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The Journal of Immunology, 2001, 167: 3367-3374.
Copyright © 2001 by The American Association of Immunologists

Complementary Antitumor Immunity Induced by Plasmid DNA Encoding Secreted and Cytoplasmic Human ErbB-21

Marie P. Piechocki*,{dagger}, Shari A. Pilon*,{ddagger} and Wei-Zen Wei2,*,{ddagger}

* Karmanos Cancer Institute, Departments of {dagger} Otolaryngology, and {ddagger} Immunology and Microbiology, School of Medicine, Wayne State University, Detroit, MI 48201


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A plasmid DNA was constructed to encode the N-terminal 505 aa of human ErbB-2 (E2, HER-2/neu) and designated as secreted ErbB-2 (secE2). Recombinant secE2 protein was detected in the transfected cells and was secreted as an 80-kDa glycoprotein. Vaccination of BALB/c mice with secE2 DNA induced both IgG1 and IgG2a ErbB-2-specific Abs and protected ~90% of mice against mouse mammary tumor D2F2, which expressed human ErbB-2 (D2F2/E2). The efficacy of secE2 vaccine was comparable with that of wild-type ErbB-2 DNA, which encodes the entire 1258 aa of ErbB-2 protein, induced only IgG2a E2-specific Abs, and stimulated greater CTL activity. Immune lymphocytes were stimulated in vitro with irradiated 3T3 cells, which expressed ErbB-2, Kd, and B7.1. CTL activity was measured by the lysis of E2-positive target cells and by intracellular IFN-{gamma} production. To enhance CTL activation, mice were immunized with a combination of secE2 and cytoplasmic E2 (cytE2); the latter encodes the 1258-aa ErbB-2 protein that was released into the cytoplasm upon synthesis. Significant increase in CTL activity was demonstrated after mice were immunized with the combined vaccines and all mice were protected from D2F2/E2 tumor growth. Therefore, secE2, which induced Th2 Ab and weak CTL, conferred similar protection as E2, which induced Th1 Ab and strong CTL. Combined vaccination with secE2 and cytE2 resulted in Th2 Ab, strong CTL, and the most effective protection against tumor growth. The strategy of coimmunization with DNA that direct Ags to different subcellular compartments may be adapted as appropriate to optimize immune outcome.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Wild-type human ErbB-2 proto-oncogene (E2)3 or HER-2/neu, a member of the epidermal growth factor receptor family with tyrosine kinase activity, is overexpressed in several human cancers, including breast, ovarian, and lung cancers (1, 2, 3, 4, 5, 6). Overexpressed ErbB-2 is associated with aggressive disease and poor prognosis (7, 8, 9). ErbB-2-specific Ab and T cells are detected in breast and ovarian cancer patients, and ErbB-2 is recognized as a target of immunotherapy (10, 11, 12, 13, 14, 15, 16).

A panel of human E2 DNA vaccines has been generated in our lab (17). E2A encodes full-length ErbB-2 with a single amino acid substitution to replace ATP-binding lysine (K) with alanine (A) and to eliminate tyrosine kinase activity. Cytoplasmic ErbB-2 (cytE2) has a truncated endoplasmic reticulum (ER) signal sequence and encodes a full-length ErbB-2 that is released into the cytoplasm upon synthesis. CytE2A is cytE2 with the K to A mutation. These mutations were created to eliminate potential oncogenic activity of the gene product if it were to be administered to patients (E2A and cytE2A) and to enhance proteasomal processing and degradation in MHC I-associated peptides (cytE2 and cytE2A). No such mutations have been observed in the human HER-2 oncogene, although recent data suggest the presence of splice variants (18). The oncogenic activity of HER-2 with respect to cancer progression appears to be from its overexpression and association with other ErbB family members that further enhance mitogenic signaling (19, 20). Overexpression and/or inappropriate expression of ErbB-2 are critical for focusing immune effector cells at E2 presented as a tumor-specific Ag.

In our hands, DNA vaccination with E2, E2A, cytE2, or cytE2A resulted in approximately 90, 60, 30, and 10% protection against D2F2/E2 tumor, respectively. These recombinant proteins all contained the same, complete structural sequence of E2, but their immunogenicity was affected by the subcellular localization, membrane stability, and signaling activity of the recombinant protein. Although native E2 is by far the strongest vaccine, the oncogenic activity prohibits its use as a vaccine for patients. A single residue substitution in E2A greatly reduced the efficacy. The mechanism for this drastic change is not yet defined. On the other hand, the weak immunogenicity of the cytoplasmic variants may be due to inadequate CD4 T cell activation. Consistent with this notion, cytE2 and cytE2A induced excellent antitumor activity when administered with plasmids encoding either GM-CSF or IL-2.4 E2-specific CTL, but not Abs, were detected in these mice, supporting the presentation of MHC I epitopes from cytE2 to CTL.

A number of HLA-restricted HER-2 peptides have been identified that are capable of generating HER-2/neu-specific CTL with cytotoxic activity against autologous and allogeneic HER2-expressing tumors (12, 21, 22, 23, 24). These HER-2-specific CTL have been derived from several sources, including mixed tumor cell/lymphocyte cultures and stimulation of PBL with autologous and peptide-loaded APC from both patients and human HLA transgenic mice (25, 26, 27). The immunodominant peptides identified in these studies are derived from amino acid sequences from the extracellular domain (ECD) and intracellular domain of HER-2. Similar peptides have been identified that are capable of inducing HER-2-specific CTL and generating protective immunity against HER-2-expressing murine tumors. Several peptides have been shown to serve as common tumor rejection Ag in mouse (H-2Kd) and human (HLA-A2402 and HLA-A24) tumors that express human HER-2 (28, 29). Therefore, there is still some debate as to which epitopes are most important for tumor rejection in murine and human models and the way in which to best present these Ags to the immune system for effective priming.

Activation of B cells and Abs may hinder CTL activation and is not desirable in many tumor systems (30, 31). The efficacy of humanized anti-ErbB-2 mAb Herceptin in advanced breast cancer patients indicates anti-ErbB-2 Abs as antitumor effectors (32, 33, 34, 35). It may be advantageous to elicit both Ab and CTL response by E2 vaccination. In this study, we generated and tested a DNA construct encoding a secreted ErbB-2 (secE2) containing the N-terminal 505 aa. SecE2 induced significant anti-ErbB-2 Ab and antitumor effect even though it does not contain the cytoplasmic domain and the associated T cell epitopes. Immunization with a combination of secE2 and cytE2 resulted in equivalent CTL activation and superior antitumor activity as compared with native E2 vaccine. More recent findings of the presence of shed ectodomain fragment of the E2 ECD in cancer patients (36, 37, 38) present an additional variable to consider when designing immunotherapies that target E2 as a tumor Ag. One study does consider the immunological consequences of the shed receptor (39), but further studies need to be developed to describe the impact of the shed receptor on autoantibody induction, immune effector priming, induction of anergy, and peripheral tolerance. Therefore, by combining DNA vaccines designed to activate different effectors, it is possible to achieve maximal immune response by activating both humoral and cellular immune responses.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Construction of secE2

A 1.5-kb NcoI/HinCII fragment containing the ER signal peptide (aa 1–21) and the amino-terminal aa 1–505 of the mature protein (encoding most of the ECD) was isolated from pCMVhuErbB-2 expression vector, and the ends were made blunt by treatment with T4 DNA polymerase. This fragment was ligated into BamHI/HinDIII-digested, Klenow-modified (blunt) pSecTag2B (Invitrogen, San Diego, CA). The orientation and integrity of the inserted sequences were screened by detailed restriction analysis and sequencing. This clone represents a fusion of the Ig{kappa} leader sequences (for Golgi targeting of the secretory signal) in frame with the 5' end of ErbB-2 ER signal peptide and first 505 aa of the E2 ECD with a 10-aa myc-tag epitope and a 6-aa polyhistidine tail. Expression of the protein was driven by the CMV promoter for high level expression in mammalian cells and can be selected with Zeocin, which is expressed under the elongation factor 1{alpha} promoter in the plasmid vector.

Cell lines

Mouse mammary tumor line D2F2 was derived from a spontaneous mammary tumor that arose in a BALB/c hyperplastic alveolar nodule line D2 (40). This tumor cell line expresses a low, but detectable level of the endogenous mouse ErbB-2 receptor. The cell line was maintained in vitro in DMEM supplemented with 5% heat-inactivated FBS (Sigma, St. Louis, MO) and 5% cosmic calf serum (HyClone, Logan, UT), 10% NCTC 1099 medium (Sigma), 2.5 mM 2-ME, 0.5 mM sodium pyruvate, 2 mM L-glutamate, 0.1 mM MEM nonessential amino acids, 100 U/ml penicillin, and 100 µg/ml streptomycin.

Transfection of D2F2 cells

LipofectAMINE Plus Reagent was purchased from Life Technologies (Gaithersburg, MD). D2F2 cells were cotransfected with XmnI-linearized test plasmid DNA, including the E2 expression vectors: pCMV/E2, pSecTagE2, and selectable plasmid DNA, pRSV/neo, at a 1:10 ratio. Transfected cells were passaged into selection medium containing 800 µg/ml G418 (Geneticin; Life Technologies) at 48 h after transfection. Individual colonies were expanded and screened for expression of the recombinant proteins by flow cytometry. Positive colonies were further cloned by limiting dilution.

Generation of 3T3 APCs

BALB/c NIH3T3 fibroblasts were cotransfected with pCMVhuErbB-2 and pRSVneo-Kd (provided by S. Ostrand-Rosenberg, University of Maryland, Baltimore, MD) at a 10:1 ratio using LipofectAMINE Plus Reagent (Life Technologies) and selected in medium containing 800 µg/ml G418 and cloned by limiting dilution. Clones expressing high, stable levels of both genes were used for a second round of cotransfection with plasmid pEXV3-murine B7.1 (provided by S. Ostrand-Rosenberg, University of Maryland) and pcDNA3.1-Zeo at a 10:1 ratio and cloned by limiting dilution under selection with 800 µg/ml Zeocin. Positive clones were identified by flow cytometry with mAb TA-1 (human ErbB-2), hybridoma supernatant SF1.1-1 (MHC I Kd), FITC-conjugated anti-murine B7.1 (CD80; Caltag Laboratories, Burlingame, CA), and hybridoma supernatant HB26 (MHC II IAd). Cells were gradually weaned from coselection with G418 and Zeocin and maintained stable expression of all genes in medium containing G418.

Flow cytometric analysis

mAbs TA-1 (AB-5) and 3B5 (AB-3), which recognize the ECD and cytoplasmic domain of ErbB-2, respectively, were purchased from Oncogene Research Products (Cambridge, MA). FITC- or PE-conjugated goat anti-mouse, anti-human, or anti-rabbit IgG was used as the secondary Ab (Jackson ImmunoResearch Laboratories, West Grove, PA). To detect the cytoplasmic domain of ErbB-2, cells were monodispersed, washed with PBS, then fixed and permeabilized with ice-cold 100% histograde methanol on ice for 10 min. Cells were washed thoroughly with PBS and blocked on ice in 2% calf serum at 4°C for 20 min. The fixed and permeabilized cells were stained with mAb 3B5 and FITC- or PE-conjugated secondary Ab. Normal mouse Ig or isotype-matched mAb were used as the negative controls. Flow cytometric analysis was performed with a FACScan or FACSCalibur (BD Biosciences, Mountain View, CA). Hybridoma supernatant HB26 recognizes MHC II IAd and IAb (American Type Culture Collection, Manassas, VA; mouse IgG hybridoma). Hybridoma SF1.1-1 produced mAb, which recognized H2-Kd. FITC-conjugated rat anti-mouse B7.1 (CD80) was purchased from Caltag Laboratories.

Immunoprecipitation and Western blot analysis

Supernatant or lysates were prepared from monolayer cultures. For lysates, cells were washed twice with ice-cold PBS, harvested by mechanical scraping, pelleted, and resuspended in ice-cold lysis buffer containing protease inhibitors (50 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mM PMSF, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM Na3VO4, and 1 mM NaF). Culture supernatant was purified by affinity chromatography using mini nickel-NTA-charged minispin columns, washed, and eluted according to the manufacturer’s instructions (Qiagen, Chatsworth, CA). The eluted his-tag protein was diluted with lysis buffer. Epitope-specific mAbs were added and incubated for 1–2 h before adding protein A/G plus agarose and rotated overnight at 4°C.

To measure intracellular secE2, secE2-transfected cells were lysed with lysis buffer, incubated on ice for 60 min with occasional mixing. After clearing the samples by centrifugation at 16,000 x g for 10 min at 4°C, protein concentrations in the supernatant were determined with a modified Lowry assay (Bio-Rad, Hercules, CA). ErbB-2 protein was immunoprecipitated from the cell lysates by incubation with mAb TA-1 at 4°C for 16–18 h. Immune complexes were recovered by incubation with protein A/G plus agarose (Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C for 16–18 h. The agarose beads were subjected to centrifugation and washed twice with lysis buffer. Proteins were eluted in 1x sample buffer and boiled for 3 min before fractionation in 6% SDS-PAGE. Proteins were electrotransferred to Immobilon-P (Millipore, Bedford, MA) polyvinylidene difluoride membranes. Membranes were fixed with methanol, then rehydrated and blocked overnight at 4°C in TBST buffer (10 mM Tris-HCl, pH 8, 50 mM NaCl, 0.1% Tween 20) with 1% BSA. ErbB-2 protein was detected by immunoblotting with mAb clone 42, specific for an epitope between residues 182 and 373 of the ECD of ErbB-2 (Transduction Laboratories, Lexington, KY). Primary Abs were used at the concentration of 0.2 µg/ml. HRP-conjugated goat anti-mouse IgG at 0.5–1 µg/ml was the second Ab (Transduction Laboratories). Immunoblots were developed with ECL reagents (Amersham, Arlington Heights, IL) and Kodak-MR film.

Inhibition of tumor growth by DNA vaccination

Female BALB/c mice (6 wk of age) were obtained from Charles River Laboratory (Frederick, MD) or The Jackson Laboratory (Bar Harbor, ME). Groups, consisting of 8–10 mice, were vaccinated three times, at 14-day intervals. Vaccines were administered i.m. and consisted of 100 µg plasmid DNA suspended in 0.1 cc 0.9% sterile saline. Approximately 0.05 cc was injected into each quadriceps. The plasmid DNAs used were pCMV5, pCMVE2, pCMVcytE2, or pSecTagE2. For the group receiving a covaccination, a mixture of 100 µg pCMVcytE2 and 100 µg pSecTagE2 was suspended in 0.1 cc and used for each vaccination. One week after the third immunization, animals were anesthetized and a blood sample was taken by retro-orbital puncture. One week later, the mice were challenged s.c. in the right flank with 2 x 105 pCMVE2-transfected mouse mammary tumor cells, D2F2/E2. Expression of ErbB-2 protein by the challenging tumor was verified by flow cytometry the day before tumor cell injection. Tumor growth was monitored by weekly palpation, and measurements were taken in two dimensions with calipers.

In vitro stimulation and amplification of immune T cells from spleens of DNA-vaccinated animals

Spleens from DNA-vaccinated BALB/c mice were aseptically removed from immunized mice. Ficoll-purified splenocytes were stimulated in vitro with genetically engineered APCs, which are NIH3T3 cells transfected with the wild-type ErbB-2, class I MHC Kd murine B7.1, or a combination of the above. APCs were irradiated (6000 R) and added to primary splenocyte cultures at a 1:10 ratio in six-well plates containing RPMI 1640 supplemented with L-glutamine, penicillin and streptomycin, nonessential amino acids, sodium bicarbonate, HEPES, 50 µM 2-ME, and 10% FCS. At 48 h poststimulation, T cells were collected and replated in fresh media containing 5–10 U/ml murine IL-7 (R&D Systems, Minneapolis, MN) and 30–60 IU/ml human IL-2 (Cetus, Emeryville, CA). Cells were cultured for an additional 5 days before they were tested in the chromium release assay and intracellular cytokine analysis.

51Cr release assay

D2F2 or D2F2E2 tumor target cells were labeled with 51Cr by incubating 1 x 106 cells with 100 µCi Na51CrO4 (NEN Research Products, Boston, MA) in 1 ml HEPES (2 mM)-buffered HBSS + 2% cosmic calf serum at 37°C for 2 h. In some experiments, the target cells were simultaneously incubated with peptide E63 (TYLPTNASL; Genemed Synthesis, South San Francisco, CA) at 200 µg/ml. The unincorporated 51Cr was removed by three washes with HBSS + 2% cosmic calf serum. Graded numbers of effector cells were mixed with 4000 labeled target cells in 200 µl RPMI plus 5% FBS in the wells of round-bottom microtiter plates. After centrifugation at 200 x g for 1 min, the plate was incubated at 37°C for 4.5 h. After the incubation, the plate was centrifuged at 480 x g for 10 min, and a 50-µl aliquot was removed from each well for counting in a 1450 MicroBeta TRILUX liquid scintillation counter (Wallac, Gaithersburg, MD). The percent lysis was calculated as: percent specific lysis = 100 x [(cpmtest - cpmmedium)/(cpmmax - cpmmedium)]. The cpmmax was determined by adding 1/6 N HCl to wells containing 51Cr-labeled target cells. Each group contained four replicates.

Measurement of intracellular cytokine production

In parallel with the chromium release assay, immune splenocytes (1–2 x 106) were plated on immobilized anti-CD3{epsilon} in RPMI supplemented with 10% FCS for 4–5 h. This is in accordance with the standard Th1/Th2 discrimination protocol (BD PharMingen, Los Angeles, CA), which uses immobilized anti-CD3 (and/or soluble CD28, phorbol ester, or calcium ionophore) as an agonist for triggering the release of cytokines from Ag-primed lymphocytes. Monensin (GolgiStop) was added to inhibit protein transportation in the Golgi following BD PharMingen Intracellular Cytokine Labeling Protocol. Cells were collected and processed for dual-colored flow cytometric analysis. Cells were treated with Fc block (BD PharMingen) for 20 min before cell surface labeling with anti-CD4 PE or anti-CD8 PE. Cell were washed, fixed, permeabilized, and stained for intracellular IL-10, IL-4, or IFN-{gamma} using FITC-conjugated mAbs, according to the manufacturer’s protocol (BD PharMingen). Samples were analyzed using a FACSCalibur flow cytometer, and data were analyzed by WinMDI 2.8 software.

Statistical analysis

The Student’s t test was used to evaluate the significant differences between treatment groups.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression and subcellular localization of secE2 compared with E2 and cytE2

To develop an ErbB-2 DNA vaccine that induces strong CD4 T cell and Ab response, plasmid DNA encoding a secreted form of the human ErbB-2 (secE2) ECD (aa 1–505) was generated, as described in Materials and Methods. Based on this strategy, secE2 protein will be secreted in vivo from the transfected muscle cells or APCs and induce CD4 T cell and Ab response. Transfected APCs should also process secE2 as an endogenous Ag and present to CD8 T cells. Therefore, CD4, CD8, and Ab responses can all be expected from a DNA vaccine encoding a secreted ErbB-2. To verify that secE2 was expressed as a secreted protein, mouse mammary tumor cell line D2F2 was transfected with secE2, and stable clones were established by limiting dilution in medium containing zeocin.

Expression and subcellular distribution of the chimeric protein were measured by flow cytometry and immunofluorescence microscopy. Expression of ErbB-2 was evaluated on live or fixed and permeabilized cells. Anti-ErbB-2 mAb TA-1, which recognized an epitope in the ECD, was used to detect transmembrane ErbB-2 on live cells. SecE2 was not detected on the cell surface, whereas D2F2/E2 cells expressing the native, transmembrane ErbB-2 demonstrated strong staining (Fig. 1GoA, top row). As expected, cytE2 in D2F2/cytE2 cells was not detected on the cell surface. Fluorescent microscopy confirmed the findings by flow cytometry. Fig. 1GoB showed the even staining of D2F2/E2 cells by TA-1, with focal aggregates representing areas of high receptor density. No staining was detected on the surface of live D2F2/secE2 or D2F2/cytE2 cells (not shown).



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  		FIGURE 1. A, Intracellular and extracellular expression of variant ErbB-2 proteins. D2F2 cells stably transfected with ErbB-2, SecE2, or cytE2 were stained with anti-ErbB-2 mAb TA-1, which recognizes an epitope in the ECD, followed by PE-conjugated anti-mouse IgG, and analyzed with a FACSCalibur (top row, shaded histogram). The clear histograms were isotype controls. To detect intracellular ErbB-2, transfected cells were permeabilized by methanol fixation before they were stained with mAb 3B5, which recognized an epitope in the intracellular domain (middle row). To block proteasome-mediated protein degradation, D2F2/cytE2 cells were incubated with proteasome inhibitor LLnL overnight before the cells were fixed and stained (bottom row). B, Localization of recombinant ErbB-2 proteins by immunofluorescent microscopy. Surface expression of ErbB-2 was detected on transfected D2F2 cells by staining live cells with anti-ErbB-2 mAb TA-1, followed by FITC-conjugated goat anti-mouse IgG Fc. D2F2 cells expressing secE2 were fixed with methanol and stained as described. D2F2 cells transfected with cytE2 were fixed and stained with mAb 3B5, which recognized an epitope in the intracellular domain. There was no detectable stain. D2F2/cytE2 cells were incubated with 100 µM LLnL for 16 h before they were fixed and stained. Stained samples were examined at x100 under oil with a Zeiss microscope equipped with a Sony 970 digital cooled camera. Fluorescence photomicrographs were imaged using MCID5+ software.

 
The intracellular accumulation of ErbB-2 was measured in fixed and permeabilized cells with mAb 3B5, which recognized residues 1234–1253 of the intracellular domain. When measured by flow cytometry, recombinant secE2 and E2 were expressed at comparable levels in the transfected D2F2 cells (Fig. 1GoA, middle row). Under the same conditions, no ErbB-2 protein was detected in D2F2/cytE2 cells. When examined under the microscope, intracellular secE2 was present in the perinuclear region with a punctuate pattern, consistent with their localization in the ER and Golgi complex (Fig. 1GoB). Consistent with our previous finding, cytoplasmic E2 was not detected in fixed and permeabilized cells by either flow cytometry (Fig. 1GoA, bottom row) or fluorescent microscopy (Fig. 1GoB) unless protein degradation by the proteasome was blocked by overnight incubation with a proteasome inhibitor such as N-acetylleucylleucylnorleucinal (LLnL).

Secreted secE2 in the culture supernatant was measured by Western blotting. A protein with the expected molecular mass of 80 kDa was immunoprecipitated and detected by anti-ErbB-2 mAb clone 42 in the supernatant of D2F2/secE2 culture (Fig. 2Go, lanes 1 and 2). The same band was also detected without immunoprecipitation (Fig. 2Go, lane 4). A slightly faster migrating band was detected in the whole cell lysate of D2F2/secE2 (Fig. 2Go, lane 3), consistent with incomplete glycosylation of the recombinant protein before secretion. Taken together, these results demonstrated unequivocally the intracellular accumulation of secE2 and its secretion from the transfected cells.



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  		FIGURE 2. Western blot analysis of recombinant secE2 protein. Supernatants were collected from D2F2/SecE2-expressing cells grown in media containing serum (lane 1), or without serum (lane 2). Whole cell lysate was prepared from D2F2/SecE2 (lane 3). Samples in lanes 1–3 were purified through a Ni+ spin column (Qiagen). The sample in lane 4 is 10x concentrated, 48-h serum-free culture supernatant without further purification. Lane 5 contains cell culture supernatant from D2F2 cells expressing a control 43-kDa prostate-specific Ag-myc-tag-secreted protein. Protein samples were denatured in Laemmli buffer, boiled and resolved in 10% SDS-PAGE, and transferred to Immobilon-P polyvinylidene difluoride membranes. Membranes were hybridized with anti-myc-HRP (9E10; Invitrogen), detected by chemiluminescence (Amersham), and recorded on Kodak MR x-ray film.

 
Induction of antitumor immunity by immunization with pCMVsecE2 or with combined pCMVsecE2 and pCMVcytE2

BALB/c mice were immunized three times at 2-wk intervals with 100 µg each of pCMV, pCMV-secE2, pCMV-cytE2, or a combination of the two plasmids, pCMV-secE2 and pCMV-cytE2, at 100 µg each. The overall amino acid identity of the full-length human and mouse ErbB-2 proteins is 88% (41). The 505-aa region corresponding to secE2 is 86% identical between mouse and human. As a comparison, a group of eight mice was immunized with pCMV-E2, which was previously found to be an effective vaccine. At 2 wk after the last vaccination, mice were challenged s.c. with BALB/c mammary tumor D2F2 expressing human ErbB-2 (D2F2/E2). All mice injected with pCMV control vector developed tumors within 2 wk (Fig. 3GoA). At 4 wk after vaccination, 7 of the 10 mice injected with pCMV-cytE2 developed tumors, consistent with our previous findings that cytE2 induced only modest antitumor activity. Of eight mice immunized with pCMV-E2, six were protected from tumor growth, i.e., ~80% protection. Of 10 pCMV-secE2-immunized mice, one developed tumor. None of the mice immunized with combined pCMV-secE2 and pCMV-cytE2 developed tumors. Therefore, pCMV-secE2 induced strong antitumor immunity, comparable with the protective effect of pCMV-E2 that we observed in repeated experiments. Combined vaccination with secE2 and cytE2 protected all mice from tumor growth. At 10 wk after tumor challenge, all tumor-free mice were rechallenged with D2F2/E2 tumors, and all mice rejected the second tumor challenge, demonstrating sustained immunity to tumor-associated Ags.



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  		FIGURE 3. A, Protective antitumor immunity induced by secE2 DNA vaccination. BALB/c mice were immunized three times with the indicated plasmid DNA, as described in Materials and Methods. There were 8–10 mice in each group. Mice covaccinated with secE2 and cytE2 received 100 µg of each plasmid DNA in a mixture. At 2 wk after the final immunization, mice were challenged with 2 x 105 D2F2-E2 cells s.c. in 0.1 ml PBS. Animals were palpated weekly for the presence of tumor. Data represent the number of tumor-bearing animals in each group at weekly intervals. Animals that remained tumor free at 10 wk post-tumor challenge were rechallenged with the same tumor in the opposite flank. Animals were palpated weekly for the presence of tumor. B, Induction of ErbB-2-specific Ab by DNA vaccination. Anti-ErbB2 Ab was measured by binding to SKBR-3 cells using flow cytometry. Sera were collected from immunized mice 1 wk after the third vaccination and used as the primary Ab at a 1/20 dilution. FITC-conjugated goat Ab against isotype-specific mouse IgG was the secondary Ab. The binding is expressed as MCF, and the channel values of individual animals are plotted. The median and SD for each group are indicated by the box-whisker plots. There were 10 animals in each group, except the pCMV-E2-immunized group, which has 26 mice.

 
Terminal analysis of sera and spleen from these tumor-free animals demonstrated an increase in anti-ErbB-2 IgG1 and IgG2a titers, as would be predicted from the Ag boost provided by the E2-expressing tumor. When these sera were screened in vitro on D2F2 and D2F2-E2 tumor cells in culture, no effects on tumor cell growth, HER-2 receptor level, or Ab-dependent cellular cytotoxicity were observed. Spleens, however, were enriched in ErbB-2-specific CTL and used to derive the BALB/c human ErbB-2-specific CTL line described below.

Maturation of DNA-immunized T cells upon Ag challenge and the development of a memory T cell repetoire were not examined. After exposure to and rejection of the tumor Ag, de novo priming of naive lymphocytes in vivo can mask the mechanisms contributing to the long-term protective effects primed by the DNA vaccination alone. Therefore, we cannot make an absolute statement regarding the relative roles of memory T cells and Abs in the second tumor rejection. However, mechanistic studies by Pilon et al. in our lab have demonstrated that covaccination with cytE2 and the cytokine gene encoding murine GM-CSF results in Ab-independent E2-tumor rejection that does not require CD4 effector T cells at the time of tumor challenge.4

Induction of anti-E2 Ab by DNA vaccination

To measure the induction of anti-E2 Ab, mice were immunized three times i.m. with plasmid DNA at 2-wk intervals. Sera were collected 2 wk after the third vaccination and diluted 1/20, and anti-E2 Ab was measured by its binding to the breast cancer cell line, SKBR3, using flow cytometry. The titer of anti-ErbB-2 Abs in the sera is a function of the mean channel fluorescence (MCF) of the Ab + FITC-labeled SKBR3 cells. The specificity and relative titer of anti-E2 Ab were verified by ELISA using immobilized, recombinant secE2 (not shown). Vaccination with secE2 induced both IgG1 (MCF of 19 ± 6.5) and IgG2a (MCF of 18 ± 7.8) Abs. E2 vaccination induced primarily IgG2a (MCF of 54 ± 12) and little or no IgG1 (MCF of 4 ± 1) (Fig. 3GoB). CytE2 did not induce anti-E2 Ab in any of the mice, as we previously reported. Covaccination with secE2 and cytE2 did not significantly alter the profile of anti-E2 Ab; the MCF for IgG2a was 22.2 ± 6.5 and IgG1 was 24.9 ± 10.1. Therefore, secE2 induced both IgG1 and IgG2a and is a shift toward Th2 response when compared with the Th1 response in E2-immunized mice.

Generation and T cell-stimulating activity of 3T3-derived APCs

To analyze CTL activity, stable Ag-presenting cell lines were generated. The syngeneic BALB/c fibroblast cell line, NIH3T3, was transfected sequentially with the following genes: ErbB-2, Kd, and B7.1. Stable clones of 3T3/E2, 3T3/E2/Kd, and 3T3/E2/Kd/B7.1 were established. Fig. 4GoA is a representative staining profile, showing strong expression of all three transgenes in 3T3/E2/Kd/B7.1 cells.



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  		FIGURE 4. A, Genetically engineered NIH3T3 as E2-specific APCs. NIH3T3 cells were cotransfected with ErbB-2 and pRSVneo-Kd at a ratio of 10:1. G418-resistant clones (800 µg/ml) were selected, and expression of transmembrane ErbB-2 and Kd was analyzed by flow cytometry. The double-positive clone was subsequently transfected with pEXV3 murine B7.1 and pcDNA3.1-Zeo at a 10:1 ratio and coselected with 800 µg/ml each of G418 and Zeocin. Expression of all three genes in this clone (NIH3T3 E2/Kd/B7.1) was routinely monitored by flow cytometry. ErbB-2 (left) was detected by mAb TA-1 and PE-conjugated goat anti-mouse IgG Fc. Kd (center) was detected by hybridoma supernatant SF1.1.1 and PE-conjugated goat anti-mouse IgG Fc. Murine B7.1 (right) was detected by FITC-conjugated rat anti-mouse B7.1, CD80. The appropriate isotype and secondary Ab controls are indicated by the clear histograms. Flow cytometric analysis was performed with a FACSCalibur. B, Relative Ag-presenting activity of genetically engineered NIH3T3 cells. Splenocytes were prepared from mice that were immunized with secE2 and had rejected D2F2/E2 tumors. Lymphocytes were stimulated in vitro with irradiated NIH3T3 cells expressing either E2 ({blacksquare}), E2/Kd ({triangleup}), or E2/Kd/B7.1 ({blacktriangleup}). At day 7 of culture, lymphocytes were collected and tested in chromium release assay at varying E:T ratios. The target cells were 51Cr-labeled D2F2 (left) or D2F2-E2 (right) cells. Each group contained three to four replicates.

 
Relative Ag-presenting activity of 3T3/E2, 3T3/E2/Kd, or 3T3/E2/Kd/B7.1 was tested. The 3T3 APC were adhered to six-well plates overnight and irradiated just before immune lymphocytes were added. Spleen cells were prepared from mice, which were immunized with pCMV-SecE2 three times and had rejected D2F2/E2 tumor challenge. Immune spleen cells were incubated for 7 days with irradiated APC at a lymphocyte-APC ratio of 10:1. CTL activity was measured by the lysis of 51Cr-labeled D2F2 or D2F2/E2 target cells. As shown in Fig. 4GoB, specific CTL activity against D2F2/E2 was demonstrated when 3T3/Kd/B7.1 were the APCs. The 3T3/E2 was not effective, and 3T3/E2/Kd induced weaker CTL than 3T3/Kd/B7.1. Therefore, 3T3/E2/Kd/B7.1 was used as the APC in this study.

Activation of CTL by secE2 and cytE2

To compare the activation of CTL by different vaccines, splenocytes were prepared from mice that were immunized three times with pCMV-E2, pCMV-secE2, or a combination of pCMV-secE2 and pCMV-cytE2 and had rejected D2F2/E2 tumors. Immune cells were cultured with irradiated 3T3 APC for 7 days, and CTL activity was tested. Fig. 5GoA shows specific lysis of ~50% D2F2/E2 cells at an E:T ratio of 40:1 using CTL from mice that were immunized with pCMV-E2 or the combined vaccines of pCMV-secE2 and pCMV-cytE2. There was marginally detectable CTL activity in pCMV-secE2-immunized mice. When the same effector cells were stimulated a second time with the same APC, enhanced CTL activity against D2F2/E2 was observed (Fig. 5GoA, left). There was no killing of D2F2 cells by any of the test effectors at any time. Therefore, 3T3/E2/Kd/B7.1 was effective at stimulating anti-E2 CTL in vitro. CTL activity was comparable in mice immunized with pCMV-E2 or a combination of pCMV-secE2 and pCMV-cytE2 after a single in vitro stimulation. The second in vitro stimulation further amplified CTL activity and allowed the detection of additional higher titer, CTL.



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  		FIGURE 5. A, ErbB-2-specific CTL activity in vaccinated animals. Splenocytes were prepared from mice that were immunized with wild-type E2 ({triangleup}, {blacktriangleup}), secE2 ({circ}, •), or secE2 plus cytE2 ({square}, {blacksquare}) DNA and had rejected D2F2/E2 tumors. Lymphocytes were stimulated in vitro with NIH3T3 cells expressing E2/Kd/B7.1. CTL activity was measured after one (left) or two (right) cycles of stimulation against 51Cr-labeled D2F2 ({square}, {circ}, {triangleup}) or D2F2-E2 ({blacksquare}, •, {blacktriangleup}) cells. Each group contained three to four replicates. B, Detection of CD8+, IFN-{gamma}-producing T cells. In parallel with the CTL assay (above), splenocytes from the in vitro stimulated cultures were plated on immobilized anti-CD3{epsilon} in the presence of monensin (GolgiStop) for 4–5 h. Cells were stained with PE-conjugated anti-CD8 mAb, and washed, fixed, permeabilized, and stained with FITC-conjugated mAb directed to IFN-{gamma}, according to the manufacturer’s instructions (PharMingen). Samples were analyzed using a FACSCalibur, and data were analyzed by WinMDI 2.8 software.

 
Production of IFN-{gamma} by anti-ErbB-2 CD8 T cells

To further characterize the CTL, IFN-{gamma} production by CTL was measured in parallel with the chromium release assays (above). CTL that were stimulated once (Fig. 5GoB, top row of density plots) or twice (Fig. 5GoB, bottom row) with 3T3 APC were incubated for 4–5 h with anti-CD3 and monensin, which blocks protein transportation in the Golgi. Treated cells were stained with PE-conjugated anti-CD8. Surface-stained cells were fixed, permeabilized, and further stained with FITC-conjugated anti-IFN-{gamma}. The majority of CD8-positive T cells produced IFN-{gamma} after either one or two in vitro stimulations (Fig. 5GoB), supporting a correlation between IFN-{gamma} production and lytic activity of CTL. Although these are physiologically different responses of an Ag-primed CTL, we have observed IFN-{gamma} production to be a reliable indicator CTL activity if assayed in parallel. The percentages of CD4 (expanded) in these cultures were low (less then 10% after the initial stimulation), and neither IL-10, IL-4, nor IFN-{gamma} was detected in the CD4 subsets (not shown).

Recognition of peptide E63 by E2-specific CTL

Peptide TYLPTNASL corresponding to aa 63–71 was reported to be a Kd-associated epitope (41, 42). Our own studies also demonstrated the immunogenicity of this peptide in BALB/c mice (our unpublished result). A CTL line was established from the spleens of immunized animals that rejected E2 tumors after a second challenge by repeated stimulation with 3T3/E2/Kd/B7.1. Recognition of E63 by ErbB-2-specific CTL was tested after 12 stimulations. At E:T ratio of 1:1, significant lysis of E63-coated D2F2 cells was detected (Fig. 6Go), supporting E63 as a dominant ErbB-2 epitope. The same CTL also lysed D2F2/E2, but not D2F2 cells. D2F2 cells coated with {beta}-galactosidase peptide were not lysed. Therefore, E63 was an E2-derived, Kd-associated peptide and was recognized by E2-specific CTL.



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  		FIGURE 6. Cytolytic activity of BALB/c anti-ErbB-2 CTL line against E2-expressing tumor cells and cells loaded with peptide E63. ErbB-2-specific CTL were incubated with 51Cr-labeled D2F2 ({circ}), D2F2/E2 (•), E63-loaded D2F2 ({blacksquare}), or {beta}-galactosidase ({beta}-gal) peptide-loaded D2F2 ({triangledown}) at E:T ratios of 30:1, 10:1, 3:1, or 1:1. Each group contained three to four replicates.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our previous studies demonstrated that DNA encoding transmembrane E2 with intact tyrosine kinase activity was the strongest antitumor vaccine compared with E2A and cytE2(A). The oncogenic activity of native E2 prohibits its use as a DNA vaccine. In this study, secE2 was generated to encode a secreted form of ErbB-2 containing the N-terminal 505 of human ErbB-2. Extensive analysis by flow cytometry, fluorescent microscopy, and Western blotting established the synthesis and secretion of the 80-kDa secE2 protein from transfected cells. Vaccination efficacy of secE2 was compared with that of E2 DNA. E2 induced primarily IgG2a and stronger CTL activity, indicating a Th1 response. SecE2 induced IgG1 and IgG2a and lower CTL activity, indicating a Th2 response. Either response was effective in controlling tumor growth. When secE2 and cytE2 were combined, CTL were induced at the same level as that by E2, and complete protection against D2F2/E2 was achieved.

Production of IgG1 by secE2 immunization follows the activation of Th2 cells, which are often correlated negatively with antitumor immunity. On the other hand, humanized anti-ErbB-2 mAb 4D5 or Herceptin was constructed with a human IgG1 C region, and has demonstrated significant survival benefit in advanced ErbB-2-positive breast cancer (43, 44). Other ErbB-2-specific mAbs of either IgG1 or IgG2a isotype have demonstrated direct tumor-inhibitory effects in mice (45). ErbB-2-positive tumors may be unique in their sensitivity to Ab-mediated toxicity. Following Ab binding to ErbB-2, there may be aberrant signaling through ErbB-2, Ab-dependent immune cell killing, accelerated degradation of ErbB-2, etc. (46). The anti-lymphoma activity of a mAb to CD20, also a transmembrane-signaling molecule, corroborates the efficacy of Abs to certain transmembrane-signaling molecules (47). Therefore, in developing E2 DNA vaccine, it may be advantageous to preserve Ab response. In this regard, secE2, which induces excellent Ab response, is free of tyrosine kinase activity, and inhibits nearly 90% of tumor growth, is potentially an excellent vaccine.

The subcellular localization and stability of the recombinant Ag dictate the type and intensity of the immune response (4). Although secE2 induced excellent Ab response and antitumor activity, it did not induce significant CTL response when compared with E2. To achieve antitumor activity in human patients who may be tolerant to E2, it will be important to mobilize all arms of the immune effectors. CTL are critical effectors even if Abs have direct effect. Several human MHC I (17, 22, 25)- and MHC II (48, 49)-associated E2 peptides have been characterized. Unfortunately, immunization with one such peptide (p369–377) associated with HLA-A2 induced peptide-specific CTL that failed to recognize ErbB-2-positive tumors (50). This may be a result of low epitope density, low CTL affinity, or other variables. By vaccination with cytE2, which contains the entire repertoire of ErbB-2 epitopes, all epitopes can be presented in vivo, and there may be a greater chance of inducing functional CTL. The elevated CTL activity and complete tumor protection in mice immunized with secE2 and cytE2 support the efficacy of this strategy.

Anti-E2 CTL line established from immunized mice that rejected D2F2/E2 tumor lysed D2F2/E2, but not D2F2 tumor cells. The same CTL lysed E63-coated target cells with high efficiency, supporting E63 as a significant E2 epitope.

Immunization with rat neu DNA was found to inhibit tumor growth in normal (51) or neu transgenic mice (51, 52, 53). DNA encoding the secreted form of rat neu was either comparable or superior to the native, transmembrane neu. Anti-neu Ab was not correlated in the study with normal mice, but was considered the major effector in neu transgenic mice (54, 55). Our findings with cytE2 and secE2 suggest that either cellular or humoral immunity can play a significant role in tumor rejection. It is most beneficial, however, to maximize both arms of immune reactivity by combining secE2 and cytE2 vaccines.

Therefore, by combining DNA vaccines designed to activate different effectors, it is possible to achieve maximal response. Although we observed excellent protection using the secE2 as a single vaccine in our model, we suspect that to get the best coverage of all potential class I and class II epitopes that can be presented by the diverse MHC in the human population, covaccination with DNA is required. To this end, we are currently evaluating a third generation of E2-based DNA vaccines that encode various truncated forms of E2, targeted to different subcellular and extracellular compartments and chimeric vaccines that fuse E2 sequences to immunogenic proteins. Wild-type, immune-depleted BALB/c and human E2 transgenic mice developed in our lab are currently being examined.


   Footnotes
 
1 This work was supported by Grants CA76340-01 and 5 T32 DC00026-12 from the National Institutes of Health. Back

2 Address correspondence and reprint requests to Dr. Wei-Zen Wei, Breast Cancer Research Program, Department of Immunology, Karmanos Cancer Institute, 429B Prentis Building, 110 East Warren Avenue, Detroit, MI 48201. E-mail address: weiw{at}karmanos.org Back

3 Abbreviations used in this paper: E2, wild-type human ErbB-2 proto-oncogene; cytE2, cytoplasmic E2; ECD, extracellular domain; ER, endoplasmic reticulum; LLnL, N-acetylleucylleucylnorleucinal; MCF, mean channel fluorescence; secE2, secreted ErbB-2. Back

4 S. Pilon, M. P. Piechocki, and W.-Z. Wei. Vaccination with cytoplasmic ErbB-2 DNA protects mice from mammary tumor growth without anti-ErbB-2 Ab. Submitted for publication. Back

Received for publication April 20, 2001. Accepted for publication July 11, 2001.


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 Materials and Methods
 Results
 Discussion
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