tumor implants for frequently overexpress EGFR (24). Finally, we also evalu- efficacy testing were s.c. in nude mice and employed tumor ated the utility of combining cetuximab with the chemo- fragments from tumors passaged at least twice in vivo. therapeutic agents irinotecan (CPT-11) or cisplatin in Quantitation of Total EGFR and Activated (pY1068) chemotherapy-refractory or poorly responding tumor EGFR Levels in HumanTumor Xenograft Samples models because positive clinical results have been reported Human tumor xenografts were established in athymic in the same setting (5, 25). Based on our collective mice and surgically removed when they reached an preclinical data, attempts were then made to identify the average tumor size of 150 to 300 mg. Resected tumors determinants that predict synergistic antitumor activity of were immediately snap-frozen in liquid nitrogen and cetuximab with conventional anticancer agents. stored at À80jC until further use. Frozen tumor tissues were then lysed in ice-cold lysis buffer that contained 10% Triton X-100, 5% glycerol, 20 mmol/L Tris-HCl (pH 7.7), Materials and Methods 0.5 mmol/L EDTA, 0.1 mol/L NaCl, 1 mmol/L sodium Compounds and Their Administrations orthovanadate, 1% Phosphatase Inhibitor Cocktail 2 Clinical-grade cetuximab was supplied by ImClone (Sigma), and 2% Complete protease inhibitors (Roche Systems (New York, NY) at a concentration of 2 mg/mL Molecular Biochemicals, Indianapolis, IN). The sample in a buffer consisting of 10 mmol/L sodium phosphate and was subsequently homogenized using a Wharton tissue 145 mmol/L sodium chloride at pH 7.2. For all efficacy homogenizer. Cell lysates were clarified by centrifugation studies requiring lower concentrations of cetuximab, (10 minutes at 10,000 Â g), and protein concentrations the stock solution was diluted with sterile PBS (pH 7.4). were determined using the MicroBCA method (Pierce). Cetuximab was administered i.p. at a constant volume of Tumor lysates from all of the samples were adjusted 0.5 mL/mouse. Clinical-grade cisplatin (Platinol) was to a concentration of 3 mg/mL protein. Equal amounts of obtained from Bristol-Myers Squibb (Princeton, NJ), and total protein (30 Ag) from each tumor sample were then irinotecan (CPT-11) was purchased commercially; they used to analyze the relative total human EGFR and were both dissolved in sterile 0.9% NaCl (saline) and activated EGFR (pY1068) levels in the different xenograft administered i.v. in a volume of 0.01 mL/g of mouse body samples as determined by ELISA assay according to the weight. CPT-11 and cisplatin were administered up to their protocol recommended by the manufacturer (Biosource maximum tolerated dose (MTD) as previously reported International). Expected values for different tumor cell (26, 27). lines, as provided by the assay manufacturer, were used Chemicals and Reagents as a guideline to classify samples as high, medium, or low Complete protease inhibitor tablets were from Roche EGFR expressers. Furthermore, we considered samples Diagnostics (Indianapolis, IN). MicroBCA reagents were with EGFR levels higher than the calculated mean for all from Pierce (Rockford, IL). Human-specific EGFR and evaluated tumor samples (f20 ng/mL) as high receptor pY1068 EGFR ELISA kits were purchased from Biosource expressers. International, Inc. (Camarillo, CA). All

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