HER-2/neu-Specific Monoclonal Antibodies Collaborate with HER-2/neu-Targeted Granulocyte Macrophage Colony-Stimulating Factor Secreting Whole Cell Vaccination to Augment CD8+ T Cell Effector Function and Tumor-Free Survival in Her-2/neu-Transgenic Mice

Matthew E. Wolpoe, Eric R. Lutz, Anne M. Ercolini, Satoshi Murata, Susan E. Ivie, Elizabeth S. Garrett, Leisha A. Emens, Elizabeth M. Jaffee and R. Todd Reilly
J Immunol August 15, 2003, 171 (4) 2161-2169; DOI: https://doi.org/10.4049/jimmunol.171.4.2161

Abstract

HER-2/neu is overexpressed in several cancers including 30% of breast carcinomas, and correlates with a poor outcome. HER-2/neu-transgenic (neu-N) mice that overexpress the non-transforming rat neu develop spontaneous mammary carcinomas and demonstrate immunotolerance to the neu protein similar to that observed in patients with neu-expressing cancers. In neu-N mice, neu-targeted vaccination induces weak T cell and negligible Ab responses sufficient to delay but not eradicate transplanted neu-expressing tumor. Here we demonstrate that passive infusion of neu-specific mAbs in sequence with whole cell vaccination significantly improves tumor-free survival over either modality alone. Importantly, treatment of neu-N mice with vaccine in combination with two distinct neu-specific Abs is particularly efficacious, preventing tumor in 70% and eradicating established tumor in 30% of neu-N mice. In vivo lymphocyte subpopulation depletion experiments demonstrate that the efficacy of Ab, alone or combined with vaccine, is dependent on both CD4+ and CD8+ T cells. Furthermore, the in vivo antitumor effects of vaccine and Ab are associated with a significant increase in the number and function of neu-specific CD8+ T cells. Collectively, these observations suggest that similarly increased efficacy could be obtained by combining neu-targeted vaccination and neu-specific Abs such as trastuzumab (Herceptin) in patients with neu-expressing cancers.

Her-2/neu or erbB-2 (neu)3 is a 185-kDa transmembrane protein and a member of the epidermal growth factor receptor family. The neu proto-oncogene is overexpressed in approximately one-third of breast carcinomas and in epithelial tumors of other origins including the pancreas, lung, ovary, and oral cavity (1, 2, 3, 4, 5). In many tumor types, it has been shown to confer a worse prognosis (4, 5, 6, 7, 8). neu-specific T cells and Abs have been isolated from the peripheral blood of breast cancer patients whose tumors overexpress this oncogene, demonstrating that neu may serve as an appropriate target for immunotherapeutic development (9, 10, 11, 12). In fact, neu-specific Ab and T cell responses have been observed after DNA and peptide-based immunizations but are often short-lived and have not been associated with improved survival (13, 14, 15). Both the lack of durable neu-specific T cell responses and simultaneous tumor progression in these cancer patients strongly point to the existence of peripheral tolerance mechanisms that are barriers to effective cancer-targeted immunization.

To develop clinically successful immune-based strategies for cancer treatment and prevention, it is necessary to evaluate new approaches in the setting of immune tolerance to the target Ag. Her-2/neu transgenic (neu-N) mice (16) express the nontransforming rat neu cDNA under a mammary specific promoter. As a consequence, these mice develop neu-overexpressing spontaneous mammary carcinomas in a stochastic manner beginning at ∼4 mo (16, 17). The tumors resemble human breast carcinoma in chronology and histology, as well as in their metastatic pattern (16, 18). In addition, neu-N mice exhibit evidence of immunological tolerance to the neu protein that appears to mimic what is encountered clinically. Specifically, Ab and T cell responses to neu have been measured in vaccinated neu-N mice in the presence of tumor progression. Several groups have reported the induction of neu-specific T cells after immunization with neu-targeted vaccines (17, 19, 20). However, these studies have failed to correlate increased T cell responses with significant tumor rejection or improved survival. To generate a more potent antitumor response, we have been interested in understanding the differences in vaccine-mediated antitumor immunity in neu-N mice compared with that seen in the parental FVB mice, the strain from which the neu-N mice are derived (i.e., in the absence of tolerance). We previously reported that vaccinated FVB mice mount more potent neu-specific T cell responses and generate higher titers of neu-specific Ab than vaccinated neu-N mice (21). In addition, adoptive transfer experiments in SCID mice demonstrated that the administration of both neu-specific CTL and neu-specific Ab was necessary for the eradication of a pre-existing neu-expressing tumor burden (21). These results suggest that neu-specific Ab may be integral for vaccine-mediated suppression of neu-expressing tumor in neu-N mice (in the setting of immune tolerance).

Drebin et al. (22, 23) have developed and characterized several neu-specific mAbs that can inhibit the growth of neu-expressing tumors in vitro and in athymic nude mice. Although Drebin et al. (23) demonstrated that these mAbs were effective as single agents, they also showed that using a combination of more than one mAb was more efficacious than using a single mAb at the same concentration. In a different model, mAbs recognizing different epitopes on the human neu extracellular domain (ECD) more effectively mediate Ab-dependent cell cytotoxicity (ADCC), tumor growth inhibition, apoptosis induction, and the inhibition of vascular endothelial growth factor secretion in addition to the in vivo suppression of tumor growth (24). However, because the in vivo antitumor activity of these neu-specific mAbs was assessed in immunocompromized mice, the effects of neu-specific Ab administration on the development of T cell-mediated immunity was not addressed.

In this report, we examined whether combining neu-specific mAbs with neu-targeted whole cell vaccination (for the generation of neu-specific cellular immunity) could generate more potent antitumor immunity in neu-N mice. Our data demonstrate that the combination of two neu-specific mAbs that recognize distinct epitopes of the neu-ECD in sequence with a neu-targeted whole-cell vaccination results in a significant increase in tumor-free survival over either mAb or vaccine treatment alone. In addition, in vivo lymphocyte subpopulation depletion studies indicate that the increased therapeutic efficacy achieved using this regimen is dependent on both CD4+ and CD8+ T cells. Finally, the ability of a given mAb preparation to interact with vaccine to achieve more potent antitumor immunity in vivo was reflected in in vitro assays of CD8+ T cell frequency and lytic function. These findings have important implications for improving immune-based therapies currently under clinical development.

Materials and Methods

Mice and cell lines

The neu-N-transgenic mouse line (line N 202) (16) was originally obtained from Dr. W. Muller (McMaster University, Hamilton, Ontario, Canada). The 3T3/GM, 3T3-neu/GM, NT5/B7-1, NT2, and 3T3-neu/B7-1 cell lines have been described previously (17). Animals were maintained under pathogen-free conditions and were treated in a humane manner in accordance with institutional and American Association of Laboratory Animal Committee policies.

mAbs

Hybridomas expressing the murine anti-rat neu mAbs 7.9.5 and 7.21.2, both of the IgG1 subtype, were generously provided by Dr. M. Greene (University of Pennsylvania, Philadelphia, PA). Drebin et al. (22, 23) showed that these two mAbs recognize distinct epitopes on the neu extracellular domain and inhibit growth of neu-expressing tumors in vitro and in athymic nude mice. The hybridomas for 7.9.5 and 7.21.2 were grown in athymic nude mice, and ascites was collected by Harlan Bioproducts for Science (Indianapolis, IN). The mAbs were purified over a T-Gel column (Pierce Biotechnology, Rockford, IL), using the Biologic LP purification system (Bio-Rad, Hercules, CA), dialyzed into PBS, and subsequently determined to be >90% pure by SDS-PAGE analysis using Criterion 4–15% Tris-HCl Pre-cast Gels and the Criterion Cell (Bio-Rad).

Tumor prevention experiments

Female neu-N mice, 8 wk old, were vaccinated with 3T3-neu/GM cells (or control 3T3/GM cells) followed 2 wk later by s.c. tumor challenge with 1 × 105 NT2 cells as previously described (17). Animals also received sterile filtered (0.22-mm pore size mStar filters, Costar, Corning, NY) neu-specific mAb i.p. or polyclonal mouse IgG i.p. (Sigma-Aldrich, St. Louis, MO) as a control, on the day of tumor challenge and weekly thereafter for a total of five injections (100 μg of IgG per injection). Animals were then monitored for the formation of palpable tumors, defined as tumors with diameters that exceeded 3 mm.

Treatment experiments

Female neu-N mice, 8 wk old, were given an NT2 challenge (1 × 105 cells) s.c. on day 0, followed by vaccination with 3T3-neu/GM cells (or 3T3/GM cells as a control) on day 3 as described previously (25). Animals also received sterile filtered (0.22-mm pore size mStar filters) neu-specific mAb i.p. or polyclonal mouse IgG i.p. (Sigma-Aldrich) as a control, on the day of vaccination and weekly thereafter for a total of five injections (100 μg of total IgG per injection). Animals were then monitored for the formation of palpable tumors, defined as tumors with diameters that exceeded 3 mm.

Depletion experiments

Female neu-N mice, 8 wk old, received either 3T3-neu/GM vaccine or control (3T3/GM) vaccine. Fourteen days later (1 wk before tumor challenge) either CD4+ or CD8+ T cells were depleted using the GK1.5 or 2.43 Ab, respectively, as described previously (25). The antibody PK136, which binds to the NK cell marker NK1.1 was used as a control Ab in these experiments, as the NK1.1 protein is not expressed on the FVB background. Depletions were confirmed by flow cytometry of spleen cells (data not shown) and maintained by twice weekly injections of appropriate mAbs. On day 21 after vaccination, animals received an NT2 challenge consisting of 1 × 105 cells given s.c. Animals were then monitored for the appearance of palpable tumors, defined as tumors with diameters that exceeded 3 mm.

ELISPOT assays

Female neu-N mice, 8 wk old, were given either vaccine or control vaccine on day −14, and an NT2 challenge (1 × 105 cells) on day 0. At the time of tumor challenge, animals were given 100 μg of control IgG, 7.9.5, 7.21.2, or a mixture of 50 μg each 7.9.5, and 7.21.2. The mice were then sacrificed 7 days after tumor challenge. Neu-specific CD8+ T cells were quantified by ELISPOT analysis as previously described with some modifications (25). Briefly, splenocytes were subjected to RBC lysis using ACK Lysis Buffer (BioSource International, Camarillo, CA) enriched for CD8+ T cells using the Spin-Sep CD8+ Isolation Kit (Stem Cell Technologies, Vancouver, British Columbia, Canada). FACS demonstrated >90% purity of CD8+ T cells using this method (data not shown). CD8+ T cells derived in this way were incubated with NT5/B7-1 cells in the presence of 20 U/well recombinant murine IL-2 (National Institutes of Health, Bethesda, MD) and were subjected to ELISPOT analysis for TNF-α production using the murine TNF-α ELISPOT Kit (R&D Biosystems, Minneapolis, MN) at E:T 10:1. Cells were incubated overnight at 37°C at 5% CO2, and the ELISPOT plates were developed according to the manufacturer’s specifications. Control wells containing T cells alone were also included. Spots were counted using the KS ELISPOT scope and software (Zeiss, Thornwood, NY). For each condition, the number of spots counted in the wells with T cells alone were averaged and subtracted from the number of spots in each of the wells with T cells plus targets.

Chromium release assays

Assays of cell lysis were performed as previously described (17) with the following modifications. Briefly, Fneu-CTL T cells, an FVB-derived neu-specific clonotypic CD8+ T cell line (21), were used as effector cells. The Fneu-CTL T cells lyse neu-expressing cell lines in an MHC class I-restricted (data not shown). NT2 target cells (2 × 106) were incubated for 2 h at 37°C, 5% CO2 with one of the following: 50 μg/m of polyclonal IgG, 50 μg/ml 7.21.2, 50 μg/ml 7.9.5, 25 μg/ml of 7.9.5 plus 25 μg/ml 7.21.2, or 3 μM geldanamycin (Sigma-Aldrich). These NT2 cells were labeled with 0.2 mCi of 51Cr for 1 h at room temperature and then washed three times with CTL buffer (17). NT2 target cells were then mixed with the T cell clone, and lysis was conducted and quantified as described (21).

Ubiquitin immunoprecipitation

The immunoprecipitation of ubiquitin and Western blot for neu were done according to Current Protocols in Immunology (26) with the following modifications. First, 1 × 107 NT2 cells were incubated with either control polyclonal mouse IgG (Sigma), 7.21.2, 7.9.5, both 7.9.5 and 7.21.2, or geldanamycin (Sigma-Aldrich). The polyclonal mouse IgG, 7.21.2, 7.9.5, or the combination of 7.9.5 and 7.21.2 were all used at a concentration of 50 μg/ml, and the geldanamycin was used at 3 μM. All incubations and dilutions were done in breast medium (17), and cells were harvested after 1, 5, 15, 30 or 60 min. The cells were then lysed and incubated with protein G-Sepharose bead slurries (Pierce, Rockford, IL) that were precoated with 1 mg of rabbit anti-ubiquitin (Dako, Carpinteria, CA) by rolling for 2 h at 4°C. The samples were then denatured using denaturing lysis buffer (18% glycerol, 5% w/v SDS, 100 mM DTT, and 300 mM Tris-HCl, pH 6.7) and heated to 80°C for 10 min. The concentrations of the protein samples were ascertained by UV OD280 using the Smart Spec 3000 Spectrophotometer (Bio-Rad). To ensure equal protein loading, 10 μg of each sample were added per lane to a 4–15% Tris-HCl gradient gel (Bio-Rad) and run at 100 V for ∼3 h using the Criterion Cell in Tris-glycine electrophoresis buffer (25 mM Trizma base, 250 mM glycine, 0.1% SDS, pH 8.3). The protein samples were then transferred to a nitrocellulose membrane (Amersham Pharmacia, Piscataway, NJ) at 400 mV for 45 min using a Transfer Blot Cell (Bio-Rad) at 4 °C. Ponceau S staining was used to verify equal protein transfer from the gel. The blots were then incubated with a polyclonal neu mAb (Cell Signaling Technology, Beverly, MA) at a 1/1000 dilution in primary Ab buffer (PBS plus 0.05% Tween 20 (Sigma-Aldrich) plus 5% w/v BSA) gently shaking overnight at 4°C. The following day, the blots were incubated with a 1/5000 dilution of donkey anti rabbit polyclonal IgG HRP (Amersham Pharmacia) in blocking solution for 1 h, rocking at room temperature. The blots were exposed to the ECL chemiluminescent solution (Amersham Pharmacia) and subsequently to ECL chemiluminescent film (Amersham Pharmacia) for 5 min according to the manufacturer’s instructions.

Statistical analyses

Kaplan-Meier curves were plotted using time to tumor formation as the outcome. Statistical differences in survival across groups were assessed using the log rank test. Statistical comparisons of mean neu-specific T cell frequencies (ELISPOT assay) and neu-specific lysis (chromium release assay) were made using an unpaired t test (two-tailed). All statistical analyses were performed using the GraphPad Prism 3 Program (GraphPad Software, San Diego, CA).

Results

Neu-specific mAbs enhance vaccine-mediated antitumor immunity

We previously demonstrated that neu-targeted vaccines induce weak neu-specific T cell responses and no measurable neu-specific IgG. This level of vaccine-mediated antitumor immunity is ineffective at preventing neu tumor growth or rejecting pre-existing neu-expressing tumors in neu-N mice (17). In contrast, the same neu-targeted vaccines induce both humoral and T cell responses against neu that are sufficient to reject large burdens of neu-expressing tumors in the parental FVB strain of mice (17, 25). Furthermore, passive administration of vaccine-induced Abs and CD8+ T cells together, but not separately, can eradicate these same neu-expressing mammary tumors when grown in SCID mice (21). We therefore examined whether combining neu-specific mAbs with a neu-targeted whole cell vaccine genetically modified to overexpress GM-CSF can overcome immune tolerance and eliminate neu-expressing tumors in neu-N mice. To accomplish this, we first examined the effectiveness of the neu-specific mAbs 7.9.5 and 7.21.2, either individually or in combination with a neu-targeted vaccine (22, 23).

In initial prevention experiments, mice were vaccinated 2 wk before NT2 challenge and mAb administration. As shown in Fig. 1⇓, mice given a neu-targeted whole cell vaccine (3T3-neu/GM) plus control Ab had a significantly longer tumor-free period when compared to mice receiving control vaccine 3T3/GM (p < 0.0001). However, all of these mice eventually developed tumors. In the treatment setting, a more stringent in vivo paradigm where vaccine is given 3 days after tumor inoculation, neu-targeted vaccination is similarly effective in delaying tumor growth, although tumors grow progressively in all mice (Fig. 2⇓). These results are consistent with data reported previously (17, 25). Treatment of neu-N mice with a single neu-specific mAb combined with the control vaccine also resulted in delayed tumor growth in neu-N mice. In the prevention setting, mice given Ab 7.21.2 had a significantly greater tumor-free survival than did control animals (Fig. 1⇓A, p < 0.0001). This effect was essentially equivalent to what was seen in mice given vaccine alone. Similarly, animals treated with the mAb 7.9.5 had a greater tumor-free survival in prevention experiments (Fig. 1⇓B, p < 0.0001) as well as in the treatment setting (Fig. 2⇓B, p = 0.02) when compared with control animals. Tumor-free survival in animals given neu-specific mAb in the context of control vaccine did not exceed that seen in mice given neu-targeted vaccine plus control IgG (Fig. 2⇓, A and B, p = 0.88 and 0.11, respectively).

FIGURE 1.
FIGURE 1.

Neu-specific Ab given in sequence with a GM-CSF-secreting neu-targeted vaccine (Vac) can prevent tumor growth in the majority of neu-N mice. Neu-N mice received neu-targeted vaccine (3T3-neu/GM, •, ▴) or control (Ctrl) vaccine (3T3/GM, ○, ▵) 2 wk before challenge with 1 × 105 NT2 cells. Ab was administered weekly beginning on the day of NT2 challenge (100 μg given i.p. for a total of 5 wk). A, Mice were treated with either vaccine plus AB 7.21.2 (▴, ▵) or vaccine plus isotype control IgG (•, ○). B, Mice were treated with either vaccine plus Ab. 7.9.5 (▴, ▵) or vaccine plus isotype control IgG (•, ○). C, Mice were treated with either vaccine plus an Ab mixture consisting of 50 μg of 7.21.2 and 50 μg of 7.9.5 (▴, ▵) or vaccine plus isotype control IgG (•, ○). Data are presented as Kaplan-Meier survival curves indicating the percentage of tumor-free animals as a function of time after NT2 challenge. Groups contained between 10 and 16 mice, and the data are combined from at least two replicate experiments.

FIGURE 2.
FIGURE 2.

The combination of neu-specific Ab and neu-targeted vaccine is potent enough to reject pre-established tumors. Neu-N mice were challenged on day 0 with 1 × 105 NT2 cells (s.c.) followed by neu-targeted vaccine (3T3-neu/GM, •, ♦) or control vaccine (3T3/GM, ▿, □) on day 3. Ab (100 μg) was administered at the time of vaccination, and weekly thereafter for a total of five injections. A, Mice were treated with either vaccine plus Ab 7.21.2 (▿) or vaccine plus isotype control IgG (•). B, Mice were treated with either vaccine plus Ab 7.9.5 (▿) or vaccine plus isotype control IgG (•). C, Mice were treated with either vaccine plus an Ab mixture consisting of 50 μg of 7.21.2 and 50 μg of 7.9.5 (▿) or vaccine plus isotype control IgG (•). The data are presented as Kaplan-Meier survival curves indicating the percentage of tumor-free animals as a function of time after NT2 challenge. Groups contained between 10 and 16 mice, and the data are combined from at least two replicate experiments.

Despite the fact that the mAbs 7.21.2 and 7.9.5 are both IgG1 and both demonstrated similar growth inhibitory effects on neu-expressing cells (20, 21), the combination of 7.9.5 with neu-targeted vaccine was much more efficacious than the combination of 7.21.2 and neu-targeted vaccine. In the prevention setting, mice given neu-targeted vaccine plus the 7.21.2 Ab had a greater tumor-free survival than animals given vaccine alone (Fig. 1⇑A, p = 0.01), but the effect was not significantly greater than that seen in animals given only 7.21.2 (p = 0.25). In contrast, mice given 3T3-neu/GM in combination with the mAb 7.9.5 showed a significantly improved tumor-free survival when compared with mice given either vaccine (p = 0.03) or the 7.9.5 mAb (p = 0.04) alone, with 40% of animals remaining tumor free beyond the 60-day endpoint of the experiment (Fig. 1⇑B). Similar results were observed in the treatment experiments. Animals given vaccine in sequence with the 7.21.2 mAb developed tumors more slowly than mice given the 7.21.2 mAb alone (Fig. 2⇑A, p = 0.04), and tumors grew more slowly in animals given vaccine in sequence with the 7.9.5 mAb than in those given the 7.9.5 mAb alone (Fig. 2⇑B, p = 0.01). In the treatment setting, however, none of the mice given neu-targeted vaccine plus a single neu-specific mAb remained tumor free beyond 45 days after tumor inoculation.

A mixture of neu-specific Abs collaborates with neu-targeted vaccine to enhance antitumor immunity in neu-N mice

Previous experiments by Drebin et al. (23) with the mAbs 7.9.5 and 7.21.2 demonstrated that both of these mAbs are able to prevent tumor formation in vitro and in athymic nude mice. However, to eradicate existing tumors, it was necessary to use two mAbs recognizing distinct epitopes of the neu ECD (23). Similarly, we found that although using a single neu-specific mAb was able to prevent tumor formation when used in combination with vaccine in a proportion of neu-N mice, this treatment was not effective at eradicating pre-established tumor in these mice. Therefore, we sought to investigate whether combining both mAbs 7.9.5 and 7.21.2 as a mixture in sequence with the neu-targeted vaccine would augment antitumor immunity and further enhance in vivo tumor rejection. In both the treatment and the prevention settings, the 7.9.5-7.21.2 mixture combined with the control vaccine produced significant delays in tumor formation when compared with mice given the control vaccine and control IgG (Fig. 1⇑C, p < 0.0001; Fig. 2⇑C, p = 0.0005). Neu-specific mAb mixture given in the context of a control vaccine was sufficient to prevent tumor growth in a small proportion of animals (Fig. 1⇑C). In the treatment setting, no animals in this treatment group remained tumor free for longer than 40 days. Strikingly, the combination of vaccine with the Ab mixture was highly efficacious in both the prevention and treatment settings. In prevention experiments, animals given neu-specific mAb mixture in the context of a 3T3-neu/GM vaccine had a significantly greater tumor-free survival than those given either mAb mixture or vaccine alone (p = 0.013 and 0.0003, respectively). Furthermore, 70% of the animals in this treatment group remained tumor free beyond the 60-day endpoint of the experiment (Fig. 1⇑C). Similarly, the combination of neu-specific mAb mixture and neu-targeted vaccination was effective in the eradication of an existing tumor burden. In the treatment setting, mAb mixture given in sequence with the neu-targeted vaccine also significantly improved tumor-free survival over either intervention alone (p = 0.01 and 0.006, respectively). Remarkably, complete eradication of tumor burden was seen in 30% of animals given neu-specific mAb mixture plus neu-targeted vaccination.

The antitumor effect induced by the combination of neu-targeted vaccine and neu-specific Ab mixture in neu-N mice requires both CD4+ and CD8+ T cells

Previously, we showed that both CD4+ and CD8+ T cells are necessary for vaccine-mediated neu-expressing tumor growth inhibition in both neu-N mice and nontolerant FVB mice (17). To assess the T cell dependence of the observed enhancement of vaccine-mediated antitumor immunity by neu-targeted mAb mixture we depleted either CD4+ or CD8+ T cell subsets after vaccine administration. Mice received a neu-targeted vaccine (or control vaccine) to prime Ag-specific adaptive immunity followed 2 wk later by depletion of CD4+ or CD8+ T cells using Abs GK1.5 and 2.43, respectively (or a mock depleting Ab, PK136). T cell depletions were verified by FACS and maintained throughout the course of the experiment. On the day of tumor challenge (21 days postvaccination), the mice were given either the 7.21.2-7.9.5 mixture or an IgG control Ab. Mice that were mock depleted and treated with the vaccine plus the control IgG survived longer than mock depleted animals given a control vaccine and control IgG (Fig. 3⇓A, p = 0.0007). This vaccine-mediated inhibition of neu-expressing tumor growth was abrogated when either CD4+ or CD8+ T cells were depleted (Fig. 3⇓A, p = 0.005 and 0.0006, respectively). These results are consistent with previous data (17). In mice given the mock depletion and treated with control vaccine plus neu-specific mAb mixture (i.e., neu-specific Ab alone), there is a statistically significant difference in tumor-free survival relative to mock depleted mice given control vaccine and control IgG (Fig. 3⇓B; p = 0.0007). However, this Ab-mediated antitumor effect is completely abrogated with the depletion of CD4+ T cells (p = 0.001) and greatly diminished in the absence of CD8+ T cells (p = 0.04) (Fig. 3⇓B). This indicates that the antitumor effect of the neu-specific mAb in neu-N mice (an immunocompetent host), even in the absence of neu-specific vaccination, is largely T cell dependent. Finally, as was seen in both the treatment and prevention settings, mock depleted mice given neu-targeted vaccine plus neu-specific mAb mixture demonstrated significant tumor protection relative to mock depleted mice treated with either the control vaccine plus neu-specific Ab mixture or neu-targeted vaccine plus control IgG (p < 0.0005). This treatment combination prevented neu-expressing tumor growth in 80% of neu-N mice (Fig. 3⇓C). However, depletion of either CD4+ or CD8+ T cells resulted in a nearly complete abrogation of the antitumor effect of neu-targeted vaccine plus neu-specific mAb mixture (p = 0.0009 and 0.0003, respectively), demonstrating that the induced tumor eradication is T cell dependent.

FIGURE 3.
FIGURE 3.

The enhancement of vaccine (Vac) mediated antitumor immunity by neu-specific mAbs is T cell dependent. Neu-N mice received neu-targeted vaccine (3T3-neu/GM) or control (Ctrl) vaccine (3T3/GM) followed 2 wk later by the initiation of T cell subset depletion. Animals were depleted of CD4+ or CD8+ T cells using GK1.5 or 2.43 Ab, respectively, as described in Materials and Methods. Undepleted mice received an irrelevant Ab (PK136). A, Effects of depletion on treatment with neu-targeted vaccine plus control Ab (i.e., vaccine alone). Kaplan-Meier survival curves are shown for undepleted mice receiving a neu-targeted (3T3-neu/GM) vaccine plus control Ab (•) vs CD4- or CD8-depleted mice (▿ and □, respectively). ♦, Tumor-free survival of mice given control (3T3-GM) vaccine plus control Ab. B, Effects of depletion on treatment with control vaccine plus neu-specific Ab mixture (cocktail) (i.e., Ab alone). Kaplan-Meier survival curves are shown for undepleted mice receiving a control vaccine plus neu-specific Ab mixture (•) vs CD4- and CD8-depleted mice (▿ and □, respectively). ♦, Tumor-free survival of mice given control vaccine plus control antibody. C, Effects of depletion on treatment with neu-targeted vaccine plus neu-specific Ab mixture, (i.e., vaccine plus Ab). Kaplan-Meier survival curves are shown for undepleted mice receiving a neu-targeted vaccine plus neu-specific Ab mixture (•) vs CD4- and CD8-depleted mice (▿ and □, respectively). ♦, Tumor-free survival of mice given control vaccine plus control Ab.

Treatment with the neu-targeted vaccine in sequence with the neu-specific Ab enhances neu-specific CD8+ T cell frequency

Machiels et al. (25) have demonstrated previously a correlation between in vivo efficacy of neu-specific vaccination and increased neu-specific CD8+ T cells as determined by ELISPOT. Here we have shown a requirement for both CD4+ and CD8+ T cells for in vivo tumor-protection by neu-targeted vaccination and neu-specific adoptive Ab therapy. In light of this T cell dependence and the previously reported results, we sought to determine by ELISPOT assay whether the observed improvement in tumor protection seen in neu-N mice treated with neu-targeted vaccine in sequence with neu-specific Ab correlated with an increase in neu-specific CTL frequency. For these experiments, mice were vaccinated on day −14 and a neu-expressing mammary tumor challenge on day 0. At the time of tumor challenge, the mice were given the control IgG, the 7.9.5, or the 7.21.2 neu-specific mAbs, or a mixture of both of these neu-specific mAbs. All animals were sacrificed on day 7 after challenge, and their spleens were removed and harvested for ELISPOT analysis. Mice that received neu-targeted vaccine alone showed a 2-fold increase in the frequency of neu-specific CD8+ T cells compared with mock treated mice (Fig. 4⇓; p = 0.009). Mice treated with neu-targeted vaccine plus the 7.21.2 Ab showed a 1.5-fold increase in neu-specific CD8+ T cells compared with mice given the 7.21.2 mAb alone (p = 0.02). However, there was no difference in neu-specific CD8+ T cell frequency in the vaccine plus 7.21.2-treated mice compared with mice given vaccine alone (Fig. 4⇓). In contrast, mice treated with neu-targeted vaccine and the 7.9.5 mAb produced more neu-specific CD8+ T cells than mice given either vaccine alone or the 7.9.5 Ab alone (Fig. 4⇓, p = 0.002 and p < 0.0001, respectively). Mice treated with neu-targeted vaccine plus the 7.21.2-7.9.5 mixture also showed an increased frequency of neu-specific CD8+ T cells. With this treatment, there was a 2-fold increase over both the vaccine only treated group (p = 0.0002, 4-fold over control-treated animals) and the mAb mixture only treatment group (p < 0.0001). Interestingly, treatment with either Ab 7.9.5 alone or the neu-specific mAb mixture alone yielded neu-specific CD8+ T cell frequencies that were equivalent to those induced with the vaccine alone. Mice treated with vaccine in sequence with the mAb mixture did not yield statistically significant increases in neu-specific CD8+ T cells over those treated with the vaccine in combination with only the mAb 7.9.5.

FIGURE 4.
FIGURE 4.

TNF-α ELISPOT demonstrates that vaccine (vac) Ab interaction improves Ag-specific CD8+ T cell priming. Neu-N mice received either neu-targeted (3T3-neu/GM) or control (ctrl; 3T3/GM) vaccine followed by NT2 challenge (1 × 105 tumor cells) 2 wk later. Mice were given either neu-specific Ab or isotype control Ab (100 μg) on the day of tumor challenge. Two weeks after tumor challenge, CD8+ T cells were isolated from these mice and pooled according to the treatment group. The frequency of neu-specific CD8+ T cells secreting TNF-α in response to neu-expressing tumor cells was assessed by ELISPOT as described in Materials and Methods. Data represent the triplicate measurement of the number of spots counted for each treatment group, less the spots counted for T cells alone. Individual measurements are indicated (○) with a horizontal bar representing the mean value for that group. The data are compiled from at least two replicate experiments for each treatment group.

Treatment of neu-expressing tumor cells with the neu-specific Abs 7.9.5 and 7.21.2 increases ubiquitination of neu

Ubiquitination is an integral step in the degradation of cellular proteins and the resultant presentation of these peptides on MHC class I molecules. Therefore, we chose to use ubiquitination of the neu protein as a surrogate marker of degradation and presentation of MHC I-restricted neu-derived peptides. Improved Ag presentation on the surface of neu-expressing tumor cells would define one potential contributing factor toward the improved tumor-free survival observed in mice treated with neu-specific Ab in sequence with the neu-targeted vaccine. Klapper et al. (27) showed previously that treatment of neu-expressing cell lines with the neu-specific Ab L26 led to increased neu protein turnover and concomitant augmentation of ubiquitination. Here we examined whether or not similarly enhanced ubiquitination also occurred with the neu-specific mAbs 7.9.5 and 7.21.2. To assess this, NT2 cells were incubated for up to 60 min with control IgG, the mAbs 7.21.2 or 7.9.5, or both 7.9.5 and 7.21.2. Geldanamycin, which has been shown in several studies to increase protein degradation and subsequent ubiquitination of neu was used as a positive control (28, 29). After exposure to these experimental conditions for 1, 5, 15, 30, or 60 min, the cells were lysed and immunoprecipitated for ubiquitin, and equivalent amounts of protein were loaded onto a polyacrylamide gel. We then performed a Western blot for neu using a neu-specific polyclonal rabbit Ab. The data demonstrated that incubating neu-expressing tumor cells with Ab 7.9.5 increased the levels of ubiquitinated neu over the control IgG and the 7.21.2 mAb (Fig. 5⇓). Furthermore, incubation of the neu-expressing cell line with both 7.9.5 and 7.21.2 led to a more rapid ubiquitination of neu than that observed with either the control IgG-treated cells or the individual Ab-treated cells. Consistent with results reported with other neu-expressing cell lines (28, 30), treatment of NT2 cells with geldanamycin resulted in rapid and sustained ubiquitination of the neu protein (28, 30).

FIGURE 5.
FIGURE 5.

Treatment of neu-expressing tumor cells with neu-targeted Ab enhances ubiquitination of neu protein. NT cells were incubated with 50 μg/ml control Ab (A), 7.21.2 (B), 7.9.5 (C), 7.9.5-7.21.2 mixture (cocktail) (D), or 3 μM geldanamycin (as a positive control; E), for 1, 15, 30, or 60 min. The tumor cells were then lysed and immunoprecipitated for ubiquitin, and Western blot was performed using a neu-specific Ab. Data are representative of two replicate experiments with virtually identical outcome.

Incubation of neu-expressing tumor cells with neu-specific Abs enhances CTL-mediated lysis

Spiridon et al. (24) demonstrated that the neu-specific mAb trastuzumab (Herceptin) was able to augment CTL-mediated tumor lysis. Similarly, Castilleja et al. (30) found that incubation of the neu-expressing human tumor cells with geldanamycin also augmented CTL-mediated lysis. The enhanced lysis observed in these studies was attributed to increased ubiquitination and degradation of neu (30). As exposure of neu-expressing tumor cells to the combination of 7.9.5 and 7.21.2 improved ubiquitination in a manner analogous to that of geldanamycin, it seemed plausible that exposure of neu-expressing tumor cells to these Abs may also improve CTL function. To address this, NT2 cells were incubated with the mAbs 7.21.2 or 7.9.5, a mixture of these neu-specific mAbs, control IgG, or geldanamycin (as a positive control) for 4 h. The tumor cells were labeled with 51Cr and incubated with a neu-specific CD8+ T cell line developed in our laboratory (17). Incubation of the target cells with the mAb 7.21.2 did not augment lysis compared with tumor cells that were treated with control IgG (Fig. 6⇓), and was equivalent to the Fneu-CTL-mediated lysis of untreated NT cells (data not shown). The incubation of the targets with the 7.9.5 mAb resulted in a modest increase in the lysis of NT2 cells, reaching statistical significance at E:T ratios of 1:5 and 1:17 (p < 0.05 for both). However, incubation of the NT2 cells with 7.9.5 plus 7.21.2 resulted in a statistically significant increase in lysis over targets treated with control IgG, 7.21.2, or 7.9.5 alone at all E:T ratios. The percent lysis of the mixture-treated NT2 cells was nearly equivalent to that seen with geldanamycin. Furthermore, the results of these in vitro studies parallel the in vivo antitumor treatment efficacy achieved when these neu-specific mAb preparations were combined with neu-targeted vaccination in neu-N mice.

FIGURE 6.
FIGURE 6.

Treatment with neu-specific Ab enhances Ag-specific CD8+ T cell effector function. NT2 cells were incubated with a total of 50 μg/ml control Ab (▵), 7.9.5 (×), 7.21.2 (•), a 7.9.5-7.21.2 mixture (□), or geldanamycin (○) for 4 h. The Ab was replenished, and these cells were labeled with 51Cr and then incubated with a neu-specific CD8+ T cells clone at E:T ratios varying from 1:5 to 1:50. The mean ± SD of triplicate measurements of neu-specific lysis are shown. Data are representative of two replicate experiments with virtually identical outcome.

Discussion

This report describes three new findings that define a potential new therapeutic approach for the treatment of patients with neu-expressing carcinomas, and it provides a new mechanism by which a vaccine given in conjunction with passively transferred Ab can lead to the generation of potent antitumor immunity in a tolerant host. First, these data demonstrate that the combination of a GM-CSF-secreting, neu-targeted whole cell vaccine in sequence with a neu-specific mAb significantly enhances tumor protection compared with either modality alone. Importantly, treatment of mice with the vaccine in combination with a mixture of two distinct neu-specific mAbs was significantly more effective at rejecting tumors in vivo than the combination of vaccine and any single neu-specific mAb tested. Second, both CD4+ and CD8+ T cells were required for the antitumor effect induced by neu-targeted vaccination given in sequence with passively transferred neu-specific mAbs. Interestingly, both CD4+ and CD8+ T cells were also required for the antitumor effect of neu-specific Ab administered as a single agent. Third, the augmented in vivo antitumor response induced by the combination of passively administered tumor-specific mAbs and vaccine correlates with increased CD8+ T cell frequency and enhanced lytic function in vitro.

To our knowledge, this is the first report to demonstrate synergy between mAb and a T cell targeted whole cell vaccination. However, previous studies in neu-N mice have demonstrated a requirement for both neu-specific CTL and neu-specific IgG for the eradication of a neu-expressing tumor burden (21). These studies have also demonstrated that after vaccination with 3T3-neu/GM there is induction of neu-specific CD4+ and CD8+ T cells (17, 25), but not neu-specific IgG (21). Neu-N mice vaccinated in this way demonstrate delayed tumor growth kinetics in both the treatment (25) and prevention (17) settings, but all of these mice ultimately develop tumors. Here, we show that the passive administration of neu-specific mAb in neu-N mice, at the time of tumor inoculation or 3 days thereafter, results in a similar delay in tumor growth but not tumor eradication. This minimal antitumor effect was seen whether mice were given a single mAb or a mixture of two neu-specific mAbs. However, the combination of neu-targeted vaccination and the passive infusion of neu-specific mAb yielded greatly improved efficacy relative to either vaccine or mAb given as a single therapeutic agent. Furthermore, Vasovic et al. (31) have demonstrated a similar requirement for the infusion of both tumor-specific mAb and CTL in a murine EL-4 lymphoma model, suggesting that the enhanced antitumor immunity obtained by combining passively infused Ab and vaccine-induced cellular immunity may also be relevant to other tumor-associated Ags.

In this report, we also show that the 7.9.5 and 7.21.2 neu-specific mAbs differ in the degree to which they interact favorably with neu-targeted vaccination, despite the fact that these two mAbs have a similar growth-inhibitory effect on neu-expressing cells grown in vitro (22, 23). The combination of 3T3-neu/GM with 7.21.2 did not increase tumor-free survival over that seen for animals given 7.21.2 plus control vaccine. In contrast, the combination of 7.9.5 with neu-targeted vaccine resulted in a significant increase in tumor-free survival (relative to 7.9.5 or vaccine alone) in both the prevention and treatment settings. However, the most potent antitumor effect was seen in mice that received neu-targeted vaccination in combination with a mixture consisting of both 7.9.5 and 7.21.2. In their original description of these neu-specific mAbs, Drebin et al. (23) observed increased tumor growth inhibition in nude mice with a combination of two mAbs that recognized different epitopes on the neu ECD. Similarly, Spiridon et al. (24) demonstrated an enhanced antitumor effect against neu-expressing tumor cell lines both in vitro and in SCID mice using a mixture of mAbs specific for the human neu ECD, relative to a single mAb. The use of multiple neu-specific mAbs increased not only tumor growth inhibition, but also the induction of apoptosis, complement-dependent cell cytotoxicity (CDCC), and ADCC (24). The data presented here provide the first evidence that an oligoclonal neu-specific mAb preparation can also enhance T cell-mediated adaptive immunity to tumor.

In the current investigation, we also confirm previous studies that vaccine-mediated antitumor immunity in neu-N mice is dependent on the presence of both CD4+ and CD8+ T cells (21). Of particular interest, however, was the observation that Ab-mediated antitumor immunity (in the absence of neu-specific vaccination) was completely abrogated in CD4+ T cell-depleted animals and greatly diminished in the absence of CD8+ T cells. The inhibition of neu-mediated signal transduction using neu-specific mAbs has been demonstrated in numerous systems (reviewed in Refs. 32 and 33). Trastuzumab (Herceptin), a Food and Drug Administration-approved humanized mAb specific for human neu that has demonstrated a survival benefit in women with neu-overexpressing breast cancer, exerts its antitumor effect in part through the inhibition of the phosphatidylinositol-3 kinase and Akt signaling cascades (34). Furthermore, trastuzumab, as well as other neu-specific mAbs, can sensitize neu-expressing tumor cells to the induction of apoptosis (24, 29, 35, 36, 37). In addition to these direct effects of Ab-tumor interaction, neu-specific Abs can exert an antitumor effect through the action of innate effector mechanisms such as CDCC and ADCC (22, 24, 38). In fact, Clynes et al. (39) demonstrated that much of the antitumor effect of trastuzumab is dependent on the presence of FcγR-bearing myeloid cells, using FcγR knockout mice on a nude background. Here we have demonstrated that in an immunocompetent host, the antitumor effect of neu-specific mAb, both alone and in combination with neu-targeted vaccination, requires the presence of both CD4+ and CD8+ T cells. It remains to be determined whether the antitumor effects of trastuzumab in patients is similarly T cell dependent.

In this report, we also show that the in vivo antitumor effects of the vaccine and the mAb mixture were associated with an increase in the number and function of neu-specific CD8+ T cells. Other investigators have demonstrated a role for Ab in inducing dendritic cell uptake and processing of Ag and subsequent enhancement of T cell priming (40, 41, 42). Rafiq et al. (41) recently reported that targeting OVA Ab immune complexes to dendritic cells resulted in efficient priming of both CD4+ and CD8+ effector T cells, leading to protection from an OVA-specific tumor challenge. Intact MHC class I and class II processing, as well as FcγR-bearing cells, was required for the observed protection. A similar mechanism of FcγR-mediated Ag acquisition and presentation may also account for the increased CD8+ T cell frequency observed in this study. However, generating neu-specific CD8+ T cells alone does not necessarily predict tumor rejection in this model. For example, there is no difference in the neu-specific CD8+ T cell frequency in mice given vaccine plus the 7.9.5 mAb compared with that in mice receiving vaccine plus mAb. However, these two treatment groups differ greatly in overall in vivo antitumor efficacy, given that the combination of neu-targeted vaccine and neu-specific mAb mixture was the only treatment combination to induce antitumor immunity potent enough to eradicate established tumors in neu-N mice. These observations also indicate that efficient tumor destruction may have further requirements beyond CD8+ T cell induction. One possible mechanism for the increased survival seen in mice given vaccine plus Ab mixture that was addressed in the present studies lies in the enhancement of CTL effector function. zum Buschenfelde et al. (43) demonstrated that the treatment of human tumor cells with Herceptin augmented the CTL-mediated lysis of these cells in a chromium release assay by ∼15–20%. Similarly, we have demonstrated that treating neu-expressing tumor cells with Ab mixture significantly enhances lysis by a neu-specific CTL clone over control treated cells. The enhancement of CTL function seen was equivalent to that observed for neu-expressing tumor cells treated with geldanamycin. This agent has been shown to destabilize neu on the cell surface by preventing the binding of factors such as heat shock protein 90 and glucose-related peptide 94 (28, 29, 44, 45). Castilleja et al. (30) found that incubation of the neu-expressing human tumor cells with this agent resulted in the augmentation of CTL-mediated lysis. They were able to attribute this effect to the increased degradation and subsequent ubiquitination of neu. Our data corroborate these findings. Interestingly, these data parallel the observed in vivo antitumor effect. This ability of neu-specific Ab to enhance CD8+ T cell effector function may be of particular importance in the context of host tolerance to neu because tolerance is thought to lead to the development of a lower avidity Ag-specific T cell repertoire.

In summary, we have utilized a clinically relevant tumor tolerance model to evaluate the efficacy of combined passive and active neu-targeted immunotherapy against neu-expressing mammary tumors. The data indicate that vaccine-mediated antitumor immunity is significantly enhanced in the presence of passively infused neu-specific mAbs. Several previously unrecognized mechanisms may account for the improved efficacy observed with the combination in vivo. Collectively, these observations suggest that similarly increased efficacy could be obtained by combining neu-targeted vaccination and neu-specific Abs such as trastuzumab (Herceptin) in patients with neu-expressing cancers.

Acknowledgments

We gratefully acknowledge Dr. Mark Greene (University of Pennsylvania) for generously providing mAbs 7.9.5 and 7.21.2.

Footnotes

  • ↵1 This work was supported by a National Institutes of Health T32 grant (to M.E.W.); an American Head and Neck Society Pilot Grant (to M.E.W.); National Institutes of Health/National Cancer Institute of Allergy and Infectious Diseases Grant 5T32AI07247-21 (to A.M.E.); Department of Defense Grant DAMD17-01-1-0282 (to A.M.E.); Maryland Cigarette Resitution Fund M020216 (to L.A.E.); Johns Hopkins University Clinician Scientist Award (to L.A.E.); National Institutes of Health/National Cancer Institute National Cooperative Drug Discovery Group Grant 2U19CA72108 (to E.M.J.); Breast Cancer Research Foundation Grant (to E.M.J.); Department of Defense Grant DAMD17-01-1-0282 (to A.M.E.); American Cancer Society Research Scholar Grant RSG-01-080-01-LIB (to R.T.R.); Susan G. Komen Foundation Grant BCTR000068 (to R.T.R.); and a Pilot Project Grant from National Institutes of Health Specialized Programs of Research Excellence in Breast Cancer (P50 CA 88843; to R.T.R.).

  • ↵2 Address all correspondence and reprint requests to Dr. R. Todd Reilly, Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins 1650 Orleans Street, CRB Room 4M08, Baltimore, MD 21231. E-mail address: reilly{at}jhmi.edu

  • ↵3 Abbreviations used in this paper: neu, HER-2/neu; ADCC, Ab-dependent cell cytotoxicity; CDCC, complement-dependent cell cytotoxicity; ECD, extracellular domain; neu-N, HER-2/neu-transgenic.

  • Received March 21, 2003.
  • Accepted June 18, 2003.

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