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Cancer Therapy: Preclinical |
Authors' Affiliations: 1 Ludwig Institute for Cancer Research, Melbourne Branch, Tumor Targeting Program, Austin Hospital, Heidelberg; 2 Ludwig Institute for Cancer Research, San Diego Branch, University of California, San Diego, La Jolla, California; 3 Epithelial Biochemistry Laboratory, Royal Melbourne Hospital, Parkville, Victoria, Australia; and 4 Ludwig Institute for Cancer Research, New York Branch, New York, New York
Requests for reprints: Terrance G. Johns, Oncogenic Signalling Laboratory, Ludwig Institute for Cancer Research, Austin Hospital, Studley Road, Heidelberg 3084, Victoria, Australia. Phone: 61-3-9496-3068; Fax: 61-3-9496-5334; E-mail: Terry.Johns{at}ludwig.edu.au.
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Abstract |
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Inhibition of the EGFR is a rational strategy for the development of new cancer therapeutics (11). Potential therapeutics include anti-EGFR antibodies (12) and small molecular weight tyrosine kinase inhibitors of the EGFR (13). A number of antibodies directed to the extracellular domain of the EGFR have now been tested in the clinic including EMD 55900 (14), ABX-EGF (15), and IMC-C225 (16), all of which have displayed some antitumor activity in patients (12). The most clinically advanced of these is IMC-C225, which is currently being tested in phase II/III clinical trials for the treatment of head and neck, colorectal, and non–small cell lung carcinomas (17) and was recently approved for use in Europe. It has been presumed that the antitumor activity of these antibodies is primarily related to their ability to block ligand binding, but further antitumor mechanisms such as immune effector function, receptor down-regulation, induction of inappropriate signaling, and interference with receptor dimerization and/or oligomerization need to be considered in more detail. One limitation of antibodies targeting the wt EGFR is that they show significant uptake in normal tissue such as the liver and skin (18–20). Whereas targeting of normal tissues seems to cause manageable side effects such as skin rash (20), it does mean that these antibodies would generate significant side effects if coupled to cytotoxic agents or radioisotopes for antitumor therapy.
Monoclonal antibody (mAb) 806 is a novel anti-EGFR specific antibody that was generated from mice immunized with de2-7 EGFR-transfected NR6 fibroblasts (21, 22). Interestingly, whereas mAb 806 clearly binds the de2-7 EGFR, it also binds to a subset of the wt EGFR (
10%) expressed on the surface of A431 cells (a vulval carcinoma cell line that overexpresses the EGFR; ref. 22). Recent analysis shows that mAb 806 recognizes a transitional form of the EGFR before it becomes activated (23, 24). Binding of mAb 806 to this transitional form of the EGFR prevents receptor activation and subsequent signaling. Unlike the wt EGFR, the de2-7 EGFR is constitutively in this transitional conformation and thus available for mAb 806 binding. Our previous studies have shown that treatment of glioma xenografts that express the de2-7 EGFR with mAb 806 causes significant inhibition of tumor growth (25). Significantly, established A431 tumor xenografts were also inhibited by mAb 806 despite the antibody only recognizing a small proportion of the EGFR expressed in these cells (25).
Given the unique properties of mAb 806, we decided to investigate its efficacy in combination with mAb 528, a prototypical anti-EGFR antibody. The 528 antibody was produced and isolated at the same time as the murine version of the C225 antibody and displays very similar properties (26). mAb 528 acts as a ligand antagonist and inhibits the growth of EGFR expressing cells both in vitro and in vivo when grown as xenografts (27). In this report, we examine the efficacy of mAbs 806 and 528 against xenografts expressing the de2-7 EGFR or overexpressing the wt EGFR in both s.c. and i.c. models.
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Materials and Methods |
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2-7 and the U87MG.DK cell line expressing a kinase inactive variant of the de2-7 EGFR, have been described previously (7, 28). The A431 cell line has also been described previously (22). The mAbs 806 and 528 have been described previously and were produced in the Biological Production Facility (Ludwig Institute for Cancer Research, Melbourne, Australia). The anti-EGFR antibody, EGFR1, was purchased from DAKO Corp. (Carpinteria, CA) and the monoclonal rat anti-mouse CD34 from Abcam (Cambridge, United Kingdom). Fluorescence-activated cell sorting analysis of receptor expression. The level of de2-7 EGFR and wt EGFR expression in cultured cells was analyzed using mAb 806 (IgG2b), mAb 528 (IgG2a), EGFR1 (IgG1) antibodies. Cells were incubated with primary antibody or a negative control antibody (IgG2b) and detected using a secondary anti-mouse FITC-conjugated antibody (Calbiochem, San Diego, CA). Analysis was done using an Epics Elite ESP (Beckman Coulter, Hialeah, FL) by observing a minimum of 20,000 events and data analyzed using EXPO (version 2) for Windows.
Subcutaneous xenograft model. Tumor cells (3 x 106) in 100 µL of PBS were inoculated s.c. into both flanks of 4- to 6-week-old, female nude mice (Animal Research Centre, Perth, Australia). All studies were conducted using established tumor models as previously reported (25). Treatment commenced once tumors had reached a mean volume indicated in the appropriate figure legend. Tumor volume in mm3 was determined using the formula (length x width2) / 2, where length was the longest axis and width being the measurement at right angles to the length. Data are expressed as mean tumor volume ± SE for each treatment group. In some experiments, survival was evaluated using the technique of Kaplan-Meier with comparisons between groups analyzed by log-rank test. This research project was approved by the Animal Ethics Committee of the Austin Hospital.
Intracranial tumor model. U87MG.2-7 (1 x 105) or U87MG.DK cells (5 x 105) in 5 µL of PBS were implanted into the right corpus striatum of nude mice brains as described previously (29). Mice were then treated systemically with normal saline, 0.5 mg of mAb 806, 0.5 mg of mAb 528 as single agents or both antibodies in combination, every other day from post-implantation days 0 to 14.
In vitro receptor down-regulation assay. Parallel flasks of U87MG.2-7 cells were treated with 100 µg/mL of mAbs 806 and 528 either alone or in combination for 30 minutes at 37°C in the presence of 10 µg/mL of fibronectin (Sigma Chemical Co., St. Louis, MO), following serum starvation for 36 hours. Cells were washed in ice-cold PBS before lysis in cold lysis buffer [30 mmol/L HEPES, 150 mmol/L NaCl, 10 mmol/L NaF, 1% Triton X-100, 200 µmol/L NaO3V, 0.4% H2O2 and the protease inhibitor cocktail set 1 (Calbiochem) containing 500 µmol/L AEBSF, 150 nmol/L aprotinin, 1 µmol/L E-64 protease inhibitor, 0.5 mmol/L EDTA, and 1 µmol/L leupeptin (pH 7.4)]. Equal volumes of each sample were mixed with SDS sample buffer and loaded onto 4% to 12% NuPage gels (Invitrogen Life Technologies, Mulgrave, Australia). Each lysis experiment and immunoblot was repeated at least thrice.
Preparation of xenograft homogenates. Xenograft homogenates of tumors excised from three different mice per treatment group were prepared by homogenization in immunoprecipitation assay buffer [50 mmol/L Tris (pH 7.5), 150 mmol/L NaCl, 5 mmol/L EDTA, 200 mmol/L NaO3V, 0.4% H2O2, 0.5% deoxycholate, 0.005% SDS, 10 mmol/L NaF, and the protease inhibitor cocktail set 1]. Homogenization was conducted in a Dounce Homogeniser before incubation for 1 hour at 4°C. Preparations were subsequently clarified by centrifugation at 14,000 x g for 30 minutes at 4°C. The protein content of the resultant supernatants was determined using the detergent-compatible protein assay (Bio-Rad Laboratories, Hercules, CA) according to manufacturer's instructions. Samples were equalized for total protein, mixed in an equal volume of SDS sample buffer and loaded onto 4% to 12% NuPage Bis-Tris gels.
Immunoblotting. After transfer to polyvinylidene difluoride membranes, separated proteins were detected by immunoblotting with anti-EGFR (Cell Signaling Technology, Beverly, MA), anti-phospho-EGFR-1173 (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-p27KIP1 (Santa Cruz Biotechnology) antibodies. To ensure equal loading, immunoblots were probed with anti-
-tubulin antibody (Santa Cruz Biotechnology) and separate gels stained with Coomassie.
Ki-67 immunostaining. Xenografts were removed, embedded in Tissue Tek ornithine carbamyl transferase compound (Sakura Finetek, Torrance, CA), frozen in liquid nitrogen, and stored at –80°C. Ten-micrometer sections were cut, fixed in acetone, and stained with sheep anti-human Ki-67 polyclonal antibody (Chemicon International, Temecula, CA) for 1 hour. After rinsing, a rabbit anti-sheep-horseradish peroxidase antibody (Chemicon International) was applied and bound antibody detected with AEC substrate solution (0.1 mol/l acetic acid, 0.1 mol/L sodium acetate, 0.02 mol/L AEC, and 0.03% H2O2). Slides were then counterstained in hematoxylin (BDH Laboratory, Poole, United Kingdom) and mounted. The percentage of reactive tumor nuclei to the Ki-67 antibody was estimated by examining an average of 17 representative high-power fields per treatment group containing a total of 8,000 cells. Data were expressed as proliferation index (PI) using the formula: (cells positive for Ki-67 staining / total cells) x 100.
Immunohistochemical analysis of microvessel density. Xenografts were removed and fixed in 10% buffered formalin and used for microvessel density detection. Sections of 4-µm thickness were cut, mounted onto Superfrost Plus slides (Menzel-Glaser, Germany), and deparaffinized in a 60°C oven for 30 minutes followed by 2 x 5 minutes xylene rinses and 2 x 5 minutes 100% ethanol rinses. Sections were then rehydrated in distilled water and microwaved for 15 minutes in EDTA buffer (pH 8.0; Lab Vision Co., Fremont, CA) for antigen retrieval. Endogenous peroxidase was quenched before blocking with 1 x Protein Blocking Solution (Immunon Thermo Shandon, Pittsburgh, PA). Sections were stained with rat anti-mouse CD34 (Abcam) for 1 hour followed by goat anti-rat IgG-Biotin-labeled secondary antibody. Sections were then incubated with horseradish peroxidase–conjugated Streptavidin (Chemicon Australia, Melbourne, Australia). Slides were then incubated with AEC, counterstained with hematoxylin (BDH Laboratory), and mounted. As a measurement of angiogenesis, the microvessel density (i.e., the number of positive vessels in a given area) was determined. Quantification of microvessel density involved counting the number of CD34-positive vessels in four to six random 390-µm2 fields at x200 magnification for each tumor (n = 3) per treatment group.
Statistical analysis. All data was analyzed for significance by Student's t test, except for the in vivo survival data, which was analyzed by log-rank Mantel-Cox test. Data from the microvessel density detection was analyzed for difference between treatment groups by a one-way ANOVA, where P < 0.05 was considered statistically significant.
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Results |
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2-7 (which expresses the de2-7 EGFR) and U87MG.DK (which express a dead kinase form of the de2-7 EGFR) cells express similar levels of the various EGFR proteins. The EGFR1 antibody only recognizes the extracellular domain of the wt EGFR and does not bind to the de2-7 EGFR or the kinase dead form of de2-7 EGFR (7). EGFR1 bound both U87MG.2-7 and U87MG.DK cells with similar intensity, confirming that these cell lines express comparable amounts of endogenous wt EGFR (Fig. 1). The mAb 528, which recognizes all of the EGFR subtypes, also stained both the U87MG.2-7 and U87MG.DK cell lines to a comparable degree, showing that they express similar levels of total EGFR (Fig. 1). Finally, mAb 806, which in these cells only recognizes the de2-7 EGFR or its dead kinase equivalent (29), bound to each cell line with similar intensity indicating that both express comparable levels of either the de2-7 EGFR or dead kinase receptor (Fig. 1). Thus, the level of EGFR expression is similar in U87MG.2-7 and U87MG.DK cell lines and therefore should not influence subsequent therapy studies.
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2-7 and A431). Using a competitive ELISA, we have previously shown that mAbs 806 and 528 do not compete and can bind the EGFR simultaneously (30). However, we have previously shown by fluorescence-activated cell sorting (22) and immunoprecipitation (31) that whereas mAb 806 binds a high proportion of de2-7 EGFR (50%), it only binds a small proportion of the wt EGFR only in cells overexpressing the receptor. Consistent with these previous observations, mAb 806 bound A431 cells, which overexpress the wt EGFR, at a lower level than mAb 528 (Fig. 1B). Likewise, mAb 528 bound more receptors on U87MG.2-7 cells compared with mAb 806 (Fig. 1C). Again, this was expected as mAb 528 not only binds the de2-7 EGFR expressed in theses cells but also the coexpressed wt EGFR, whereas mAb 806 can only bind the de2-7 EGFR in these cells.
Treatment of established glioma xenografts with monoclonal antibodies 806 and 528. We examined whether two mAbs with differing specificity for the EGFR displayed increased antitumor activity against glioma tumor xenografts when used together. Treatment of established U87MG.2-7 glioma xenografts with mAbs 806 and 528 in combination displayed significantly higher antitumor activity than either antibody alone (Fig. 2A). Whereas treatment with mAb 806 or 528 as single agents at day 20 after inoculation resulted in a reduction in overall tumor growth rate compared with treatment with vehicle (P < 0.005 and P < 0.003, respectively), combined treatment with both antibodies resulted in at least additive tumor inhibition that was significantly higher than mAb 528 (P < 0.001) and mAb 806 (P < 0.001) alone. The average tumor volume on the final day of therapy (day 20 after inoculation) was 1,850, 880, 800, and 30 mm3 for the vehicle, mAb 806, mAb 528, and combination treatment groups, respectively. Furthermore, 30% of U87MG.2-7 xenografts underwent complete regression in the combination therapy group and had not recurred at day 30 after inoculation. No complete regressions were seen in any of the other treatment groups (Fig. 2A).
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Previous reports have shown that the efficacy of EGFR-specific antibodies is reduced when used to treat large xenografts (32). Therefore, the efficacy of mAbs 806 and 528 combination therapy was examined against large established U87MG.2-7 xenografts with a mean volume of 550 mm3 (Fig. 2C). Only xenografts treated with mAbs 806 and 528 in combination displayed a significant reduction in xenograft growth when compared with tumors treated with vehicle (P < 0.01; Fig. 2C). No significant difference in tumor growth rate was observed following treatment with mAb 806 (P = 0.40) or mAb 528 (P = 0.30). Indeed, the average tumor volume on day 26 after inoculation was 2,140, 2,080, 1,990, and 1,210 mm3 for the vehicle, mAb 806, mAb 528, and combination treatment groups, respectively (Fig. 2C). These results suggest that the combination of mAbs 806 and 528 results in greater than additive antitumor activity.
Mice bearing established U87MG.DK xenografts were also treated with mAbs 806 and 528 as single agents or in combination to ascertain whether the antibodies mediated the antitumor activity through immune effector function or signaling effects. On day 32 after inoculation, the growth rate of tumors treated with mAbs 806 and 528 as single agents or in combination did not differ from that of vehicle-treated xenografts. The average tumor volume was 1,170, 1,110, 1,260, and 1,220 mm3 for vehicle, mAb 806, mAb 528, and combination therapy groups, respectively (Fig. 2D). Because both antibodies bind U87MG.2-7 and U87MG.DK cells equivalently (Fig. 1), this result indicates that both mAbs 806 and 528 initiate their antitumor activity independent of immune effector function in this model.
Survival analysis for mice treated with antibodies. We also analyzed the data from the combination antibody therapy studies (Fig. 2A and C) with respect to survival. When used as single agents (three 0.5 mg injections per week for 2 weeks) to treat small established U87MG.2-7 xenografts (i.e., mice shown in Fig. 2A), both mAbs 806 and 528 had a small benefit on survival (P < 0.004 and 0.01, respectively) with median survival being days 23 and 22, respectively, compared with day 20 for the control group (Fig. 2E). All mice receiving a combination antibody treatment survived to day 31 (P < 0.0001 versus control, P < 0.0003 versus mAb 528 alone, and P < 0.001 versus mAb 806 alone; Fig. 2E), at which point the experiment was terminated for ethical reasons. Thus, combination therapy significantly improved survival in all mice with small established tumors compared with either antibody alone.
We also analyzed survival in the cohort of mice that were treated with antibody once their U87MG.2-7 xenografts had reached an average of 500 mm3 (i.e., mice shown in Fig. 2C). Consistent with the growth curves (Fig. 2C), neither mAb 806 nor mAb 528 used alone had any effect on survival (Fig. 2F) with median survival being day 22 for all three groups. In contrast at day 26, when the experiment with large xenografts had to be terminated for ethical reasons, 90% of mice had survived in the combination group. Therefore, combination antibody treatment greatly enhanced survival even in mice with large established xenografts providing further evidence of the synergistic nature of these two antibodies.
Mechanism of synergistic antitumor activity. To determine a mechanism for the synergistic antitumor activity of mAbs 806 and 528 in combination, we examined whether either antibody alone or in combination down-regulated the expression of de2-7 EGFR on the cell surface. U87MG.2-7 cells were incubated at 37°C with antibodies for 30 minutes, lysed, and analyzed for de2-7 EGFR levels by immunoblotting. Whereas mAbs 806 and 528 alone had no effect on de2-7 EGFR expression, when used in combination, there was a dramatic decrease in the total de2-7 EGFR (Fig. 3A, top), suggesting that in combination the antibodies induce rapid down-regulation of the receptor. Whereas no decrease in the level of phosphorylation at Tyr1173, the major activation site for the EGFR, was detected following treatment with mAb 806, a noticeable decrease was observed following mAb 528 treatment (Fig. 3A, second). However, the level of phosphorylation at Tyr1173 was greatly reduced in the presence of both antibodies (Fig. 3A, second), compared with treatment with either antibody alone, which correlates with the decrease in total de2-7 EGFR. Consistent with the lack of synergistic antitumor activity seen when mAbs 806 and 528 were used together against U87MG.DK xenografts, this combination of antibodies did not induce down-regulation of the dead kinase de2-7 EGFR (Fig. 3A, third). As expected, there was virtually no phosphorylation of Tyr1173 with the dead kinase receptor (Fig. 3A, bottom). Thus, the synergistic activity of mAbs 806 and 528 in combination seems related to their ability to cause extensive down-regulation of the de2-7 EGFR.
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2-7 and U87MG.DK xenografts 1 day after the third administration of mAbs 806 and 528. These experiments were done as described for Fig. 2B, where the total amount of antibody given was identical in all groups. The down-regulation of total de2-7 EGFR and decreased phosphorylation of Tyr1173 following treatment with mAbs 806 and 528 in combination was observed in antibody-treated U87MG.2-7 xenografts (Fig. 3B, top and second). In contrast to the in vitro assay, mAb 806 alone did block the phosphorylation of Tyr1173 in treated xenografts, suggesting that its effects on signaling inhibition maybe more pronounced in vivo.
p27KIP1 is a negative regulator of G1-S phase transition that functions by inhibiting cyclin-Cdk kinase activity (33). Treatment of U87MG.2-7 xenografts with mAb 806 or 528 alone produced a measurable increase in p27KIP1 expression compared with tumors treated with PBS (Fig. 3B, third). Treatment with both antibodies in combination caused an additive increase in the level of p27KIP1, consistent with the down-regulation of total de2-7 EGFR. As expected, U87MG.DK xenografts (Fig. 3C) treated with the combination of antibodies did not show down-regulation of de2-7 EGFR (Fig. 3C, top) or changes in the level of p27KIP1 (Fig. 3C, third) compared with xenografts treated with PBS or either antibody alone.
To determine if antibody mediated down-regulation of the de2-7 EGFR combined with the up-regulation of p27KIP1 has an antiproliferative effect on xenografts, we analyzed the percentage of Ki67-positive cells from treated mice. These experiments were also done as described for Fig. 2B, where the total amount of antibody given was identical in all groups. U87MG.2-7 xenografts treated with vehicle had a PI of 34.6% (Fig. 4A). In contrast, U87MG.2-7 xenografts treated with either mAb 806 or 528 had lower PI of 20.1% and 19.6%, respectively (P < 0.001). U87MG.2-7 xenografts treated with both mAbs 806 and 528 in combination had a PI of 11.3%, which was significantly lower than the vehicle, mAb 806 or 528 treatments (P < 0.001 for all groups; Fig. 4A). Consistent with its slower growth rate, the PI of U87MG.DK xenografts treated with vehicle was 18.7% (Fig. 4A). Treatment with mAb 806 (PI = 18.8%) or 528 (PI = 18.3%) had no effect on the proliferation of U87MG.DK xenografts. Significantly, the combination treatment did not have any effect (PI = 19.1%) on the U87MG.DK xenografts (Fig. 4A). Interestingly, analysis of the apoptotic index through terminal deoxynucleotidyl transferase–mediated nick-end labeling staining (28) showed an extremely low level of apoptosis that did not differ between treatment and control groups at this early time point (data not shown).
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2-7 xenografts treated as in Fig. 2B, was assessed by immunostaining xenograft sections for CD34 (Fig. 4B). The number of CD34-positive micovessels in xenografts treated with PBS was 34.2 ± 10.8. Following treatment with mAbs 806 or 528 as single agents, the number of mean CD34-positive microvessels was 35.2 ± 4.8 and 37.6 ± 8.5, respectively (Fig. 4B). These values were not statistically different to that observed for PBS-treated tumors. However, following combined mAbs 806 and 528 therapy, there was a statistically significant reduction of 46% in the number of CD34-positive vessels compared with PBS-treated tumors (18.4 ± 5.8; P < 0.0001; Fig. 4B), indicating that the inhibition of U87MG.2-7 tumors in vivo following combination antibody therapy is partly the result of the blockade of angiogenic progression. Both the subclass control sections and the negative control sections did not stain positive for CD34.
Treatment of established A431 xenografts with monoclonal antibodies 806 and 528. Given the efficacy of antibody combination therapy against U87MG.2-7 xenografts, we elected to examine its efficacy against A431 tumors. A431 cells contain an amplification of the wt EGFR gene and overexpress the receptor on the cell surface. Combination therapy with mAbs 806 and 528 displayed at least additive antitumor activity against established A431 xenografts. At day 20 after tumor inoculation, the A431 xenografts treated with both antibodies had a mean tumor volume of 15 mm3 which was significantly lower than vehicle (1,016 mm3, P < 0.001), mAb 806 alone (473 mm3, P < 0.001), and mAb 528 alone (372 mm3, P < 0.001) treatment groups (Fig. 5A and B). Furthermore, combined treatment with mAbs 806 and 528 caused complete regression in 60% of the tumors, which again is indicative of a synergistic antitumor effect (Fig. 5B). Histologic examination at day 28 after inoculation revealed the complete absence of tumor cells in the majority of regressed xenografts (data not shown). No complete regressions were seen in any of the other treatment groups.
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515 mm3 at which time antibody therapy commenced. Treatment with mAb 806 alone did not inhibit A431 tumor growth rate when compared with vehicle-treated controls at day 26 after inoculation (P = 0.19), whereas treatment with mAb 528 alone did have an antitumor effect (P < 0.001). However, treatment with both antibodies in combination resulted in a greater than additive reduction in A431 xenograft growth rate compared with vehicle (P < 0.001), mAb 806 alone (P < 0.001), and mAb 528 alone (P < 0.01) treatment groups. At day 26, when vehicle-treated animals were sacrificed, the mean tumor volume was 1,810, 2,150, 850, and 360 mm3 in the vehicle, mAb 806, mAb 528, and combination treatment groups, respectively (Fig. 5C).
Systemic treatment of intracranial glioma xenografts with monoclonal antibodies 806 and 528. To further test the efficacy of mAbs 806 and 528 in combination, we treated mice bearing i.c. U87MG.2-7 glioma xenografts with i.p. injection of antibody (Fig. 6A). Animals treated with vehicle had a median survival of 14.8 days, whereas systemic treatment with either 0.5 mg mAb 806 or 528 increased the median survival to 19.2 (P < 0.003) and 17.6 (P < 0.009) days, respectively (Fig. 6A). Treatment of animals with 0.5 mg of mAbs 806 and 528 in combination significantly prolonged survival to a median of 25.7 days (P < 0.001 compared with all groups). Animals bearing i.c. U87MG.DK xenografts treated with vehicle had a median survival of 24.2 days, whereas those treated with mAbs 806 and 528 in combination as above had a median survival of 23.8 days (P = 0.76; Fig. 6B). Thus, as in the s.c. xenograft models, the combination antibody therapy was ineffective against xenografts expressing the dead kinase de2-7 EGFR.
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2-7 xenografts were histologically analyzed at the completion of antibody therapy (i.e day 14 after inoculation). Consistent with the survival data, combined treatment with mAbs 806 and 528 dramatically reduced the volume of i.c. U87MG.2-7 tumors in comparison to those treated with vehicle, mAb 806 alone, or mAb 528 alone (Fig. 6C). Combination antibody treatment of U87MG.DK i.c. tumors did not produce a reduction in tumor volume compared with treatment with vehicle (Fig. 6D). |
Discussion |
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2-7 xenografts was not directly mediated through its effect on the endogenous wt EGFR coexpressed in these cells, as mAb 528 did not inhibit the growth of parental U87MG5 or U87MG.DK xenografts both of which express similar levels of the wt EGFR. Furthermore, the lack of mAb 528 activity against U87MG.DK xenografts suggests that antibody effector function does not have a significant role in initiating the antitumor activity. To the best of our knowledge, this is the first report demonstrating that an antibody to the wt EGFR also has direct antitumor activity against de2-7 EGFR–expressing xenografts and partially addresses the concern that expression of de2-7 EGFR may cause resistance to EGFR antibody therapy.
Treatment of established U87MG.2-7 xenografts with a combination of mAbs 806 and 528 resulted in additive, and in some cases synergistic, tumor inhibition leading to complete regression of some of these aggressive tumors. Furthermore, the almost complete resistance of large U87MG.2-7 xenografts to either antibody alone was overcome by using the antibodies in combination. Significantly, this increased efficacy seen with combination antibody therapy was also observed in our i.c. model. Whereas the increase in survival was relatively modest (i.e., 11 days), it should be noted that i.c. U87MG.2-7 tumors grow extremely rapidly with control animals dying just 15 days after inoculation with 100,000 cells. Indeed, combination therapy should be more effective against less aggressive tumors, as was the case when we evaluated mAb 806 as a single agent (29). Importantly, the antitumor effect of both antibodies was seen following systemic administration, extending our previous data with mAb 806 alone (29). This observation suggests it maybe possible to use systemic antibody therapy in glioma patients with advanced disease as there is usually localized disruption of the blood/brain barrier in these patients.
The in vivo consequences of EGFR blockade by mAbs such as 528 has not been fully elucidated, although recent reports suggest that these antibodies switch off key EGFR signaling pathways leading to decreased proliferation, increased apoptosis, and the inhibition of proangiogenic growth factors, all of which can contribute to the antitumor effects seen (38). Analysis of mAb 806– and mAb 528–treated U87MG.2-7 xenografts showed that these antibodies caused a significant reduction in tumor cell proliferation compared with PBS-treated tumors which correlates with the in vivo growth rate of tumors treated with both antibodies in combination. This is most probably due to the significant down-regulation and degradation of de2-7 EGFR, which was observed following combination antibody therapy both in vitro and in vivo. This observation is in line with a recent report showing similar effects following treatment of ErbB2-overexpressing breast cancer cells with a combination of the anti-ErbB2 antibodies Herceptin and 2C4 (39). Combination Herceptin-2C4 treatment reduced ErbB2 levels within 24 hours to a greater degree than either agent alone (39). As both the ErbB2 and de2-7 EGFR do not bind ligand and are internalized respectively at a slower rate (40–42) or not at all (43) compared with wt EGFR, down-regulation of these receptors may contribute to signal attenuation and subsequent growth inhibition. Indeed, our data shows that combination therapy with both antibodies leads to down-regulation and degradation of de2-7 EGFR, which would lead to attenuation of the constitutive de2-7 EGFR signal.
A reduction in the level of receptor signaling would in turn have consequences on the proliferative capability of cells. Indeed, previous reports, investigating the ability of anti-EGFR agents to block EGFR signaling through blockade of ligand binding or kinase activation, have shown that these agents reversibly arrest cells in G1 phase of the cell cycle by modifying the expression of key proliferative/antiproliferative signaling molecules such as p27KIP1, leading to blockade of proliferation (44–48). In particular, the expression of p27KIP1 is thought to be required for growth inhibition when the EGFR pathway is blocked in cell lines dependent on the EGFR for their viability (48). Similarly, the enhanced tumorigenicity of U87MG.2-7 cells was directly related to de2-7 EGFR-mediated down-regulation of p27KIP1, through activation of the phosphatidylinositol 3-kinase/Akt pathway (49), identifying a potential pathway for therapeutic intervention. Indeed, U87MG.2-7 xenografts treated with either mAb 528 or 806 showed specific increase in p27KIP1 expression compared with PBS-treated xenografts. Combination antibody therapy also led to enhanced up-regulation of p27KIP1, which was greater than that observed following single-agent therapy. Furthermore, as mAb 806 does not bind to the endogenous wt EGFR expressed in these cells, increased p27KIP1 expression following mAb 806 therapy would be directly attributable to the interaction between this antibody and the de2-7 EGFR. Therefore, the additive up-regulation of p27KIP1 following combination antibody therapy would not occur simply through dual targeting of the wt EGFR but rather from specific targeting of both subsets of receptor.
Taken as a whole, the down-regulation of de2-7 EGFR and concurrent up-regulation of p27KIP1 following combination antibody therapy led to an additive antiproliferative effect and decreased intratumoral microvessel formation. Furthermore, the lack of down-regulation of the dead kinase variant of the de2-7 EGFR following combination antibody therapy, together with no changes in p27KIP1 expression or proliferative index correlate with the lack of therapeutic efficacy in vivo and highlight the importance of de2-7 EGFR kinase activity in mediating the inhibitory effects of these antibodies.
Finally given the significant decrease in proliferation and inhibition of blood vessel formation in U87MG.2-7 xenografts treated with mAbs 806 and 528 in combination, together with the up-regulation of p27 that inhibits G1-to-S phase transition, it is interesting that we do not see significant differences in apoptosis between treatment groups. However, given that these observations represent mechanisms of tumor cell inhibition early in the therapy cycle (1 day after the third injection of antibody), it is likely that changes in apoptotic level may become evident following prolonged therapy. Indeed, our unpublished data show that continued antibody therapy is able to cause a significant increase in apoptosis compared with control-treated tumors.6 Similarly, although our data do not show a difference in microvessel density between xenografts treated with mAb 806 or 528 as single agents compared with PBS, it is possible that such observations would be more likely following prolonged antibody therapy. A previous report by Mishima et al. reported a 30% decrease in tumor vascularity in mAb 806–treated tumors compared with control tumors (29). However, this occurred after prolonged therapy, where antibody was given for 14 consecutive days again highlighting the possibility that such events may become more evident following sustained therapy over longer time frames.
Greater than additive antitumor activity was also observed following treatment of established A431 xenografts with a combination of mAbs 806 and 528. This observation is somewhat remarkable given that mAb 528 recognizes the majority of EGFR expressed on A431 cells and mAb 806 binds <10% of the receptors (22). Recently, we have shown that mAb 806 recognizes a transitional form of the EGFR and prevents the formation of signaling-capable EGFR dimers (23, 24). Thus, whereas mAb 806 only binds a low percentage of the EGFR at any given time, over an extended period of time, it would be capable of inhibiting a substantial proportion of EGFR signaling which in turn generates an antitumor effect in vivo. Thus, the antitumor mechanism mediated by mAb 806 is clearly distinct to the ligand-inhibiting activity of mAb 528. Therefore, these antibodies inhibit wt EGFR signaling by different modes of action, which in the case of A431 xenografts, results in greater than additive tumor inhibition.
The most important outcome of our experiments is that single EGFR therapeutics do not inhibit all the functions associated with the de2-7 or the overexpressed wt EGFR; thus, it may be necessary to target the EGFR with multiple therapeutics. Indeed, our data suggests that the development of novel therapeutics to the EGFR should continue to identify new agents with unique properties. Given that we and others recently determined the crystal structure of the extracellular domain of the EGFR, it should be possible to identify regions of the receptor that represent potential targets (50, 51). Finally, if there are some aspects of EGFR function that are not inhibited by antibodies such as mAb 528, it may explain why some EGFR therapeutics have had less clinical success than anticipated (52). Therefore, combination therapy with mAb 806 and agents such as IMC-C225 or Iressa may increase the response rate seen in patients. Consequently, we are currently developing a chimeric version of mAb 806 for use in clinical trials.
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Footnotes |
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Received 12/22/04; revised 5/ 9/05; accepted 6/28/05.
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. Cell 2002;110:763–73.[CrossRef][Medline]
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), 0.5 mg of mAb 806 (
), or mAb 528 (
) as single agents or 0.5 mg of each antibody in combination (
), thrice weekly for 2 weeks on days 9, 11, 13, 16, 18, and 20 when the starting tumor volume was 115 mm3. B, mice (n = 5) bearing U87MG.
), mAb 528 (
), mAb 806 (
), both antibodies (
). F, the mice described in (C) were analyzed for survival by Kaplan-Meier. In this experiment, mice had to be sacrificed once both xenografts had reached 1,500 mm3, which was therefore considered the end point for each individual xenograft. Ethical considerations also only allowed this analysis until day 26.
