Targeting Multiple Her-2 Epitopes with Monoclonal Antibodies Results in Improved Antigrowth Activity of a Human Breast Cancer Cell Line in Vitro and in Vivo

Cells.

The human breast cancer cell line BT474 (American Type Culture Collection, Manassas, VA) was maintained in monolayer culture in MEM (Life Technologies, Inc., Grand Island, NY) supplemented with 1% vitamins, 2 mm l-glutamine, 0.1 mm nonessential amino acids, 1 mm sodium pyruvate, 1% HEPES buffer (all from Life Technologies, Inc.), and 10% heat-inactivated FCS (Hyclone, Logan, UT). Cultures were grown at 37°C and 5% CO₂, and cells were passaged when they were 70% confluent. Before passage, cells were removed from the flasks after a 5-min incubation with trypsin/EDTA (Life Technologies, Inc.) at room temperature and were washed twice in CM.

MAbs.

Mouse anti-Her-2 MAbs were generated in our laboratory by immunizing BALB/c mice s.c. with the EC domain of Her-2.4 Spleen cells from the immunized mice were harvested and fused with the myeloma cell line SP2/0. The hybridomas were subcloned and assayed by ELISA for their ability to secrete immunoglobulin. Immunoglobulin-containing SNs from positive clones were tested by ELISA for reactivity against the EC domain of Her-2. Positive SNs were then tested by indirect fluorescence on a panel of breast cancer cell lines and analyzed on a FACS (Becton Dickinson, Franklin Lakes, NJ). The MAbs were affinity purified on protein G-Sepharose (Pharmacia, Piscataway, NJ) and further characterized.

Characterization of MAbs.

Twelve MAbs recognizing nine different epitopes on Her-2 were characterized for binding to BT474 cells using indirect immunofluorescence assays and for epitope specificity by cross blocking in direct immunofluorescence assays. Indirect immunofluorescence assays were carried out on BT474 cells suspended at 1 × 10⁵/ml in CM and treated with 0.01–1 μg/ml MAbs for 30 min on ice. The excess MAbs were washed out with CM containing 0.05% sodium azide, and the cells were then treated with 2 μl of the secondary FITC-labeled goat antimouse immunoglobulin and analyzed on a FACScan to determine the percentage of cells that are stained and their MFI.

Direct immunofluorescence assays were carried out with FITC-anti-Her-2 MAbs to determine the amount of MAbs needed to self-block and to cross-block. BT474 cells were treated as described above with saturating concentrations of unlabeled anti-Her-2 MAbs, washed twice, and then treated with various amounts of FITC-labeled anti-Her-2 MAbs. The percentage of cross-blocking was determined by comparing the percentage of positive cells treated with unlabeled anti-Her-2 MAbs followed by FITC-anti-Her-2 MAbs versus the percentage of positive cells treated with FITC-anti-Her-2 MAbs alone. Cross-blocking was considered positive if the binding of the FITC-anti-Her-2 MAbs was reduced by at least 50%.

The isotype of each of the anti-Her-2 MAbs was determined using the ISOStrip Mouse Monoclonal Antibody Isotyping kit (Boehringer Manheim, Indianapolis, IN) according to the manufacturer’s directions. All of the MAbs were of the IgG1k isotype.

Of these 12 MAbs, three IgG1 MAbs (HER-50, HER-66, and HER-70), which recognize three different epitopes on the EC domain of Her-2, were selected for in vivo evaluation because of their superior ability to inhibit the growth of the Her-2-overexpressing BT474 cell line in vitro.

Growth of BT474 s.c. Tumors in SCID Mice.

Male CB.17-SCID mice were used. The sera from all of the mice were screened by ELISA for the presence of mouse immunoglobulin. If serum levels exceeded 10 μg/ml, the mice were considered “leaky” and were not used because tumor-take in these mice is less reliable (26) . The mice were then irradiated with 150 cGy and given a single s.c. injection of 4 × 10⁶ BT474 cells/mouse. Tumor nodules were allowed to grow for 10 weeks or until they reached 400 mm³. The diameter of s.c. tumors was measured twice a week using Venier calipers, and the volumes were calculated according to the formula: volume (mm³ ) = length (mm) × width (mm²).

IHC.

s.c. tumors were removed from mice at the end of the experiment after anesthetizing the animals and perfusing them with heparinized saline followed by 4% paraformaldehyde. Mouse tumors were fixed in 10% formalin for 24 h and then embedded in paraffin. Three-μm paraffin sections were prepared at the U. T. Southwestern Pathology Laboratory. Paraffin-embedded tumors were stained with 5 μg/ml affinity-purified polyclonal rabbit antihuman c-erbB-2 antibody (Dako Co., Carpinteria, CA), followed by 2.5 μg/ml biotinylated goat antirabbit IgG (27, 28, 29) . The same sections were also immunostained with 20 μg/ml HER-50, HER-66, or HER-70 MAbs, or the isotype-matched (negative control) anti-CD25 MAb, RFT5, followed by 2.5 μg/ml biotinylated horse antimouse IgG. The endothelial cells were immunostained with 5 μg/ml rat antimouse CD31 (PECAM-1), followed by 2.5 μg/ml biotinylated rabbit antirat IgG. Positive reactions were visualized by horseradish peroxidase-conjugated streptavidin-biotin complex and diaminobenzidine. Sections were counterstained with hematoxylin.

ADCC Assays.

ADCC assays were performed as previously described (30) with some modifications. The peripheral blood mononuclear cells used as human effector cells were separated from the blood of healthy donors on Ficoll-Hypaque density gradients. Cells were washed once in PBS and then incubated with the green fluorescence cytoplasmic dye, CFSE (Molecular Probes, Inc., Eugene, OR), using 25 μl of a 10-μm solution/10⁶ cells for 7 min at 37°C in a water bath. Murine effector cells were obtained from the spleens of BALB/c mice and were stimulated with concanavalin A (2.5 μg/1 × 10⁶ cells) for 24 h. BT474 (target) cells were harvested, washed, and incubated with different dilutions of either a single anti-Her-2 MAb (HER-50, HER-66, and HER-70), the MAb mixture, Herceptin, or isotype-matched murine or human IgGs (controls). After 30 min at 4°C, excess MAbs were washed out with medium containing 1% FCS plus 0.05% sodium azide. Target cells (BT474) were mixed with either murine or human effector cells at various E:T cell ratios (10:1, 50:1, and 100:1) and incubated for 7 h in a 37°C, 5% CO₂ incubator. PI was used at 50 μg/ml to stain dead cells before analyzing the cells on a Becton Dickinson FACS.

CDC Assays.

The CDC assay was similar to the ADCC assay with some modifications (30) . For the murine MAbs, mouse serum (Sigma) was used as a source of complement. For Herceptin, human serum isolated from freshly drawn blood was used as one source of complement and mouse serum (Sigma) was used as another. BT474 target cells, treated as described previously, were mixed with either mouse or human sera diluted 1/10, 1/50, or 1/100, and the cells were incubated for 4 h in a 37°C, 5% CO₂ incubator. PI was used at 50 μg/ml to stain dead cells before FACS analysis.

Measurement of VEGF in the SNs of BT474 Cells by ELISA.

5 × 10⁵ BT474 tumor cells/well were seeded in 24-well plates (Falcon). Cells were allowed to adhere overnight, and they were then treated with different concentrations of either HER-50, HER-66, HER-70, the MAb mixture, or Herceptin. Cells were removed by centrifugation at different time points, and the SNs were filter sterilized using a 0.22-μm pore size filter. VEGF levels were measured using the commercially available competitive ELISA kit from Cytimmune Sciences, Inc. The amount of VEGF present in the SNs was extrapolated from the VEGF standard curve and expressed in ng/ml. The levels of VEGF that could be detected in this assay ranged from 0.195–200 ng/ml.

Growth Inhibition Assay.

BT474 cells, at 2.5 × 10⁶ cells/ml in 10% MEM, were plated in triplicate (100 μl/well) in 96-well microtiter plates and were allowed to adhere overnight. The cells were then treated with 100 μl of different concentrations of HER-50, HER-66, or HER-70, a combination of all three (mixture), or Herceptin diluted in the same medium. Nine wells of untreated cells were included in each plate as controls. The plates were incubated for 72 h at 37°C in a 5% CO₂ incubator and then pulsed for 6 h with 1 μCi/well [³H]thymidine. Plates were harvested using a Skatron cell harvester, and the radioactivity was measured using a liquid scintillation counter (LKB Wallac). Inhibition of growth was determined by plotting the percentage of [³H]thymidine incorporation compared with untreated cells that were taken as 100%. The IC₅₀s (m) concentration, which inhibited [³H]thymidine incorporation by 50%, were compared.

Annexin-V Assay.

This assay measures early events in apoptosis (31) . BT474 cells were plated at 2.5 × 10⁵/ml in 24-well Falcon plates, allowed to adhere overnight, and treated with 100 μg/ml of the individual anti-Her-2 MAbs using amounts determined in preliminary experiments to inhibit the growth of BT474 cells by 50% as determined by [³H]thymidine incorporation. The MAb mixture was used at the same final concentration. Untreated cells were used as negative controls, and sodium azide was used as a positive control. After a 4-h incubation, the cells were harvested by trypsinization and washed and stained with FITC-Annexin-V and PI according to the manufacturer’s instructions. A Becton Dickinson FACS was used to quantify Annexin⁺ (apoptotic) cells and PI⁺ (necrotic) cells.

Characterization of the Three Anti-Her-2 MAbs.

We have developed a panel of murine anti-Her-2 MAbs that recognize nine different epitopes on the EC domain of Her-2. All of the MAbs were of the IgG1k isotype as determined using the ISOStrip Mouse Monoclonal Antibody Isotyping kit. The MAbs were tested for their ability to bind to specific epitopes on the EC domain of Her-2, and the epitopes recognized were determined by cross-blocking (32 , 33) .

Each MAb and Herceptin could self-block but none could cross-block, suggesting that they recognized four different epitopes on the Her-2 molecule (Table 1)⇓ . BT474 cells were stained with each of the anti-Her-2 MAbs followed by FITC-labeled goat antimouse immunoglobulin to determine the percentage of positive cells and the density of Her-2 epitopes recognized by the MAbs (MFI). Over 90% of the cells stained positively with HER-50 or HER-66, and >80% of the cells stained positively with HER-70 or Herceptin. However, the epitopes recognized by HER-70 were expressed at the highest density (highest MFI), followed by the epitopes recognized by HER-66 and HER-50. When the relative-binding affinities of all MAbs were determined (by plotting the percentage of positive cells versus the concentration and calculating the concentration required to reach 50% saturation), Herceptin and HER-50 showed the highest relative-binding affinities on BT474 cells. HER-70 had the lowest relative-binding affinity, and HER-66 was intermediary.

Growth Inhibition and Apoptotic Effect of the Anti-Her-2 MAbs on BT474 Cells.

We next determined whether the 3 MAbs would inhibit [³H]thymidine incorporation in BT474 cells. The MAbs were incubated for 72 h with BT474 cells, and the levels of [³H]thymidine incorporation in the MAb-treated cells were measured and compared with those of untreated cells to determine the IC₅₀. As shown in Fig. 1⇓ and Table 2⇓ , the IC₅₀ of the MAb mixture was decreased 3–200-fold as compared with any one of the three MAbs alone or to Herceptin. Table 2⇓ summarizes the means ± SD of three experiments.

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Fig. 1.

Killing of BT474 cells by the anti-HER-2 MAbs. BT474 cells were treated with varying concentrations of HER-50 (•), HER-66 (○), HER-70 (▾), combination of the three MAbs (▪), or Herceptin (▿) for 72 h, then pulsed with [³H]thymidine for 6 h. Growth inhibition was calculated by comparing [³H]thymidine incorporation in treated versus untreated cells. The difference between the IC₅₀s of the MAb mixture and the individual MAbs is statistically significant with Ps < 0.05. This is one representative experiment of three performed.

We also determined whether the MAb mixture would induce apoptosis of BT474 cells more effectively than the individual MAbs. Table 2⇓ shows the means ± SD of three experiments. As measured by Annexin-V staining, the MAb mixture induced the highest percentage of apoptotic BT474 cells, i.e., 20–27% higher than that induced by individual MAbs and 34% higher than that induced by Herceptin. Cell death, as determined by trypan blue exclusion and PI staining, was similar for the 3 MAbs and Herceptin and marginally superior for the MAb mixture. Levels of apoptosis and necrosis in the untreated controls were negligible. The positive control, sodium azide, induced significant apoptosis. Because the percentage of apoptotic cells induced by the MAbs surpassed the percentage of necrotic cells, it is possible that apoptosis is the major type of cell death induced by the MAbs.

The Therapeutic Effect of Anti-Her-2 MAbs in SCID Mice with s.c. BT474 Tumors.

We next determined whether HER-50, HER-66, and HER-70 would inhibit the growth of the BT474 tumor in mice. The in vivo model used to evaluate therapy has been described previously (34) ; in this model, there is 100% tumor-take and consistent tumor growth after s.c. injection of BT474 cells into preirradiated male SCID mice. Because the growth of BT474 cells is estrogen independent, (35, 36, 37) male mice could be used. This is important because estrogen can down-regulate Her-2 expression on tumor cells (38 , 39) . Individual variations of tumor nodules were included in the mean values ± SD for the five mice in each treatment group.

To determine the dose of the MAbs required to inhibit the growth of established s.c. BT474 tumors in SCID mice, we first studied the blood levels that could be achieved by injecting different amounts of ¹²⁵I-HER-66. Doses that gave levels that reached an IC₅₀ in vitro were chosen (data not shown). Biodistribution of ¹²⁵I-HER-66 was also determined after i.p. injection of ¹²⁵I-HER-66 into both normal and s.c tumor-bearing SCID mice. We observed that 1.5% of the injected dose was taken up by the s.c. breast tumor nodule as compared with 0.7% by the kidneys and liver, 0.4% by the lungs, and 0.09% by the spleen (data not shown). The MAb persisted at the tumor site for at least 4 days.

Therefore, SCID mice with advanced s.c. BT474 tumors measuring 400 mm³ were then treated with 15 μg/g mouse of the individual anti-Her-2 MAbs, the MAb mixture, or Herceptin. The MAbs were administered as three i.p. injections at 3-week intervals.

As shown in Fig. 2A⇓ , mice treated with the MAb mixture showed continuous regression of tumor nodules starting after the first course of treatment. After the third course, tumor nodules became nonpalpable for a total of 3 weeks. Individual MAbs induced 75–90% reductions in tumor volume after 2 weeks, and these reductions were maintained over the next 4.5 weeks, followed by relapse. Herceptin was inefficient initially, then induced a 40% reduction in tumor volume after the third week and progressively reduced tumor volumes to 60% of their original size in the following 3.5 weeks, followed by relapse. The reasons for this delay are unclear but may be related to poor ADCC/CDC initially, followed by delayed antigrowth activity. Fig. 2B⇓ shows that the MAb mixture also prolonged the survival of SCID/BT474 mice more effectively than either the individual MAbs or Herceptin. Thus, 100% of mice treated with the MAb mixture survived for 7 weeks after the first injection, whereas 40–60% of mice treated with individual MAbs and 5% of the mice treated with Herceptin survived for the same length of time. Untreated mice were sacrificed when tumor burdens exceeded 1000 mm² volume or 10% of their total body weight.

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Fig. 2.

BT474 cells are killed most effectively in vivo by the MAb mixture. A, preirradiated male SCID mice with 400 mm³ s.c. BT474 breast tumors were injected i.p. with three equal doses of 300 μg (15 μg/g mouse) 10 weeks after tumor inoculation on days 0, 24, and 48. The following treatments were administered to groups of five mice: HER-50 (•); HER-66 (○); HER-70 (▾); combination of the three MAbs (▪); Herceptin (▿); and control (injected with saline; □). Tumors were measured twice a week using calipers, and the mean tumor volume (mm³) ± SD for the five mice in each group was plotted. This represents one experiment of three carried out. The difference between the effects of each treatment versus the control, and between the MAb mixture and each individual MAb are significant with Ps < 0.00715 and < 0.05, respectively. B, one hundred percent of mice treated with the combination of three MAbs survived 7 weeks after the first dose injected. A total of 40–60% of mice treated with individual MAbs and 5% of Herceptin-treated mice survived after 7 weeks when the experiment was terminated. Animals were euthanized when tumor burden exceeded 1000 mm³ or 10% of total body weight.

When mice with smaller, 200 mm³ tumors were treated with the MAb mixture, tumors became nonpalpable 2 weeks after a single dose (data not shown). Remissions were sustained for 4 weeks and all of the mice treated with the mixture survived for 7 weeks after treatment. None of the individual MAbs or Herceptin induced 100% inhibition of s.c. tumor growth, nor did they prolong survival of mice as long as the MAb mixture. Histopathological examination indicated that none of the treated or untreated mice had metastases in their lungs, livers, spleens, or kidneys (data not shown). These results confirm earlier findings that metastases can only be detected when s.c. tumors are >800 mm³ (34) . In all treated animals, tumors resumed growing 2–3 weeks after the last treatment. However, the regressions induced by the MAb mixture generally lasted 1 week longer.

These experiments demonstrate that the MAb mixture is more effective in treating larger tumors than either the individual MAbs alone or Herceptin. When smaller tumors were treated, significant growth inhibition effect was achieved by only one dose of the mixture and was more rapid and long lived.

The Effects of MAb Treatment on the Expression of Her-2 Epitopes on Surviving Tumor Cells.

One mechanism by which anti-Her-2 MAbs act is to induce receptor down-modulation (40, 41, 42) . Therefore, we determined whether the tumor cells which grew back after therapy was discontinued, lacked the Her-2 epitopes recognized by the MAbs used for treatment. Tumors were removed from mice at the end of the experiment, fixed in 10% formalin, embedded in paraffin, and immunostained with HER-50, HER-70, or HER-66 MAbs, the isotype-matched anti-CD25 antibody, RFT5 (negative control), or the anti-c-erbB-2 polyclonal antibody (Dako Co.). As shown in Fig. 3⇓ , tumor nodules removed from relapsed mice expressed the same levels of Her-2 as the original tumor. This indicates that tumor cells that grew back either had not down-regulated Her-2 or that down-regulation was reversible. These findings suggest that the relapsed tumors do not lack the targeted epitope, but rather escape killing by the MAbs for other reasons, e.g., inaccessibility, mutations in signaling pathways, and so on.

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Fig. 3.

Relapsed tumors show similar expression of the Her-2 epitopes recognized by each MAb used for treatment. IHC was performed on formalin-fixed, paraffin-embedded s.c. tumors. Tumors were removed from untreated and treated mice and immunostained with HER-50, HER-66, and HER-70 versus the rabbit antihuman c-erbB-2 polyclonal antibody and the isotype-matched control anti-CD25 MAb RFT5. Dark brown areas are indicative of Her-2 expression.

The Ability of the Anti-Her-2 MAbs to Mediate ADCC and CDC.

Although the MAb mixture was highly effective at inhibiting cell growth in vitro, it has been documented that in vivo the Fc portion of MAbs can be of major importance in efficacy (43, 44, 45) . Furthermore, the binding of MAbs to three different epitopes on Her-2 might be more efficient in mediating effector functions because of higher concentrations of total antibody bound. This should contribute to a superior performance of the MAb mixture in vivo. To explore this possibility, we compared the effector functions of the individual MAbs to those of the MAb mixture in vitro. As shown in Fig. 4A⇓ , the individual MAbs induced 10–20% lysis of target cells, whereas the MAb mixture induced 30% lysis at an E:T cell ratio of 100:1. Herceptin mediated potent ADCC in the presence of human peripheral blood mononuclear cells at an E:T cell ratio of 100:1, inducing up to 50% lysis of target cells. However, Herceptin mediated only 10% lysis in the presence of murine effector cells (data not shown), at least partially explaining its inferior activity in mice as compared with the murine MAbs. As shown in Fig. 4B⇓ , the MAbs also fixed complement, resulting in dose-dependent lysis of the target cells. At the same dilution of complement, the MAb mixture induced 2-fold more lysis than HER-50 or HER-66 alone and 10% more lysis than Herceptin or HER-70. Herceptin induced only 10% lysis in the presence of mouse serum, again partially explaining why it was less effective in mice. The isotype-matched negative controls did not mediate either ADCC or CDC.

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Fig. 4.

The MAb mixture induces ADCC (A) and CDC (B) against BT474 breast cancer cells in vitro. Flow cytometric analysis was carried out on the CFSE-stained effector cells (peripheral blood mononuclear cells or mouse spleen cells) using PI to stain target BT474 cells treated with 10 ng/1 × 10⁴ cells of HER-50 (•), HER-66 (○), HER-70 (▾), a combination (▪), RFT5 (□; IgG1-isotype-matched control), Herceptin (▿), or Human IgG (♦; IgG1-isotype-matched control). A, the E:T cell ratios were 10:1, 50:1, and 100:1. B, target BT474 cells treated with 100 ng/1 × 10⁵ cells of the same MAbs were mixed with mouse serum diluted 1/10-, 1/50-, and 1/100-fold (as source of complement for the CDC assays). Relative to the isotype-matched control, the effect of each treatment is statistically significant with Ps < 0.05. Both figures illustrate one representative experiment of three performed.

The Effect of Anti-Her-2 MAbs Treatment on VEGF Secretion by Tumor Cells in Vitro.

As described by others (15) , treatment with anti-Her-2 MAbs may also exert an antiangiogenic effect in vivo by down-regulating the production of one or more proangiogenic factors produced by the tumor cells. We have documented the presence of newly formed vessels in the s.c. breast tumor nodules by immunostaining tumors with MAbs that react with the endothelial cell marker PECAM-1 (CD31; data not shown). All tumors contain a high density of vessels, which are immunoreactive with the anti-CD31 antibody. These results suggest that angiogenesis is involved in the growth of s.c. BT474 tumors.

Therefore, we used a competitive ELISA assay to determine whether the three anti-Her-2 MAbs could suppress VEGF production by BT474 cells in vitro. We measured VEGF levels in cells treated with either the individual MAbs, the MAb mixture, or Herceptin. As shown in Fig. 5⇓ , untreated BT474 cells secreted ∼50 ng VEGF/ml/5 × 10⁵ cells over 48 h. Increasing concentrations of the anti-Her-2 MAbs induced a dose-dependent decrease in VEGF production. The MAb mixture completely inhibited VEGF production at a concentration of 1 μg/ml. HER-50 and Herceptin induced appreciable but not complete inhibition of VEGF secretion, whereas HER-66 and HER-70 had marginal activity at the same concentration. These results indicate that all of the MAbs block the secretion of VEGF but that the MAb mixture is superior.

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Fig. 5.

HER-50, HER-66, HER-70, and the MAb mixture inhibit the secretion of VEGF by BT474 cells in vitro. Cell supernatants were harvested from 5 × 10⁵ BT474 cells treated with various concentrations of HER-50 (•), HER-66 (○), HER-70 (▾), combination of the three MAbs (▪), and Herceptin (▿). Untreated BT474 cells secrete ∼50 ng VEGF/ml/ 5 × 10⁵ cells over 48 h (□). Relative to the untreated control, the effect of each treatment is statistically significant with Ps < 0.001. VEGF level was measured using a competitive ELISA kit. This assay can detect 0.195–200 ng/ml VEGF. This is a representative experiment of three performed.