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. Author manuscript; available in PMC: 2009 May 4.
Published in final edited form as: Clin Cancer Res. 2008 Apr 1;14(7):2180–2189. doi: 10.1158/1078-0432.CCR-07-1392

Treatment of Human Colon Cancer Xenografts with TRA-8 Anti-death Receptor 5 Antibody Alone or in Combination with CPT-11

Patsy G Oliver 1, Albert F LoBuglio 2,4, Kurt R Zinn 3, Hyunki Kim 3, Li Nan 1, Tong Zhou 2,4, Wenquan Wang 2, Donald J Buchsbaum 1,4
PMCID: PMC2676875  NIHMSID: NIHMS110297  PMID: 18381960

Abstract

Purpose

This study was designed to evaluate the in vitro cytotoxicity and in vivo efficacy of TRA-8, a mouse monoclonal antibody that binds to the DR5 death receptor for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L), alone and in combination with CPT-11 against human colon cancer cells and xenografts.

Experimental Design

DR5 expression was assessed on human colon cancer cell lines using flow cytometry, and cellular cytotoxicity following TRA-8 treatment, alone and in combination with SN-38 was determined by measuring cellular ATP levels. Tumor growth inhibition and regression rates of well-established s.c. COLO 205, SW948, HCT116 and HT-29 colon cancer xenografts in athymic nude mice treated with TRA-8 or CPT-11 alone and in combination were determined. 99mTc-TRA-8 was utilized to examine tumor localization of TRA-8 in animals bearing each of the 4 xenografts. In addition, whole body biodistribution and imaging was carried out in COLO 205 bearing animals using in vivo SPECT imaging and tissue counting.

Results

DR5 expression was highest on HCT116, intermediate on SW948 and COLO 205 cells, and lowest on HT-29 cells. COLO 205 cells were the most sensitive to TRA-8-induced cytotoxicity in vitro, SW948 and HCT116 cell lines were moderately sensitive, and HT-29 cells were resistant. Combination treatment with TRA-8 and SN-38 produced additive to synergistic cytotoxicity against all cell lines compared to either single agent. The levels of apoptosis in all cell lines, including HT-29, were increased by combination treatment with SN-38. In vivo, combination therapy with TRA-8 and CPT-11 was superior to either single agent regimen for three of the xenografts; COLO 205, SW948, and HCT116. COLO 205 tumors were most responsive to therapy with 73% complete regressions following combination therapy. HT-29 cells derived no anti-tumor efficacy from TRA-8 therapy. Tumor xenografts established from the 4 colon cancer cell lines had comparable specific localization of 99mTc-TRA-8.

Conclusions

In vitro and in vivo effects of TRA-8 anti-DR5 monoclonal antibody on four different colon cancer cell lines and xenografts were quite variable. The HT-29 cell line had low surface DR5 expression and was resistant to TRA-8 both in vitro and in vivo. Three cell lines (COLO 205, SW948, and HCT116) exhibited moderate to high sensitivity to TRA-8 mediated cytotoxicity which was further enhanced by the addition of SN-38, the active metabolite of CPT-11. In vivo, the combination of TRA-8 and CPT-11 treatment produced the highest anti-tumor efficacy against xenografts established from the three TRA-8 sensitive tumor cell lines. All 4 colon cancer xenografts had comparable localization of 99mTc-TRA-8. These studies support the strategy of TRA-8/CPT-11 combined treatment in human colon cancer clinical trials.

INTRODUCTION

The discovery of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL, also known as Apo-2L), and the finding that TRAIL induces apoptosis in human cancer cells to a greater extent than in normal cells (1-6), generated considerable interest in the potential therapeutic use of this cytokine. Five receptors for TRAIL have been identified: DR4 (TRAIL-R1) and DR5 (TRAIL-R2), which contain cytoplasmic death domains capable of transmitting apoptosis-inducing signals, and decoy receptors DcR1 (TRAIL-R3), DcR2 (TRAIL-R4), and osteoprotegerin that are incapable of inducing apoptosis (4, 5). Early concerns about potential hepatotoxicity of TRAIL (1, 7) and its pharmacokinetics led to the development of agonistic antibodies specific for DR4 or DR5 which are capable of inducing apoptosis in human cancer cells (8-12).

Previously, we reported on the antitumor efficacy of an agonistic murine anti-DR5 mAb (TRA-8) in a xenograft model of human breast cancer (10). TRA-8 bound to DR5 on the surface of most human breast cancer cell lines, but there was considerable variation in the sensitivity of these cell lines to TRA-8-induced apoptosis in vitro. In vivo studies using s.c. xenografts of 2LMP cells, an aggressive subclone of the MDA-MB-231 breast cancer cell line, demonstrated significant enhancement of TRA-8 antitumor efficacy using combination chemotherapy with paclitaxel or adriamycin with or without concurrent radiotherapy (10).

The purpose of the present study was to evaluate the antitumor efficacy of TRA-8 using in vitro cytotoxicity assays and xenograft models of human colon cancer. We and others have demonstrated that DR5 is expressed in tumors of the colorectum (13-15). The cytotoxicity of TRA-8 alone or in combination with SN-38, the active metabolite of CPT-11, against human colon cancer cell lines of varying sensitivity to TRA-8 was investigated. Binding, cytotoxicity and mechanism studies were used to examine the relationship between in vitro sensitivity to TRA-8 and CPT-11, alterations in apoptotic signaling pathways, and the ability to predict in vivo efficacy of TRA-8 and CPT-11 against xenograft models of colon cancer. We hypothesized that combination treatment with CPT-11 may increase TRA-8 signaling by engaging the intrinsic apoptotic pathway though caspase 8-mediated Bid activation and down-regulation of anti-apoptotic proteins of the Bcl-2 and IAP families. In vivo studies using s.c. colon cancer tumor models in athymic nude mice demonstrated patterns of anti-tumor efficacy of TRA-8, CPT-11, and the combination which were unique for each cell line. This work provides a rationale for the investigation of TRA-8 and chemotherapy in patients with colon cancer.

MATERIALS AND METHODS

Cell lines and reagents

All cell lines were obtained from the American Type Culture Collection (Manassas, VA) and grown in RPMI 1640 medium supplemented with 4.5 g/l glucose, 10 mM HEPES, 1 mM sodium pyruvate and 10% FBS (COLO 205 and HT-29), DMEM with 10% FBS (SW948), or McCoy’s medium with 10% FBS (HCT116). All cell lines were maintained in antibiotic-free medium at 37°C in a 5% CO2 atmosphere and routinely screened for Mycoplasma contamination.

Purified TRA-8 (IgG1) mAb used for in vitro studies was produced and purified as previously described (9) while Sankyo Co., Ltd. (Tokyo, Japan) provided the preparations used for in vivo studies. Isotype-specific IgG1 control antibody and phycoerythrin-conjugated goat anti-mouse IgG1 were obtained from Southern Biotechnology Associates (Birmingham, AL). CPT-11 (irinotecan hydrochloride, Camptosar; Pharmacia and Upjohn, Kalamazoo, MI), oxaliplatin (Eloxatin, Sanofi Aventis, Bridgewater, NJ), topotecan (Hycamtin, SmithKline Beecham Pharmaceuticals, Philadelphia, PA) and docetaxel (Taxotere, Aventis Pharmaceuticals Inc, Bridgewater, NJ) were obtained from the University of Alabama at Birmingham Hospital Pharmacy (Birmingham, AL) and diluted in 0.9% sterile saline (in vivo studies) immediately before use. SN-38 was obtained from Toronto Chemical Co. (Toronto, Canada). Cell Stripper was from Mediatech (Herndon, VA). Collagenase type 11 and protease inhibitor cocktail were from Sigma Chemical Co. (St. Louis, MO). Lowry DC protein assay reagents and HRP-conjugated goat anti-mouse IgG and anti-rabbit IgG were from Bio-Rad (Hercules, CA). Antibodies for Western blot analysis were obtained from the following vendors: caspase 3, caspase 8, and PARP (BD Pharmingen, San Jose, CA); Bax (Southern Biotechnology Associates); caspase 9, Bid, Bcl-xl, survivin and Akt (Cell Signaling Technologies, Beverly, MA); FLIP and p53 (Calbiochem, San Diego, CA); XIAP (Stressgen, Ann Arbor, MI); actin (Sigma Chemical Co.). ECL enhanced chemiluminescence reagents were from GE Healthcare (Piscataway, NJ).

Indirect immunofluorescence and flow cytometry analysis of DR5 expression

DR5 expression on colon cancer cells was analyzed as described previously (16) using FACScan and Cell Quest software (Becton Dickinson, San Jose, CA). To examine the effect of SN-38 on DR5 cell surface expression, colon cancer cell lines were treated with SN-38 for 24 h at concentrations selected from their SN-38 dose response curve then analyzed for DR5 expression as described above.

Cell viability assays using ATPLite

Cell cultures were trypsinized, replated in complete culture medium and incubated overnight at 37°C before addition of drugs and/or antibody. For combination treatments, cells were pretreated with chemotherapy drugs for 24 h before adding TRA-8 antibody for an additional 24 h. Other studies examined the efficacy of 24 h concurrent treatment with TRA-8 and SN-38. Cell viability was assessed after 24 h exposure to TRA-8 by measurement of cellular ATP levels using the ATPLite luminescenc assay (Perkin Elmer Biosciences, Meriden, CT). All samples were assayed in quadruplicate and are reported as the mean ± SD from a minimum of 2 independent experiments.

We screened a number of chemotherapy agents in terms of their ability to enhance TRA-8 mediated cytotoxicity to colon cancer cell lines. In general, 5-FU and gemcitabine produced inferior enhancement as compared to CPT-11. Topotecan, docetaxel, oxaliplatin and Adriamycin produced similar enhancement as CPT-11 on colon cancer cell lines including the TRA-8 resistant cell line HT-29 (Supplementary Figure 2). However, their combination with TRA-8 in vivo produced little or no enhancement above the antitumor effects of chemotherapy drug alone (examples in Supplementary Figure 3). Since CPT-11 is commonly used for treatment in colorectal cancer and performed in a fashion superior to or comparable to other agents, we used this agent to characterize chemotherapy — TRA-8 combination therapy in the studies described below.

Statistical analysis of in vitro cytotoxicity of TRA-8 and chemotherapy drugs

A non-linear model: y=Min+(Max-Min)/(1+dose/β)α (17, 18), was applied to calculate IC50, where y is the response, the parameter β represents IC50, the parameter α is used to scale concentration for proper transformation, and Min and Max represent the minimum and the maximum of response. A SAS procedure NLIN was used for the computation (SAS Institute Inc., Cary, NC). The cytotoxicity data were evaluated to assess whether the combination cytotoxic effects were additive, less than additive (antagonistic), or greater than additive (synergistic). The dose response relationships for the agents alone and in combination were modeled using a second-order response surface model with linear, quadratic and interaction terms for each of the 4 cell lines (19). A significant interaction term was classed as either synergistic or antagonistic depending on whether the interaction term was negative with more than additive cytotoxicity, or positive with less than additive cytotoxicity. If the interaction term was not significant, then the relationship between TRA-8 and CPT-11 would be considered additive, provided the additive terms were significant.

Western blot analysis of colon cancer cells treated with TRA-8 and SN-38

Cell lines were trypsinized, replated, and incubated overnight at 37°C before starting treatments. Cells were treated with SN-38 (0.1 or 1 μM) for 21 h at 37°C, then TRA-8 was added. After 2.5 h (COLO 205, SW948 and HCT116) or 4 h (HT-29) treatment with TRA-8, whole cell lysates were prepared in RIPA buffer (150 mM NaCl, 50 mM Tris, pH 7.4, 1% sodium deoxycholate, 1% Triton X-100, 0.1% SDS, 1% protease inhibitor cocktail, 10 mM sodium orthovanadate). Proteins (15-25 μg) were separated by SDS-PAGE, transferred to PVDF membranes (Millipore, Bedford, MA), and probed with primary antibodies followed by HRP-conjugated secondary antibodies. Actin levels were compared to control for protein loading.

Radiolabeling and binding of TRA-8

A fresh 1.8 mM solution of succinimidyl 6-hydrazinonicotinate (HYNIC, courtesy Dr. Gary Bridger, AnorMED, Inc., Langley, British Columbia) in dimethylformamide was prepared. Forty picomoles was transferred to glass vials, followed by freezing at -90°C, then the solutions were vacuum dried using an Advantage Benchtop Freeze Dryer (Virtis Co Inc., Gardiner, NY) with the shelf temperature at -75°C and trap at -90°C. The vials were sealed under vacuum, and kept frozen at -80°C until use. Each vial was reconstituted with 1 mg of TRA-8 (HYNIC:TRA-8 molar ratio = 6) in 1 ml sodium phosphate buffer (0.15 M, pH 7.8) (20). After a 3 h incubation at room temperature, the mixture was dialyzed (10,000 MW cut-off) against PBS (pH 7.4) overnight at 4°C. The HYNIC-modified TRA-8 was labeled with 99mTc using SnCl2/tricine as the transfer ligand (21), and unbound 99mTc was removed by G-25 Sephadex size-exclusion chromatography. Protein concentrations were measured by Lowry assay. The level of 99mTc binding to TRA-8 was always greater than 96%, as measured by thin-layer chromatography using separate strips eluted with saturated saline and methyl ethyl ketone. The isotype control antibody (IgG1k, eBioscience, Inc., San Diego, CA) was iodinated with 125I using the Iodogen method. The average specific activity of 99mTc-TRA-8 and 125I-isotype control antibody were 348.6 and 34.2 kBq/μg respectively.

TRA-8, CPT-11, or combination therapy in athymic nude mice bearing colon cancer xenografts

Athymic nude mice were injected s.c. with 1 or 2 × 107 COLO 205, SW948, HCT116 or HT-29 colon cancer cells. Tumors were measured in the two largest dimensions and the tumor surface area (length × width in mm2) was calculated. Treatment was initiated when tumors reached ∼6-8 mm in diameter (8-14 days after tumor cell injection for COLO 205, 14-19 days with SW948, 10 days with HCT116, and 14 days for HT-29), and the growth of tumors was monitored over time. A dose response study of single agent TRA-8 in COLO 205 tumor bearing animals showed similar regression rates with 60 or 200 μg doses given 2 times/week × 3 weeks. We chose the 200 μg dose schedule since the other cell lines were less sensitive to TRA-8 in vitro. In subsequent studies, animals bearing s.c. colon tumors were injected i.v. with 200 μg TRA-8 beginning on treatment day one followed by five additional injections given twice weekly for 3 weeks. For animals receiving CPT-11 therapy (33 mg/kg), either alone or in combination with TRA-8, the drug was administered by i.v. injection on treatment day 2 followed by 5 additional injections given every 3-4 days for 3 weeks. Animals receiving combination treatment with TRA-8 and CPT-11 were injected with the drug 24 h after each TRA-8 administration. We used a twice weekly schedule to assure a continuous circulating level of TRA-8 (plasma T1/2 of approximately 5 days) which would limit any pretreatment effects of CPT-11. Tumors were measured three times weekly with Vernier calipers and tumor growth and regression rates were determined. We next investigated whether a second cycle of treatment with TRA-8 + CPT-11 (33 mg/kg, twice weekly × 3 weeks for each cycle) initiated 10 days after the first cycle of treatment would enhance the anti-tumor effect against SW948 tumor xenografts. Two cycles of TRA-8 + CPT-11 were more effective (3/8 complete regressions) than a single cycle of TRA-8 + CPT-11 (0/8 complete regressions) as shown in Supplementary Figure 4). Animal studies were approved by the Institutional Animal Care and Use Committee.

Animal imaging experiments

Two imaging experiments were conducted with athymic nude mice (Frederick Cancer Research Facility, Bethesda, MD) implanted subcutaneously in the right thigh with 1 × 106 COLO 205 cells. In vivo tumor imaging and biodistribution studies were performed approximately 5 weeks later. Mice were injected i.v. with 99mTc-TRA-8 (Experiment 1) or a mixture of 99mTc-TRA-8 and 125I-labeled isotype control antibody (Experiment 2). The average weight of the tumors was 0.7 g for both experiments. The mean dose of 99mTc-TRA-8 used in Experiments 1 and 2 was 9.25 MBq (15 μg) and 11.10 MBq (29 μg) respectively, while that of 125I-isotype control antibody was 0.67 MBq (16 μg). Doses of radioactivity were confirmed by measuring the syringes before and after dosing with a dose calibrator (AtomLab™ 100, Biodex Medical Systems, Inc., Shirley, NY).

Images of mice bearing COLO 205 xenografts were acquired using X-SPECT, a SPECT/CT dual-modality imaging instrument (Gamma Medica, Inc., Northridge, CA) to monitor detailed 99mTc-TRA-8 distribution in the tumor in vivo. For SPECT imaging, the focal length was 41 mm with 0.7 mm spacing between slices. A total of 64 projections were acquired with a 40 second acquisition time per projection at 20 h after dose injection, using a pinhole collimator with a 1-mm pinhole insert. The mice were induced and maintained with isoflurane gas anesthesia, and monitored continuously to allow the lowest dose (typically 1-1.5%) to prevent movement. The energy window to collect photons from 99mTc was 126-161 keV, and therefore excluded any contribution from 125I.

Biodistribution of 99mTc-TRA-8 in tumor-bearing athymic nude mice

Ten groups of mice were used; groups 1-4 bore COLO 205 tumors, groups 5 and 6 bore SW948 tumors, groups 7 and 8 bore HCT116 tumors, and groups 9 and 10 bore HT-29 tumors, subcutaneously. Groups 1 (n=8), 3 (n=5), 5 (n=5), 7 (n=5), and 9 (n=5) were injected with 99mTc-TRA-8, while groups 2 (n=3), 4 (n=5), 6 (n=5), 8 (n=5), and 10 (n=5) were injected with 125I-labeled isotype control antibody, intravenously. All mice were sacrificed at 24 hours post dosing, and the tumor, blood and organs from each mouse of groups 1 and 2 were collected and weighed, while only tumor and blood samples were collected for the other groups. The radioactivity was measured using a gamma-ray counter (MINAXIγ Auto-gamma® 5000 series Gamma Counter, Packard Instrument Company, Grove, IL). The % of injected dose per gram for each tissue was averaged for all mice in each group.

Statistical analysis

Fisher’s exact test was used to compare the regression rate between groups. For tumor growth analyses, linear regression was performed on each animal where the percentage of tumor size compared to the original tumor size was the dependent variable. Based on the linear regression, a tumor doubling time was calculated for each animal. For animals that did not have tumor doubled at the end of the study, the study period was assigned to them as the tumor doubling time. Wilcoxon rank sum test based on normal approximation was used to compare the tumor doubling time between groups. All tests had a significance level at 0.01 based on Bonferroni’s adjustment. One-way analyses of variance (ANOVA) (22) was carried out using SAS®, version 8.2 (SAS Institute Inc., Cary, NC) to compare biodistribution of 99mTc-TRA-8 and 125I-isotype control antibody.

RESULTS

DR5 expression and in vitro cytotoxicity of TRA-8 alone and in combination with chemotherapy

DR5 expression as detected by TRA-8 binding on four human colon cancer cell lines is depicted in Figure 1A. DR5 was detected on all cell lines at expression levels that ranged from weakly positive (HT-29 cells) to strongly positive (HCT116 cells). TRA-8-mediated cytotoxicity against the four colon cancer cell lines produced varying degrees of cell killing, as shown in Figure 1B. COLO 205 cells were very sensitive to TRA-8, with an IC50 value of 2.4 ng/ml, whereas SW948 and HCT116 cells were moderately sensitive with IC50 values of 9.2 and 27.2 ng/ml. In contrast, HT-29 cells were relatively resistant to TRA-8 with 58% viable cells remaining at 1,000 ng/ml TRA-8. For these cell lines, TRA-8 resistance correlated with DR5 expression (HT-29) while the cell line with greatest DR5 expression was only moderately sensitive to TRA-8 cytotoxicity (HCT116).

Figure 1.

Figure 1

Figure 1

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DR5 expression and TRA-8 cytotoxicity against colon cancer cell lines. (A) Flow cytometry analysis of DR5 cell surface expression in human colon cancer cell lines. Colon cancer cells were harvested and stained with 5 μg/ml TRA-8 for 1 h at 4°C followed by PE-conjugated goat anti-mouse IgG1, then analyzed using FACScan and CellQuest software. Thick histograms, TRA-8 staining; thin histograms, cells stained with mouse IgG1 isotype control antibody. (B) Cytotoxicity of TRA-8 alone and in combination with SN-38 treatment of human colon cancer cell lines. Cells (1,000/well) were treated at 37°C for 24 h with TRA-8 at concentrations ranging from 1 to 1,000 ng/ml. Other cells were pretreated with SN-38 for 24 h followed by 24 h concurrent treatment with TRA-8 and SN-38 or were treated with SN-38 alone for 48 h. Cell viability was assessed by measuring ATP levels. Values represent the mean ± SD derived from 3-4 independent experiments each done in quadruplicate and are reported relative to untreated control cells. (C) Effect of SN-38 on DR5 expression on colon cancer cells. Cells were treated for 24 h with 0 to 10 μM SN-38, then cells were harvested and DR5 levels were analyzed using TRA-8 binding and flow cytometry. Thick histograms, TRA-8 staining; thin histograms, cells stained with mouse IgG1 isotype control antibody.

The cytotoxic effects of a combination of SN-38 (active metabolite of CPT-11) and TRA-8 are also presented in Figure 1B. Pretreatment of TRA-8 resistant HT-29 cells with SN-38 followed by TRA-8 addition produced synergistic cell killing compared to either single agent (P = 0.0012). Synergistic killing of SW948 cells was also observed with combination treatment (P = 0.0362) using 1 μM SN-38, a concentration that had no effect on cell viability. Combination treatment resulted in additive cytotoxicity against HCT116 cells (P = 0.481). SN-38 enhanced TRA-8 mediated cytotoxicity to COLO 205 cells but analysis for synergism was hindered by their extreme sensitivity to TRA-8. Concurrent treatment of COLO 205 and HT-29 cells with SN-38 and TRA-8 for 24 h produced a stronger synergistic effect (P <0.0001) as shown in Supplementary Figure 1, but higher doses of SN-38 were required to achieve synergistic killing compared to the drug pretreatment schedule. The IC50 of cell lines for single agent SN-38 was 0.02 μM (HCT116), 0.5 μM (HT-29), 1 μM (COLO 205), and >10 μM (SW948) as shown in Figure 1B. It was interesting that the SN-38 resistant cell line (SW948) underwent significant synergistic enhancement of SN-38 mediated cytotoxicity by the addition of small amounts of TRA-8 (5 ng/ml).

Combination treatment with TRA-8 and SN-38 produced enhanced cytotoxicity against other TRA-8 resistant colon cancer cell lines, including SW1116, LoVo and T84 cells (data not shown). The effect of combination treatment with TRA-8 and other chemotherapeutic drugs, including topotecan, docetaxel, oxaliplatin and adriamycin, was also examined, as shown in Supplementary Figure 2. All four drugs produced additive or greater than additive cytotoxicity against TRA-8 resistant HT-29 cells.

The effect of SN-38 on DR5 cell surface expression was investigated by flow cytometry. Increased DR5 levels were detected on HCT116 and COLO 205 cells after 24 h treatment with 0.01 μM and 10 μM SN-38, respectively, as shown in Figure 1C. DR5 expression was unchanged in SW948 and HT-29 cells after SN-38 treatment. SN-38 treatment for 48 h did not further upregulate DR5 expression in any of the cell lines (data not shown).

Western blot analysis of colon cancer cell lines treated with TRA-8 and SN-38

The 4 colon cancer cell lines were used to examine alterations in apoptosis-related proteins produced by in vitro treatment with TRA-8 alone, SN-38 alone, or the combination. As seen in Fig. 2A, all 3 cell lines that were sensitive to TRA-8 mediated cytotoxicity had TRA-8 induced cleavage of caspases 3, 8, and 9, PARP and Bid with minimal or no effects in HT-29 cells. These changes were not seen with SN-38 alone treatment. The combination treatment had a more pronounced but similar pattern as TRA-8 alone for TRA-8 sensitive cell lines. Combination treatment induced dose dependent cleavage of caspases 3, 8, and 9, PARP, and Bid in the TRA-8 resistant cell line (HT-29) at 0.1 and 1 μM SN-38.

Figure 2.

Figure 2

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Western blot protein analysis of colon cancer cell lines treated in vitro with TRA-8 alone and in combination with SN-38. COLO 205, SW948 and HCT116 cells were treated with 0.1 μM SN-38 for 21 h followed by treatment with TRA-8 plus SN-38 for an additional 2.5 h or were exposed to TRA-8 alone for 2.5 h. HT-29 cells were treated with 0.1 or 0.1 μM SN-38 for 21 h followed by treatment with TRA-8 plus SN-38 for an additional 4 h or were exposed to TRA-8 alone for 4 h. Whole cell lysates were then prepared, and equivalent amounts of protein were analyzed by Western blot using antibodies specific for caspase 3, 8 or 9, PARP, Bid, Bcl-xl, Bax, p53, XIAP, survivin, FLIP and Akt. Actin levels were compared to control for protein loading.

As seen in Fig. 2B, the cell line most sensitive to SN-38 (HCT116) had a dramatic SN-38 mediated induction of p53 (wild-type) as well as the p53 regulated protein Bax. SN-38 induced a modest increase in p53 (mutated) and Bax in COLO 205 while the p53 and Bax levels were unaffected in SW948 (p53 absent) or HT-29 cells (high p53). As regards inhibitors of apoptosis, survivin levels increased in the presence of SN-38 and decreased with the addition of TRA-8 (COLO 205, SW948, and HCT116). HT-29 cells had a dramatic increase in survivin due to the presence of SN-38 which was not affected by the addition of TRA-8. XIAP levels were reduced by TRA-8 alone or in combination for all 3 TRA-8 sensitive cell lines but only reduced by TRA-8/SN-38 combination in HT-29 cells. Bcl-xl levels were high and unchanged by drug exposure except for HT-29, where high levels were modestly reduced by TRA-8, moderately reduced by SN-38, and dramatically reduced by the combination.

In vivo antitumor activity of TRA-8, CPT-11, or the combination against colon cancer xenografts

We examined the in vivo anti-tumor efficacy of TRA-8, CPT-11, or the combination in animals bearing COLO 205, SW948, HCT116, or HT-29 xenografts. Table 1 provides our aggregate experience in terms of tumor doubling time and complete tumor regressions with each treatment regimen. Single agent TRA-8 or CPT-11 produced moderate and significant inhibition of COLO 205 tumor growth (P < 0.001) while the combination produced dramatic inhibition of tumor growth compared to no treatment (P < 0.001), TRA-8 alone (P = 0.0002), or CPT-11 alone (P = 0.009). Combination therapy produced complete regression of COLO 205 tumors in 73% of animals, and half of these animals had no tumor recurrence over 93-270 days of observation. Single agent TRA-8 or CPT-11 produced 17% and 20% complete regressions, respectively, with about half of these animals having no recurrence. A typical experiment with COLO 205 is depicted in Figure 3A. The animals were administered multiple doses of CPT-11 and TRA-8, which circulates for several days, so that both agents are present throughout the treatment cycle and the potential benefit of treating with CPT-11 before TRA-8 is expected to be limited.

Table 1.

Effect of TRA-8, CPT-11, and the Combination on Colon Xenograft Tumor Doubling Time and Complete Regressions

Treatment # of Animals Mean Tumor Doubling
Time		 Complete Regressions
Total (%) No relapse (%)
COLO 205
No treatment 39 13 ± 14 0 (0%) 0 (0%)
TRA-8 42 78 ± 73 7 (17%) 3 (7%)
CPT-11 19 64 ± 16 4 (20%) 3 (15%)
TRA-8+CPT-11 33 138 ± 79 24 (73%) 12 (36%)
SW948
No treatment 25 13 ± 26 0 (0%) 0 (0%)
TRA-8 24 10 ± 5 0 (0%) 0 (0%)
CPT-11 24 37 ± 33 1 (4%) 1 (4%)
TRA-8+CPT-11 25 67 ± 39 9 (36%) 3 (12%)
HCT116
No treatment 7 21 ± 1 0 (0%) 0 (0%)
TRA-8 7 37 ± 24 1 (14%) 1 (14%)
CPT-11 7 56 ± 4 0 (0%) 0 (0%)
TRA-8+CPT-11 7 78 ± 10 0 (0%) 0 (0%)
HT-29
No treatment 8 11 ± 3 0 (0%) 0 (0%)
TRA-8 8 12 ± 2 0 (0%) 0 (0%)
CPT-11 8 26 ± 5 0 (0%) 0 (0%)
TRA-8+CPT-11 8 26 ± 5 0 (0%) 0 (0%)
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Figure 3.

Figure 3

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The effect of TRA-8, CPT-11, and the combination on tumor growth in nude mice bearing well-established human colon cancer xenografts. Colon cancer cell lines were injected s.c. into female athymic or BALB/c nude mice on day 0 using (A) COLO 205; (B) SW948, (C) HCT116, or (D) HT-29 cells. Treatments began when tumors reached ∼6-8 mm in diameter. Schedule of TRA-8 was 200 μg twice/week × 3 weeks and CPT-11 (33 mg/kg) was given i.v. twice/week × 3 weeks. When treatment was combined, the CPT-11 was given one day after the TRA-8. One group of mice received no treatment. Data are expressed as the average change in tumor size (surface area equal to the product of two diameters) relative to tumor size at the start of treatment.

The SW948 pattern of anti-tumor efficacy was quite different than COLO 205 in that TRA-8 alone had no beneficial effect while single agent CPT-11 significantly inhibited tumor growth (tumor doubling time = 37 ± 33 days) as compared to control (P < 0.001) or TRA-8 treated animals (P < 0.001). Although TRA-8 alone had no benefit, its combination with CPT-11 produced further inhibition of tumor growth (67 ± 39 days) as compared to CPT-11 alone (P = 0.0016). The benefit of combination therapy was further supported by an incidence of 36% complete regressions as compared to 4% for CPT-11 alone and none in TRA-8 treated or control animals. A typical experiment is presented in Figure 3B.

HCT116 tumor bearing animals treated with TRA-8 alone had a modest inhibition of tumor growth (doubling time of 37 ± 24 days; P = 0.002). Treatment with CPT-11 alone produced a moderate inhibition of tumor growth (56 ± 4 days; P = 0.002 compared to untreated). Combination therapy produced a further inhibition of tumor growth (78 ± 10 days) as compared to TRA-8 alone (P = 0.002) or CPT-11 alone (P = 0.02). There was only one complete regression which occurred in the TRA-8 alone treatment group. The experiment is depicted in Figure 3C.

Finally, HT-29 tumor cells, which were very resistant to TRA-8 induced cytotoxicity in vitro, formed tumors that were not inhibited by TRA-8 therapy alone (doubling time of 12 ± 2 days compared to untreated, 11 ± 3 days) but there was modest efficacy of CPT-11 alone (26 ± 5 days; P = 0.0009 compared to untreated). Further, TRA-8 in combination with CPT-11 had no increment in efficacy compared to CPT-11 alone (P = 0.87). The experiment is depicted in Figure 3D. In vivo therapy studies were also conducted to examine the efficacy of TRA-8 in combination with topotecan or docetaxel against HT-29 xenografts, as shown in Supplementary Figure 3. Little tumor growth inhibition occurred in mice treated with either drug alone, and combination therapy with TRA-8 and drug produced no benefit in this model compared to drug alone.

Excessive toxicity was not observed using the present TRA-8 and CPT-11 dosing schedule, and mean weight loss was less than 15% in mice treated with CPT-11 alone or in combination with TRA-8. However, this treatment regimen approaches the MTD for CPT-11, since 12.6% of mice exhibited greater than 15% weight loss during treatment, and 2 deaths occurred out of 167 mice (1.2%) treated in preliminary studies and those reported here. Some animals were also subjected to a second treatment cycle with TRA-8 and CPT-11, which produced an increase in the time to tumor doubling in the SW948 xenograft model (Supplementary Fig. 4) with no enhancement of toxicity compared to one cycle of treatment.

In summary, xenografts of three colon cancer cell lines which had high or intermediate sensitivity to TRA-8 mediated cytotoxicity in vitro achieved optimal antitumor efficacy with a combination of TRA-8 and CPT-11, while the single cell line that was TRA-8 resistant derived no benefit from TRA-8 alone or when added to CPT-11, topotecan or docetaxel. It is possible that the levels of TRA-8 binding to HT-29 cells may not reach the threshold required to induce apoptosis in vivo.

Tumor localization, biodistribution, and imaging of 99mTc-TRA-8 in tumor xenograft models

The uptake of 99mTc-TRA-8 in tumor and blood was investigated in the 4 colon cancer xenograft models at 24 h after injection and compared to the uptake of 125I-labeled isotype control antibody, as shown in Figure 4A (groups 3-10). Similar levels of 99mTc-TRA-8 in tumor were found in the 4 tumor models after injection (9.5 to 10.9% ID/g), which were greater than the uptake of control antibody (3.0 to 4.2% ID/g). The blood levels ranged from 13.5 to 16.1% ID/g and 10.9 to 12.9% ID/g for 99mTc- TRA-8 and 125I-IgG1 control antibody, respectively. Figure 4B presents the mean and standard error of radiolabeled TRA-8 uptake (%ID/g) in each tissue of nude mice bearing human COLO 205 s.c. tumor xenografts 24 hours after injection of 99mTc-TRA-8 or 125I-isotype control antibody (groups 1 and 2). The 99mTc TRA-8 localization in tumor was significantly higher than that seen with 125I-isotype control (P < 0.05). The lower level of 125I-isotype control in liver and spleen presumably reflects the reticuloendothelial system dehalogenation of the control antibody. Figure 4C shows a series of SPECT images (transaxial view, 0.7 mm between slices) of a mouse bearing a human COLO 205 tumor xenograft subcutaneously on its right thigh. The image acquisition was started at 20 h after dosing and shows higher levels of 99mTc-labeled TRA-8 retention in tumor (approximate center is white dotted circle) compared with background. These representative images of the tumor in cross-section (slices 26-37) demonstrate the pattern of specific localization of 99mTc-TRA-8 in the tumor.

Figure 4.

Figure 4

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Distribution of 99mTc-TRA-8 in athymic nude mice bearing human colon tumor xenografts. (A) Tumor and blood levels of 99mTc-TRA-8 and 125I-IgG1 isotype control antibody were quantitated 24 h after injection in mice bearing COLO 205, SW948, HCT116, or HT-29 tumor xenografts. Values are mean and SD in tumor and blood from 3 mice per tumor model. Black bars are 99mTc-TRA-8 and gray bars are 125I-IgG1 isotype control antibody. (B) Biodistribution of 99mTc-TRA-8 and 125I-isotype control antibody in tissues of athymic nude mice bearing COLO 205 human colon tumor xenografts. Data are presented as means (±SE): n=8 for 99mTc-TRA-8 and n=3 for 125I-isotype control antibody. (C) SPECT images (transaxial view) of 99mTc-TRA-8 distribution in COLO 205 tumor bearing mice were obtained 20 h after i.v. injection with radiolabeled TRA-8. The focal length for SPECT images was 41 mm with 0.7 mm spacing between slices. White dotted circle indicates the tumor region.

DISCUSSION

We and others have examined the sensitivity of various human tumor cell lines to TRAIL and death receptor agonistic antibodies as single agents and in combination with chemotherapy agents (4, 5, 10). In this study, we compared the sensitivity of 4 different colon cancer cell lines to an agonistic anti-DR5 monoclonal antibody (TRA-8), either alone or in combination with SN-38 (active metabolite of irinotecan), both in vitro and in vivo. We also examined the response of TRA-8 resistant HT-29 cells and xenografts to TRA-8 in combination with additional chemotherapeutic drugs. These observations illustrate both similarities as well as substantial differences in the biologic responses of these cell lines after exposure to TRA-8.

Using flow cytometry analysis, all 4 cell lines expressed DR5 on their tumor cell membranes, although the weakest expression was observed on the HT-29 cells (Fig. 1A). All 4 cell lines exhibited TRA-8 mediated cytotoxicity but their sensitivity or resistance to cytotoxicity was widely disparate with COLO 205 having a high sensitivity (IC50 = 2.4 ng/ml) and HT-29 relatively resistant (IC50 ≥ 1,000 ng/ml). SW948 and HCT116 had intermediate sensitivity with IC50 values of 9.2 and 27.2 ng/ml respectively (Fig. 1B). The 3 cell lines which were sensitive to TRA-8 mediated cytotoxicity were found to exhibit tumor cell cleavage of caspases 3, 8, and 9, PARP and Bid at cytotoxicity inducing dose levels, reflecting their apoptotic mechanism of cell death (Fig. 2A). Similar evidence for apoptosis has been presented for a variety of cell lines treated with TRAIL or death receptor antibodies (9, 11, 12, 23, 24). HT-29 cells had a minimal level of caspase cleavage even with exposure to 1,000 ng/ml of TRA-8.

Sensitivity of the individual cell lines to single agent SN-38 cytotoxicity varied greatly and was different than their sensitivity to TRA-8 (Fig. 1B). The IC50 values were 0.02 μM (HCT116), 0.5 μM (HT-29), 1 μM (COLO 205), and > 10 μM (SW948). When cells were exposed to the combination of TRA-8 and SN-38, all 4 cell lines had enhanced cytotoxicity as compared to either agent alone (Fig. 1B). Interestingly, the TRA-8 resistant cell line (HT-29) exhibited synergistic cytotoxicity with the addition of low doses of SN-38, while the SN-38 resistant cell line (SW948) also had synergistic cytotoxicity when as little as 5 ng/ml of TRA-8 was added. Thus, combination of TRA-8 and SN-38 achieved optimal in vitro cytotoxicity with all 4 colon cancer cell lines. This was reflected in the enhanced cleavage of caspases, PARP, and Bid elicited by combination therapy (Fig. 2A). The chemotherapy enhancement of death receptor mediated apoptosis has been reported with a variety of cell lines and drugs (3, 10, 11, 23-30). As also illustrated in Fig. 2A, SN-38 as a single agent did not trigger apoptosis as evidenced by the absence of cleaved caspases, PARP or Bid.

We attempted to explore potential mechanisms playing a role in the enhanced in vitro effects of combination therapy. Other studies have suggested that chemotherapy agents can enhance death receptor-mediated cytotoxicity by enhancing expression of tumor DR5 expression (3, 23, 26, 27). Our analysis of SN-38 effects on tumor cell DR5 expression at drug levels used in the cytotoxicity assays were unimpressive (Fig. 1C). HT-29 cells, which were TRA-8 resistant and had synergistic cytotoxicity with addition of SN-38, had no change in their minimal level of DR5 expression with 24 or 48 h of drug exposure. Similarly, SW948, which exhibited synergistic cytotoxicity with combination therapy, had no significant change in DR5 expression. The two cell lines that demonstrated either a modest increase (COLO 205) or moderate increase (HCT116) in DR5 expression (Fig. 1C) did not have synergistic effects from combination therapy.

A number of prior studies have proposed that the enhancement of cytotoxicity observed with combinations of chemotherapy and death receptor signaling is due to chemotherapy modulation of molecules that either enhance or inhibit the apoptotic pathway (4, 5). We surveyed a number of such regulatory molecules in each of the 4 colon cancer cell lines at baseline and following TRA-8 and SN-38 exposure (Fig. 2B). The results of this survey were quite variable among the 4 cell lines. In all 4 cell lines, the combination treatment appeared to reduce levels of XIAP from baseline. This is interesting since a recent report described the ability of a small molecule inhibitor of XIAP to dramatically enhance cytotoxicity by TRAIL (31). All 4 cell lines had induction of survivin on exposure to single agent SN-38, especially HT-29 cells. This would presumably inhibit apoptosis and yet SN-38 produced synergistic cytotoxicity when combined with TRA-8 in HT-29 cells. Bcl-xl was prominent baseline in all 4 cell lines and levels were decreased in HT-29 by the combination of TRA-8 and SN-38. These studies provided no consistent mechanism for SN-38 enhancement of TRA-8 mediated cytotoxicity. Each cell line appears to have its own baseline and treatment-related pattern of response of these factors.

We then examined the effects of TRA-8 and CPT-11 on xenografts of each of these 4 colon cancer cell lines, as depicted in Table 1 and Fig. 3. Each of the cell lines had its own pattern of TRA-8 and CPT-11 anti-tumor efficacy. The TRA-8 sensitive COLO 205 tumors had substantial efficacy from single agent TRA-8 or CPT-11 (P < 0.001), including 17% and 20% complete regressions. The combination of TRA-8 and CPT-11 had impressive efficacy greater than single agent TRA-8 (P = 0.0002) or CPT-11 (P = 0.009), with 73% complete regressions, and 36% of animals who had no relapse. This high degree of efficacy in COLO 205 tumors may reflect their in vitro sensitivity to SN-38, modest enhancement of DR5 expression, and moderate inhibition of XIAP and survivin by combination therapy.

In contrast, the TRA-8 resistant HT-29 tumors derived no benefit from treatment with TRA-8. Animals treated with single agent CPT-11 had a modest prolongation of tumor doubling time (26 ± 5 vs. 11 ± 3 days) which was significant (P = 0.0009). Combination therapy was no better than single agent CPT-11, suggesting that the synergistic effects of the combination in vitro did not translate to the in vivo setting. This finding was also true for HT-29 xenografts treated with TRA-8 and topotecan or docetaxel, despite the enhanced in vitro cytotoxicity produced by combination treatments with these drugs (Supplementary Fig. 2 and Fig. 4). The low cell membrane DR5 expression may have been a contributing factor to the poor effects of TRA-8 in this model.

The results of therapy in SW948 xenografts were unexpected. This cell line had an appreciable sensitivity to TRA-8 in vitro (IC50 of 9.2 ng/ml) and was resistant to SN-38 mediated cytotoxicity (IC50 > 10 μM). However, SW948 tumors were unresponsive to TRA-8 treatment and CPT-11 had moderate efficacy (P < 0.001). Despite lack of efficacy by TRA-8, the combination of TRA-8 and CPT-11 was superior to CPT-11 alone (P = 0.0016), including 36% complete regressions. These observations reflect the discrepancies often noted in correlating in vitro with in vivo therapeutic strategies.

In vitro, HCT116 cells had moderate sensitivity to TRA-8 (IC50 = 27.2 ng/ml) and high sensitivity to SN-38 (IC50 = 0.02 μM). In vivo, HCT116 tumors had a moderate response to TRA-8 with a doubling time of 37 ± 24 days (P = 0.002 compared to 21 ± 1 day with no therapy) and an even better response to single agent CPT-11 (doubling time of 56 ± 4 days with P = 0.002 vs. TRA-8 alone). Combination therapy effects (doubling time of 78 ± 10 days) was more effective than single agent TRA-8 (P = 0.002) or CPT-11 (P = 0.02).

These in vivo observations correlated reasonably well with the in vitro studies. We also examined TRA-8 localization to xenograft tumors of each of these cell lines as an additional variable in the treatment strategy. Radiolabeled TRA-8 was found to have specific and comparable radiolocalization to each of the cell line tumors, including HT-29, despite its modest tumor cell membrane expression of DR5. These studies illustrate the considerable in vitro and in vivo variation observed in tumor cell line responsiveness to an anti-DR5 agonistic monoclonal antibody (TRA-8) alone, CPT-11 alone, or the combination. However, it is clear that combination therapy with TRA-8 and CPT-11 was superior to either single agent alone in 3 out of the 4 tumor cell lines. These 3 cell lines shared a high or moderate sensitivity to TRA-8 in vitro, moderate to high cell surface expression of DR5, and inhibition of XIAP and survivin following in vitro exposure to the combination of TRA-8 and CPT-11. The poorly responsive HT-29 cell line was resistant to TRA-8 in vitro, had minimal surface membrane expression of DR5, and no inhibition of survivin with in vitro exposure to the combination of TRA-8 and CPT-11. These studies further emphasize the need to utilize the combination of chemotherapy with death receptor targeting in future clinical trials.

Supplementary Material

Figures
Legends

ACKNOWLEDGEMENTS

The authors acknowledge the expert technical assistance of Charlotte Hammond and the assistance of Sally Lagan in the preparation of this manuscript. This work was supported by NIH grant 5 P30 CA013148 and Daiichi Sankyo.

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