Effects of Palifermin on Antitumor Activity of Chemotherapeutic and Biological Agents in Human Head and Neck and Colorectal Carcinoma Xenograft Models

Home , Palifermin

Rachael Brake,1 Charlie Starnes,2 John Lu,2 Danlin Chen,2 Suijin Yang,3 Robert Radinsky,2 and Luis Borges4

1Amgen, Inc., Cambridge, Massachusetts; 2Amgen, Inc., Thousand Oaks, California; 3Pfizer, Inc, San Diego, California; and 4Amgen, Inc., Seattle, Washington

Abstract Introduction Damage to the gastrointestinal mucosa is a common The clinical utility of many anticancer agents is limited by dose-limiting toxicity of several anticancer therapies. their toxic effects on normal tissues, particularly epithelial Until recently, adequate control of oral mucositis was tissues. The high proliferative rate of the gastrointestinal considered a significant unmet medical need, with most epithelium makes it particularly susceptible to damage by available treatments providing only palliative benefits chemotherapy or radiotherapy (1), resulting in a pathologic without protecting the gastrointestinal epithelium from condition known as mucositis. The direct cytotoxic effects of the damaging effects of cancer therapy. In 2005, therapy can set in motion a cascade of pathologic events within palifermin [recombinant human keratinocyte growth the submucosal and epithelial layers that leads to progressive factor (KGF)] was approved to decrease the incidence cell damage and death, mucosal atrophy, thinning of the and duration of severe oral mucositis in patients with epithelium, ulceration, and a breakdown of the mucosal barrier hematologic malignancies receiving myelotoxic therapy function (2-4). Mucositis can be extremely painful, puts patients requiring hematopoietic stem cell support. Current trials at risk of potentially life-threatening infections, increases are investigating the use of palifermin in solid tumor overall health care use, and has been associated with increased settings. The objective of this study was to determine overall mortality in cancer patients (4-9). It is considered one of whether combining palifermin with different the most common dose-limiting toxicities of some anticancer chemotherapeutic or biological agents affected the therapies and can force dose reductions or interruptions in antitumor activity of these agents in human head and cancer treatment that may contribute to suboptimal clinical neck (FaDu) and colorectal (HT29) carcinoma xenograft outcomes (6, 10-14). models. Nude CD1mice were injected with 1 Â 107 of The incidence of mucositis ranges from 5% to 40% in either FaDu or HT29 cells, which express both KGF and patients receiving chemotherapy for solid tumors such as epithelial growth factor receptors. Animals were colorectal cancer (15-17) to near 100% in patients receiving treated with palifermin in various combinations with combined radiation and chemotherapy for head and neck cancer chemotherapeutic (5-fluorouracil and cisplatin) and/or (18, 19). The chemotherapeutic agents 5-fluorouracil (5-FU) biological (bevacizumab, cetuximab, and panitumumab) and cisplatin are associated with particularly high rates of agents. Palifermin alone had no effect on either FaDu mucositis (11, 20). Until very recently, most of the treatments or HT29 tumor growth. Palifermin did not affect the for mucositis provided only palliative benefits and did little if therapeutic efficacy of 5-fluorouracil, cisplatin, anything to interfere with the damaging effects of cancer cetuximab, bevacizumab, or panitumumab in any of the treatments on the gastrointestinal epithelium (21). two- or three-way drug combinations tested in either Keratinocyte growth factor (KGF) has been shown to model. The results of this study showed that palifermin stimulate proliferation, migration, differentiation, and survival did not promote the growth of two carcinoma cell lines of epithelial cells (22). It is a member of the fibroblast growth that express functional KGF receptors and did not factor family, and KGFR (FGFR2IIIb) is predominantly protect these tumor cells from the antitumor effects of expressed by epithelial tissues (23). Numerous in vitro and several chemotherapeutic and biological agents. in vivo studies have shown that KGF can have cytoprotective (Mol Cancer Res 2008;6(8):1337–46) and regenerative effects on epithelial tissues exposed to a variety of insults, such as radiation (24, 25), hypoxia (26), and

Received 11/5/07; revised 4/22/08; accepted 5/5/08. chemotherapy (25, 27, 28). In 2005, a truncated form of The costs of publication of this article were defrayed in part by the payment of recombinant human KGF (palifermin, Kepivance, Amgen, Inc.) page charges. This article must therefore be hereby marked advertisement in was approved to decrease the incidence and severity of oral accordance with 18 U.S.C. Section 1734solely to indicate this fact. Requests for reprints: Luis Borges, Hematology and Oncology Research, mucositis in stem cell transplant patients suffering from Amgen, Inc., 1201 Amgen Court West, Seattle, WA 98119. Phone: 206-265- hematologic malignancies (29), but the safety and efficacy of 7884; Fax: 206-216-5929. E-mail: [email protected] Copyright D 2008 American Association for Cancer Research. palifermin in patients being treated for nonhematologic doi:10.1158/1541-7786.MCR-07-2131 malignancies has not been established.

The use of any KGF-based therapy in patients with solid the therapeutic agents that were relevant to the clinical settings. tumors must be considered with caution as many solid tumors Any candidate model had to be responsive to the five antitumor are epithelial in origin and have the potential to express KGFRs therapeutic agents selected (5-FU, cisplatin, bevacizumab, (30). Several nonclinical studies, however, suggest that the cetuximab, and panitumumab) as single agents, and this KGF/KGFR axis does not seem to be a critical pathway significantly limited our choices. promoting tumorigenesis or tumor growth (30-36). In addition, The expression of KGFR and EGFR transcripts by FaDu preliminary long-term follow-up data from a clinical study of and HT29 cell lines was maintained in vivo in both xenograft palifermin in the treatment of oral mucositis in patients suffering models (Fig. 1B and C). In vitro, both FaDu and HT29 cell lines from metastatic colorectal cancer found no evidence that expressed functional KGFR and EGFR, which could be palifermin had any effect on the cancer outcomes in patients stimulated by the addition of KGF and EGF. Activation of both followed for a median of 14.5 months (27). Nonetheless, a more receptors by their respective ligands resulted in significant thorough evaluation of the potential of palifermin to stimulate phosphorylation of protein kinase B (PKB, Akt) and extracellular solid tumor growth or protect tumors from the cytotoxic effects signal-regulated kinase (Erk; Fig. 2). Although these experi- of chemotherapy or targeted biological agents is needed. ments were run under serum-free conditions, which are unlikely The purpose of the present study was to evaluate the effects to be of physiologic relevance, they were designed in this way to of palifermin on the antitumor activity of various chemother- enhance the sensitivity of this assay. This was deliberately done apeutic and biological agents in human head and neck (FaDu) in an attempt to confirm protein expression of both receptors and and colorectal (HT29) carcinoma xenograft models expressing provide the greatest opportunity to detect an intracellular signal. functional KGFRs and epithelial growth factor receptor (EGFR). The experimental design took into account current Effects of Palifermin in Combination with Cisplatin and clinical practice in the treatment of head and neck and Bevacizumab on the Growth of FaDu Human Head and colorectal cancers, evaluating several cytotoxic and antibody- Neck Carcinoma Xenografts based therapies currently used to treat these cancer types. These Xenografts of FaDu carcinoma cells were implanted included the cytotoxic agents cisplatin and 5-FU, among the subcutenously in nude mice and allowed to grow for 11 days most common treatments for head and neck cancer (37, 38) and before the start of treatment. Palifermin (5 mg/kg) was colorectal cancer (39, 40), respectively. Three new antibody- administered for 3 days before and after cisplatin dosing. The based therapies were also evaluated and they included 5 mg/kg dose is efficacious in protecting mice from bevacizumab, a recombinant humanized monoclonal IgG1 chemotherapy and radiation-induced gastrointestinal injury antibody to human vascular endothelial growth factor (Avastin, (25, 52) and the 3-plus-3 dosing regimen was chosen to mimic Genentech), cetuximab (recombinant human/mouse chimeric that used in stem cell transplant patients (53-55). Bevacizumab monoclonal antibody to human epidermal growth factor was dosed twice a week throughout the duration of the receptor; Erbitux, ImClone Systems, Inc.), and panitumumab experiment based on our previous experience with this dosing (recombinant human IgG2n monoclonal antibody to human regimen in this xenograft model (Fig. 3A). Tumor growth was EGFR; Vectibix, Amgen). These are all used to treat colorectal significantly less in animals treated with either cisplatin, cancer (41-48); cetuximab is also used to treat head and neck bevacizumab, or the combination of cisplatin and bevacizumab cancer (38). Moreover, some of these antibody-based therapies than in animals treated with vehicle (P < 0.0001). The addition are indicated for use in combination with cytotoxic agents such of palifermin had no effect on the antitumor efficacy of any of as cisplatin and 5-FU (49-51). The present study was designed these treatment regimens (P = 0.9803; Fig. 3A). to determine whether palifermin had any effect on the antitumor In animals treated with palifermin alone, the FaDu tumor activities of these agents when administered in various growth was similar to that seen in animals treated with vehicle combinations. The direct effects of palifermin on tumor growth (P = 0.9905), indicating that palifermin did not promote tumor and gastrointestinal epithelia were also evaluated. growth in this KGFR-expressing carcinoma model (Fig. 3A). In addition, the effect of palifermin on esophageal epithelial Results growth was evaluated histologically in each series of experi- KGFR and EGFR Expression in Human Carcinoma Cell ments with both xenograft models to confirm the biological Lines activity of palifermin under these experimental conditions. In In the process of identifying the best candidates for the all experimental series, the esophageal epithelium was xenograft models, we first selected cell lines that were deemed substantially thicker in mice treated with palifermin compared most relevant to ongoing and proposed clinical investigations with those treated with vehicle alone. A representative with palifermin in patients being treated for solid tumors histologic panel from FaDu tumor-bearing animals treated with (colorectal and head and neck cancers). From among the vehicle or palifermin is shown in Fig. 4. The data are not shown appropriate candidates, cell lines were selected that expressed for the other experiments due to space limitations. both the KGFR and EGFR. KGFR transcripts were expressed in 15 of 22 human carcinoma cell lines tested and EGFR transcripts were present in 19 of these same cell lines. The Effects of Palifermin in Combination with Cisplatin and the FaDu (hypopharyngeal carcinoma) and HT29 (colon carcino- Anti-EGFR Antibody Cetuximab on the Growth of FaDu ma) cell lines expressed both KGFR and EGFR transcripts Human Head and Neck Carcinoma Xenografts (Fig. 1A) and were selected for further evaluation in the tumor The FaDu xenograft model was also used to study the effect xenograft models. These tumor lines showed a good response to of palifermin on the antitumor activity of the anti-EGFR

FIGURE 1. A. Expression of KGFR and EGFR transcripts in vitro. B. In vivo expression of KGFR in FaDu and HT29 xenograft models. C. In vivo expression of EGFR in FaDu and HT29 xenograft models. Expres- sion in vivo was evaluated only for the FaDu and HT29 tumor xenografts. Three representative animals from each xenograft are shown and the expression level for the cell line grown in vitro is also shown for comparison purposes. The expression of EGFR and KGFR transcripts was analyzed by quantitative reverse transcription- PCR (see Materials and Methods) and normalized relative to the housekeep- ing gene b-actin. Transcript expression levels are shown as a fold change relative to the expression by A427 cells used as a reference.

FIGURE 2. Functional activation of KGFR and EGFR in FaDu (A) and HT29 cells (B) in the presence of ligand as shown by phosphorylation of Akt and extracellular signal-regulated kinase (Erk). The phosphorylation of Akt and extracellular signal-regulated kinase was analyzed using phospho-specific antibodies and the Meso-Scale Discovery technology and expressed relative to total protein for that analyte (see Materials and Methods).

antibody cetuximab. Carcinoma cells were implanted 9 to palifermin to either 5-FU or bevacizumab treatment had no 10 days before treatment began and dosed as shown in Fig. 3B. effect on the antitumor activity of these agents (P = 0.9991). As expected, there was significantly less tumor growth in Similarly, the addition of palifermin to the combination of animals treated with the combination of cisplatin and cetuximab 5-FU and bevacizumab also had no effect on antitumor activity than in animals treated with vehicle (P < 0.0001) and the (P = 0.8861; Fig. 5A). addition of palifermin had no effect on the antitumor efficacy of In animals treated with palifermin alone, HT29 tumor this combination (P = 0.7730; Fig. 3B). Similar findings were growth was similar to that seen in animals treated with vehicle observed in the FaDu xenograft model when another anti- (P > 0.9999), indicating that palifermin did not promote tumor EGFR antibody (panitumumab) was combined with palifermin growth in this model (Fig. 5A). and cisplatin (data not shown). In these experiments, the tumor growth in animals treated Effects of Palifermin in Combination with 5-FU and Anti- with palifermin alone was similar to that seen in animals treated EGFR Therapeutic Antibodies on the Growth of HT29 with vehicle (P = 0.9993; Fig. 3B), showing no effect of Human Colon Carcinoma Xenografts palifermin on tumor growth in vivo. In these experiments, HT29 carcinoma cells were implanted 9 to 11 days before the start of treatment and the dosing Effects of Palifermin in Combination with 5-FU and regimens followed the same pattern as described above (Fig. 5B Bevacizumab on the Growth of HT29 Human Colon and C). When used alone, both 5-FU and cetuximab Carcinoma Xenografts significantly decreased tumor growth compared with vehicle- Xenografts of HT29 carcinoma cells were implanted treated animals (P < 0.0001 and P = 0.0318, respectively) and subcutaneously in nude mice and allowed to grow for 14days the antitumor efficacy of these agents was unaffected by the before treatments were started. Palifermin was given for 3 days addition of palifermin. There was no statistically significant before and after 5 days of dosing with 5-FU. Bevacizumab difference in tumor growth in animals treated with 5-FU or was dosed twice a week for the duration of the experiment cetuximab alone compared with either of these agents (Fig. 5A). In the animals treated with 5-FU and/or bevacizu- combined with palifermin (P = 0.9985; Fig. 5B). The mab, tumor growth was significantly less than that seen in combination of 5-FU and cetuximab led to a significant vehicle-treated animals (P < 0.0001; Fig. 5A). The addition of reduction in tumor size (P < 0.0001) and the efficacy of

this combination was unaffected by the addition of palifermin mucositis in cancer patients can interfere with speech, (P = 0.9883). Animals treated with panitumumab in combina- the intake of food and medication, and overall quality of life tion with 5-FU also had significantly less tumor growth than did (4, 57-59). It creates an avenue for infection (2), lengthens vehicle-treated animals (P < 0.0001; Fig. 5C). As was observed hospital stays, increases the need for parenteral narcotics and for cetuximab, the efficacy of the combination of panitumumab nutrition (2), and can interfere with anticancer treatment and 5-FU was unaffected by the addition of palifermin (P = 0.9851). regimens (11, 56). A meta-analysis from 2004found that In both of these xenograft series, the tumor growth in among patients treated with standard-dose chemotherapy animals treated with palifermin alone was similar to that seen in regimens who developed grade 3 to 4mucositis, 70% required animals treated with vehicle (P = 0.9995 and 0.9545, feeding tubes to maintain adequate nutrition, f60% had fever, respectively; Fig. 5B and C). and 62% required hospitalization (11). This same analysis found that 35% of patients with grade 3 to 4mucositis experienced delays in their next cycle of chemotherapy, 60% Discussion had to have their dose reduced, and 30% had to have their There is a strong unmet need for more effective treatments treatment discontinued altogether (11). In 2006, a study of for chemotherapy-induced mucositis in patients with a wide patients with head and neck carcinoma found that unplanned range of cancer types (21, 56), including head and neck (18, 19) interruptions or delays in chemotherapy occurred in 25% and colorectal cancer (16). The pain and discomfort of of patients with severe mucositis; 47% of these patients

FIGURE 3. The effect of palifermin on the antitumor activity of cisplatin and biological agents in mice bearing FaDu human pharyngeal carcinoma xenografts. A. FaDu model: effect of palifermin on cisplatin and bevacizumab. B. FaDu model: effect of palifermin on cisplatin and cetuximab. Tumor cells were implanted subcutaneously and allowed to grow for 9 to 11 d before treatments were started. The treatment protocols are indicated on the figure.

FaDu human hypopharyngeal or HT29 human colon xenografts from the antitumor effects of either cytotoxic (5-FU or cisplatin), anti–vascular endothelial growth factor (bevacizumab), or anti-EGFR (cetuximab or panitumumab) therapies or from various combinations of these drugs used together. Palifermin also did not promote tumor growth in either model when it was used alone despite the fact that both of these carcinoma xenograft models expressed functional KGFRs. Moreover, despite the lack of an effect on tumor cells, palifermin did produce dramatic increases in esophageal thickness, showing that it was biologically active in these animal models. Despite these effects on normal epithelial cells, there was no evidence of enhanced tumor growth. This suggests that any potential effects that palifermin might have had on the tumor stroma did not result in any increased tumor cell growth. If palifermin had favored tumor growth by an indirect effect on the tumor microenvironment, these effects would have likely been observed in our experiments in the control groups that were treated with palifermin alone and/or any other treatment FIGURE 4. Effect of palifermin on esophageal thickness. Tissue sections from two vehicle-treated animals and two palifermin-treated groups that included the combination with palifermin. That was animals are shown. Magnification, Â20. never observed, suggesting that under the current experimental conditions, palifermin did not stimulate tumor cell growth either directly or indirectly through the stroma. experienced interruptions or delays in radiation therapy (56). In The results of the present study are supported by earlier patients with head and neck cancer, unplanned interruptions in in vitro and in vivo animal studies that showed that several therapy can reduce local control rates, decrease 5-year relapse- tumor cells that express functional KGFRs exhibit little or no free survival rates, and generally increase the risk of treatment response to KGF (30). For example, in an HT29 xenograft failure (60). Consequently, the successful prevention of model similar to the one used in the present study, KGF had no mucositis in patients with solid tumors may not only improve effect on tumor growth and did not protect the xenograft from patient comfort and reduce the risk of complications but may the cytotoxic effects of 5-FU (25). Moreover, in a small also allow physicians to maintain aggressive anticancer preliminary dose-escalation study of the effects of palifermin on treatment protocols, which, in turn, may improve the overall patients treated with the combination of 5-FU and leucovorin patient prognosis. for metastatic colorectal cancer, palifermin seemed to have no In clinical studies done to date on patients with hematologic effect on the time to disease progression, overall patient malignancies, palifermin has been shown to be safe to signi- survival time, or progression-free patient survival time (27). ficantly reduce the incidence and duration of mucositis (53), to In conclusion, the results of the present preclinical study improve patient comfort and functioning, to decrease the length showed that palifermin did not promote the growth of human of hospital stays, and to reduce the need for chemotherapy dose colon or hypopharyngeal xenografts and did not interfere with reductions secondary to chemotoxicity (27, 54, 55). Conse- the antitumor efficacy of several different types and combina- quently, there is a strong desire to investigate the use of tions of antitumor agents currently used to treat head and neck or palifermin in patients receiving cancer therapy for nonhemato- colorectal cancers. The lack of an effect of palifermin on the logic malignancies such as colorectal and head and neck cancer. efficacy of two different cytotoxic agents, two different anti- These investigations must proceed with care, however, because EGFR antibodies, and one anti–vascular endothelial growth certain solid tumors express KGFRs (30), leading to a factor antibody suggests that palifermin may have no drug theoretical concern that a KGF-based therapy, such as interactions with other members of these drug classes. Nothing palifermin, may promote tumorigenesis or protect tumors from about the clinical safety or efficacy of palifermin in patients with the effects of anticancer agents. Preclinical studies can only head and neck or colorectal cancers can be inferred from the provide limited guidance into the use of palifermin in solid results of this animal study, but the results do suggest that further tumor settings and they cannot unequivocally predict outcomes clinical investigations will be worthwhile and such studies are in cancer patients who will have to be carefully monitored for currently under way. At this time, however, palifermin should any safety signals. only be used in these patient populations during controlled The present study was designed to determine whether clinical trials in which safety variables are carefully monitored combining palifermin with different chemotherapeutic (5-FU both during and after the trial to ensure that palifermin did not and cisplatin) or biological agents (bevacizumab, cetuximab, have any negative effects on tumor outcomes. and panitumumab) currently used to treat head and neck or colorectal cancer had any effect on the antitumor activity of Materials and Methods these agents in human head and neck (FaDu) and colorectal Assessing Expression of KGFR and EGFR Genes (HT29) carcinoma xenograft models. The most important Twenty-two human carcinoma cell lines and 1 human finding from this study was that palifermin did not protect glioblastoma cell line were obtained from the American Type

FIGURE 5. The effect of palifermin on the antitumor activity of 5-FU and various biological agents in mice bearing HT29 human colon carcinoma xenografts. A. HT29 model: effect of palifermin on 5-FU and bevacizumab. B. HT29 model: effect of palifermin on 5-FU and cetuximab. C. HT29 model: effect of palifermin on 5-FU and panitumumab. Tumor cells were implanted subcutaneously and allowed to grow for 9 to 14 d before treatments were started. The treatment protocols are indicated on the figure.

Culture Collection (ATCC). Cells were grown in standard 10% cells per mouse in a 0.2 mL volume over the left flank. Nine to serum containing medium as outlined by the ATCC. Gene 14d thereafter, when tumors had reached between 200 and expression of KGFR and EGFR was assessed by quantitative 400 mm3 in volume, animals were randomized into groups reverse transcription-PCR (QRT-PCR) using the Taqman (n = 10 per group) and treatment was begun as illustrated in platform (Applied Biosystems). RNA was extracted using the Figs. 3 and 5. Tumor volumes as established by caliper RNeasy kit (Qiagen) and was then converted to cDNA using measurements (length Â width Â height) were recorded twice the Taqman cDNA kit (Applied Biosystems) using random per week, along with body weights as an index of toxicity. All prime oligonucleotides. experiments were conducted in a blinded fashion in which the The expression of KGFR and EGFR transcripts was assessed individuals making the tumor measurements were unaware of the using QRT-PCR along with a control PCR for b-actin gene treatment that the animals had received. expression. Primers and probes were designed using the Primer Express software (Applied Biosystems). Transcript expression Histology levels were expressed relative to the A427 human lung For all tumor xenograft studies, four mice from the vehicle- carcinoma cell line. The SD for each test gene was expressed treated group and four mice from the palifermin-treated group ÀDCt as a ratio relative to b-actin using the formula 2 . To ensure were sacrificed 4d after the initiation of treatment. The that receptor expression was maintained during in vivo growth, esophagus was excised, fixed for 24to 36 h in Z-FIX (3.7% QRT-PCR analyses on representative tumor samples excised formaldehyde, ionized zinc, in buffer; Anatech), and embedded from tumor xenograft–bearing animals were processed and into paraffin blocks using a Tissue Tek VIP paraffin tissue analyzed in a similar way. processor (Sakura USA). Sections (4 Am thick) were cut and mounted on Superfrost Plus slides (Fisher Scientific). All In vitro Signal Transduction sections were deparaffinized, rehydrated, and stained with H&E The FaDu human hypopharyngeal carcinoma and HT29 (Lerner Laboratories). Sections were viewed with a Nikon FXA human colon carcinoma cell lines were also analyzed for their upright compound microscope using a 20Â PlanApo Nikon ability to initiate signal transduction when stimulated by KGF lens. Images were recorded with a Nikon DXM1200 digital 6 or EGF in vitro. In this analysis, 1 Â 10 cells were serum camera under standard bright-field Kohler illumination. starved for 48 h before treatment with either KGF or EGF at 100 ng/mL, a pharmacologic relevant dose of KGF (palifermin) Reagents in humans (61, 62). Palifermin, panitumumab, human IgG2, and human IgG1 At various times between 5 and 90 min, the medium was were provided by Amgen. Bevacizumab and cisplatin were removed and cells were washed, scrapped, centrifuged, and lysed obtained from Burt’s Pharmacy. Cetuximab was obtained from in NP40 lysis buffer. The protein lysate was then assayed for Vertrieb (distribution: Merck). phosphoprotein signal using the Meso-Scale Discovery kits to PBS was used as the dilution buffer for all study drugs. assess pS473 on the Akt protein or the pT202, pY204, pT185, Palifermin (5 mg/kg) was administered subcutaneously in a Erk and pY187 sites on the kinase 1/2 protein. These kits dosing volume of 0.25 mL; antibodies and 5-FU were simultaneously detect both the phosphoprotein as well as the total administered i.p. at the indicated dose in a 0.2 mL volume. analyte. The data presented in Fig. 2 express phosphoprotein For treatment with cisplatin, mice were weighed and dosed relative to the total loading control. according to body weight; therefore, the exact volume of administration (10 AL/g) was variable but f0.2 mL. To control Xenograft Models for the effects of injections alone, all animals in each individual nu/nu Female CD1 mice (Charles River Laboratories) were study received the same number of injections according to the housed in sterilized caging, five animals per cage with the cages same dosing schedule. If the animals in a particular treatment changed twice weekly. Harlan Teklad Sterilizable Rodent Diet group were not designated to receive a particular study drug, 8656 and reverse-osmosis water from the Amgen water supply they were treated with vehicle or IgG1 instead according to the ad libitum system were supplied . Room temperature was same dosing schedule; human IgG1 was used as the control for j j maintained between 68 F and 72 F, and relative humidity was bevacizumab and cetuximab; human IgG2 was used as the maintained between 34% and 73%. The laboratory housing the control for panitumumab. cages provided a 12-h light cycle and met all International Association for the Assessment and Accreditation of Labora- Data Analysis and Statistical Methods tory Animal Care specifications. The protocols were done at the Data were expressed as means F SE and plotted as a function Amgen, Thousand Oaks site, which is accredited by the of time. The statistical significance of observed differences International Association for the Assessment and Accreditation between tumor growth curves was evaluated by repeated of Laboratory Animal Care. measures ANOVA followed by Scheffe post hoc testing for The FaDu and HT29 cell lines were originally obtained from multiple comparisons. All statistical calculations were made the ATCC. The cells were maintained and expanded under through the use of StatView software v5.0 (SAS Institute, Inc.). routine tissue culture conditions at 37jC in DMEM high glucose/ 5% fetal bovine serum, 1Â nonessential amino acids, and 2 mmol/L L-glutamine (Life Technologies). Cells were generally Disclosure of Potential Conflicts of Interest All the authors are employees of Amgen, Inc., with the exception of Suijin >90% viable at the point of transplant. Mice between 4and 8 wk Yang who was employed by Amgen, Inc. at the time of the study, but who is now 7 of age were challenged by subcutaneous injection of f1 Â 10 affillated with Pfizer, Inc. No other potential conflicts of interest were disclosed.

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