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10.1245/ASO.2005.06.028
Annals of Surgical Oncology 12:517-525 (2005)
© 2005 Society of Surgical Oncology
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Original Article

A Tat Fusion Protein–Based Tumor Vaccine for Breast Cancer

Carsten T. Viehl, MD1,2, Michelle Becker-Hapak, BA, BS3, Jason S. Lewis, PhD4,5, Yoshiyuki Tanaka, MD, PhD1, Udaya K. Liyanage, MD1, David C. Linehan, MD1,5, Timothy J. Eberlein, MD1,5 and Peter S. Goedegebuure, PhD1,5

1 Department of Surgery, Washington University School of Medicine, 660 S. Euclid Avenue, Box 8109, St. Louis, Missouri 63110
2 Department of Surgery, Divisions of General Surgery and Surgical Research, University of Basel, Spitalstrasse 21, 4031 Basel, Switzerland
3 Division of Oncology, Alvin J. Siteman Cancer Center, Washington University School of Medicine, Box 8007, 660 S. Euclid Avenue, St. Louis, Missouri 63110
4 Mallinckrodt Institute of Radiology, Washington University School of Medicine, Box 8225, 510 South kingshighway Boulevard, St. Louis, Missouri 63110
5 Alvin J. Siteman Cancer Center, Washington University School of Medicine, 660 S. Euclid Avenue, St. Louis, Missouri 63110

Correspondence: Address correspondence and reprint requests to: Peter S. Goedegebuure, PhD, Department of Surgery, Washington University School of Medicine, 660 S. Euclid Avenue, Box 8109, St. Louis, MO 63110, USA; E-mail: goedegep{at}wustl.edu.


   ABSTRACT
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Background: We recently reported that dendritic cells (DCs) transduced with a fusion protein between Her2/neu and the protein transduction domain Tat (DC-Tat-extracellular domain [ECD]) induced Her2/neu-specific CD8+ T cells in vitro. This study tested the in vivo efficacy of DC-Tat-ECD in a murine breast cancer model.

Methods: FVB/N mice received one or two weekly intraperitoneal immunizations with syngeneic DC-Tat-ECD followed by a tumor challenge with syngeneic neu+ breast cancer cells, and tumor development was monitored. To test for Her2/neu specificity, CD4+ and CD8+ cells were isolated through magnetic bead separation and analyzed for specific interferon {gamma}release.

Results: Intraperitoneally injected DCs migrated to secondary lymphoid organs, as evidenced by small-animal positron emission tomography studies. Immunized mice developed palpable tumors significantly later than control mice injected with DC-Tat-empty (P =.001 and P <.05 for two immunizations and for one immunization, respectively) or mice that received no DCs (P =.001 and P <.05). Similarly, immunized mice had smaller resulting tumors than mice injected with DC-Tat-empty (P <.05 and P <.01) or untreated mice (P <.001 and P <.001). Significantly more tumor-specific CD8+ splenocytes were found in twice-immunized mice than in untreated animals (P <.001). Similarly, a T-helper type 1 CD4+ T-cell response was observed.

Conclusions: Protein-transduced DCs may be effective vaccines for the treatment of cancer.

Key Words: Tat fusion protein • Her2/neu • Cancer vaccine • Breast cancer • Animal study


   INTRODUCTION
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Recent confirmation that tumor development is controlled by both innate1 and adaptive2 immunity has renewed interest in tumor vaccines. Clinically useful vaccines are safe, well tolerated, easy to generate and administer, and applicable to all patients. Additionally, they should induce potent and long-lasting antitumor immunity. Numerous strategies have been developed to generate vaccines, ranging from simple DNA immunizations to strategies that involve modification of antigen-presenting cells to express tumor antigens (see review3). The latter strategy has been our focus, and we recently reported on protein transduction of dendritic cells (DCs) as a means of expressing tumor antigens by major histocompatibility complex (MHC) class I and class II.4 Protein transduction is a relatively novel technology5–7 that involves the generation of a fusion protein between a protein transduction domain—e.g., from the human immunodeficiency virus (HIV) Tat protein—and the protein of interest. The protein transduction domain permits rapid penetration of virtually all mammalian cells with high efficiency.5,8 When applied to DCs, the fusion protein enters the cytosol, where it enters the MHC class I pathway of processing and presentation.9 Consequently, antigen-specific CD8+ T cells can be induced, as has been demonstrated by several groups.4,10–12 In addition, the standard antigen uptake mechanisms of DCs, such as endocytosis, permit antigen processing and presentation by MHC class II11,13,14. Thus, protein transduction offers the possibility of introducing multiple CD4 and CD8 epitopes into antigen-presenting cells and is applicable to virtually all patients regardless of their HLA phenotype.

In addition to the vaccination strategy, the choice of the target antigen is important. Human epidermal growth factor receptor 2 (Her2/neu) is a member of the epidermal growth factor receptor family.15 Amplification of the Her2/neu oncogene leads to overexpression of its gene product in approximately 30% of breast cancer patients.16 Overexpression of Her2/neu has also been shown in a variety of other human neoplasms of epithelial origin, including ovarian, lung, and head and neck cancers.15 Preexisting humoral and cellular immunity has been demonstrated in patients with Her2/neu-expressing tumors.17–19 Several MHC class I and II–restricted epitopes of Her2/neu have been identified20–23 that induce a Her2/neu-specific CD8+ and CD4+ T cell response, respectively, in vitro.

We recently reported on the generation of a Tat-Her2/neu fusion protein that contains most of the extracellular domain (ECD) of Her2/neu. DCs transduced with this fusion protein induced Her2/neu-specific CD8+ T cells in vitro that recognized and efficiently lysed Her2/neu+ breast cancer cells.4 In contrast, T cells stimulated with recombinant Her2/neu without the Tat protein transduction domain only poorly recognized the same cell lines.4 This study was undertaken to test the in vivo efficacy of Tat-Her2/neu–transduced DCs in a murine breast tumor model.


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Cell Lines
The neu+ breast cancer cell line NT5 (H-2q) was provided by Dr. E. M. Jaffee (Johns Hopkins University of Medicine, Baltimore, MD),24 and the neu+ esophageal cancer cell line third ESO (H-2b) was provided by Dr. R. J. Battafarano (Washington University School of Medicine, St. Louis, MO). The neu–cell line NIH-3T3 (H-2q) was purchased from the American Type Culture Collection (Manassas, VA). NT5 was cultured in RPMI-1640 medium (Cellgro, Herndon, VA) supplemented with 20% (v/v) fetal bovine serum (FBS; Valley Biomedical, Winchester, VA), 1% penicillin/streptomycin (Cellgro), 1% glutamine (Cellgro), 1% 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; Cellgro), 1% nonessential amino acids (Gibco, Grand Island, NY), 1% Na-pyruvate (Gibco), and.2% bovine insulin (Gibco). Third ESO was cultured in RPMI-1640 medium supplemented with 10% FBS, 1% penicillin/streptomycin, 1% glutamine, and 1% HEPES. NIH-3T3 was cultured in Dulbecco% s modified Eagle medium with 4.5 g of glucose per liter (Cellgro) supplemented with 10% FBS, 1% penicillin/streptomycin, 1% glutamine, 1% HEPES, 1% nonessential amino acids, and 1% Na-pyruvate.

In Vitro Generation of Murine DCs
Bone marrow was isolated from the thighs of female 8-week-old FVB/N and C57BL/6 mice (Jackson Laboratories, Bar Harbor, ME). The cells were cultured for 9 days in RPMI-1640 medium supplemented with 5% FBS, 1% penicillin/streptomycin, 1% glutamine, and 50 µmol/L of 2-mercapto-ethanol (Sigma, St. Louis, MO) in the presence of 10 ng of murine granulocyte-macrophage colony-stimulating factor per milliliter (BioSource, Camarillo, CA) to induce differentiation of DCs. The medium was changed every 2 days, and new cytokines were added. On day 9, maturation of DCs was induced by adding 6 µg of CpG 1826 per milliliter (Coley, Wellesley, MA). On day 10, the cells were harvested and used as mature DCs. The phenotype of the harvested cell population was analyzed by flow cytometry on a FACSCalibur (BD Biosciences, San Diego, CA) after fluorescently labeled antibody staining (all antibodies from BD): >88% of the cells were DCs, as evidenced by the presence of CD11c and the absence of markers for T, B, and natural killer cells or granulocytes (TCR{alpha}/beta;, B220, NK1.1, and Gr1.1, respectively); >92% of the DCs showed the mature phenotype, as evidenced by upregulation of MHC class II and B7.2 (data not shown).

Generation and Purification of Tat Fusion Proteins
The bacterial expression vector pTat-HA (provided by Dr. S. F. Dowdy, San Diego, CA) contains six histidines followed by the HIV Tat domain (YGRKKRRQRRR), a hemagglutinin (HA) tag, and a multicloning site.6 Two Tat fusion proteins, Tat-ECD and Tat-empty, were used and purified as described previously.4 Briefly, for Tat-ECD, the NcoI-EcoRI fragment (base pairs 174–1475) of the Her2/neu ECD was excised by digestion of the Her2/neu expression vector, pSV2-Her2/neu.25 The fragment was subcloned into the NcoI-EcoRI site of pTat-HA. For Tat-empty, the pTat-HA plasmid was used without an insert. Tat fusion proteins were expressed in Tuner(DE3) competent cells (Novagen, Madison, WI). Bacteria were sonicated in 8 mol/L of urea, and Tat fusion proteins were purified from the bacterial supernatant on a Ni-NTA column (Qiagen, Valencia, CA) under denaturing conditions. Proteins were refolded, and urea was removed by dialysis. Finally, purity and concentration were determined by gel electrophoresis and Coomassie staining, and the identity of the purified protein was confirmed by immunoblot with an anti-HA antibody (Covance, Berkeley, CA).4,26 To neutralize possible residual endotoxin, Tat fusion proteins were pretreated for 1 hour with 50 µg of polymyxin B sulfate per milliliter (Sigma) before addition to the DCs.

DC Trafficking Studies With Small-Animal Positron Emission Tomography
Radiochemistry
Copper-64 (64Cu) was produced on a CS-15 biomedical cyclotron (Cyclotron Corp., Napa, CA) at Washington University in St. Louis, MO, by using previously reported methods,27,28 and 64Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) (64Cu-PTSM) with >98% radiochemical purity (1 x 10–2 MBq/µg) was produced by methods similar to those described in the literature.29,30 All chemicals were purchased from Sigma-Aldrich Chemical Corp. (Milwaukee, WI), and all solutions were prepared by using distilled deionized water.

Cell Labeling
Mature, syngeneic DCs (C57BL/6) were incubated with 300 µCi of 64Cu-PTSM per 1 x 107 DCs for 30 minutes at 4°C, followed by three washes with phosphate-buffered saline (PBS). The three PBS washes were performed over a 30-minute period to ensure efflux of any unbound 64Cu. A total of 3 x106 DCs (labeled with approximately 40–50 µCi) were injected intravenously (IV), subcutaneously (SC), or intraperitoneally (IP) into C57BL/6 mice and imaged on small-animal positron emission tomography (PET). To control for viability, some labeled DCs were kept at room temperature; viability was 95.8% and 80.5% after 1 and 5 hours, respectively, as evidenced by trypan blue exclusion.

Small-Animal PET Imaging
All imaging was performed in a temperature-controlled imaging suite with close monitoring of the physiological status of the animals. PET was performed on a microPET-R4 system (Concorde Microsystems, Knoxville, TN). The microPET-R4 has a field view of 8 cm axially by 11 cm transaxially and is capable of a spatial resolution of 2.3 mm and an absolute sensitivity of 1020 cps/µCi in the middle of the field of view. Images were generated from three-dimensional sinogram data and rebinned to two-dimensional format by the Fourier rebinning (FORE) algorithm, followed by two-dimensional filtered back-projection.31 For imaging studies on mice, the mice were anesthetized with 1% to 2% isoflurane before scanning and were positioned supine and immobilized in a custom-prepared cradle. Two mice were imaged side by side and remained in the same bed position for all time points. Data collection consisted of 10-, 15-, or 20-minute static collections at the selected time points. The depicted coronal PET slices are approximately 1 mm thick.

Murine Immunization Studies
Mature, syngeneic DCs (FVB/N) were transduced for 4 hours with 500 µmol/L of Tat-ECD and Tat-empty at 37°C and washed twice in PBS. A total of 1 x 106 viable transduced DCs per mouse were reconstituted in 100 µL of PBS and injected IP.

Female 8-week-old FVB/N mice (H-2q) were separated into four groups: group 1 (2x DC-Tat-ECD) was immunized twice with Tat-ECD–transduced DCs at days –14 and –7; group 2 (1x DC-Tat-ECD) was immunized once with Tat-ECD–transduced DCs at day –7; (control) group 3 (DC-Tat-empty) was injected with Tat-empty–transduced DCs at day –7; and (control) group 4 (no DCs) received no DCs at all. All groups were challenged with the syngeneic neu+ breast cancer cell line NT5 at day 0 (5 x106 viable cells per mouse injected SC on the left inner thigh). The mice were monitored twice weekly for the first appearance of palpable tumors and were followed up for tumor growth. When the first tumors reached the ethically maximal tolerable size of 2 cm in one diameter, all mice were killed. The tumors were explanted to measure the two largest axes by using a Vernier caliper and to calculate the tumor volume: major axis xminor axis x.5236).32

For functional studies on CD4+ and CD8+ T cells, 10 FVB/N mice were separated into 2 groups—2x DC-Tat-ECD and no DCs, respectively—that were treated as described previously. The mice were killed 2 weeks after tumor challenge. Unmanipulated CD4+ and CD8+ T cells were isolated from spleens and tumor-draining lymph nodes (TDLNs) by negative selection by using magnetic bead T-cell isolation kits (Miltenyi Biotec, Auburn, CA) according to the manufacturer’s protocol.

Animals were housed under pathogen-free conditions. All experimental protocols were approved by the institutional Animal Studies Committee in accordance with American Association of Laboratory Animal Committee policies, and all experiments were subsequently conducted in compliance with the institutional guidelines for the care and use of research animals.

Interferon {gamma}Enzyme-Linked Immunosorbent Spot Assay
CD8+ T cell–mediated antigen-specific cytokine release was assessed with a commercially available murine interferon (IFN)-{gamma}enzyme-linked immunosorbent spot assay (ELISPOT; BioSource). A total of 200,000 CD8+ T cells per well were cocultured at 37°C with 100,000 cells per well of the neu+ haplotype-matched (H-2q) NT5, the neu–haplotype-matched (H-2q) NIH-3T3, the neu+ haplotype-mismatched (H-2b) third ESO cell line, or medium only. Every sample was run in triplicate. The target and control cell lines were pretreated with 250 U of IFN-{gamma}per milliliter for 24 hours to upregulate MHC class I expression and underwent three freeze-thaw cycles between liquid nitrogen and room temperature to reduce their adherence to the wells. After 20 hours of coculture, the plate was developed according to the manufacturer’s protocol, and the spots were counted under a dissection microscope at x40 magnification.

IFN-{gamma}Enzyme-Linked Immunosorbent Assay
CD4+ T cell–mediated T-helper type 1 type responses were assessed with a commercially available murine IFN-{gamma}enzyme-linked immunosorbent assay (BioSource). A total of 100,000 CD4+ T cells per well were cocultured at 37°C with 50,000 syngeneic mature DCs per well. Before coculture, DCs were transduced for 4 hours with 500 µmol/L Tat-ECD and with Tat-empty, respectively, and were washed twice. Unpulsed DCs and medium only served as controls. After 24 hours of coculture, supernatants were harvested and analyzed for the presence of IFN-{gamma} by following the manufacturer’s instructions.


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Kaplan-Meier curves were compared by a log-rank test; tumor volumes and ELISPOT results were analyzed with Student’s t-test. P <.05 was considered significant.


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Protein Transduction of DCs With Tat-ECD Fusion Protein Is Time and Serum Dependent
Some Tat fusion proteins are known to transduce more efficiently than others.6 Therefore, we first set out to determine conditions for protein transduction. Mature mouse DCs were transduced with equimolar amounts of Tat-ECD fusion protein for different lengths of time in serum-free and serum-supplemented medium. Equal numbers of lysed DCs were analyzed for the presence of intracellular Tat fusion protein by Western blot (Fig. 1Go) with an anti-HA antibody, which detects the Tat fusion protein–associated HA tag. We found protein transduction to be serum dependent, because intracellular levels of Tat-ECD after transduction in serum-free medium were lower than after transduction in serum-supplemented medium. Whereas detectable concentrations of Tat fusion protein were found within the DCs after minutes, lengthening the pulsing time up to 4 hours increased the concentration of intracellular Tat-ECD. We therefore chose a 4-hour transduction time in medium supplemented with 5% FBS as a standard for the subsequent experiments.


Figure 1
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  		FIG. 1. Protein transduction (PT) of dendritic cells (DCs) with Tat-extracellular domain (ECD) is time and serum dependent. Mature mouse DCs were transduced with equimolar amounts of Tat-ECD fusion protein for different lengths of time and with and without the addition of fetal bovine serum (FBS) to the pulsing medium, as specified. Equal numbers of transduced DCs for each condition were analyzed by Western blot by using an anti-hem-agglutinin (HA) antibody and an anti-actin antibody as a control.

 
IP-Injected Mature Mouse DCs Migrate to Secondary Lymphoid Organs
The induction of a T cell–mediated adaptive immune response requires migration of antigen-presenting cells to secondary lymphoid organs (peripheral lymph nodes and spleen), where naive T cells can be stimulated upon antigen presentation.33 To determine the route of DC migration, mature DCs were radioactively labeled with 64Cu-PTSM, and 2 x106 DCs were injected IV, SC, or IP. At 15 and 30 minutes and at 2, 6, and 24 hours after injection, small-animal PET was performed on the mice to follow DC migration (Fig. 2Go). Intravenous injection was associated with DC detection primarily in the pulmonary system for the first 2 hours (Fig. 2AGo) and later in the hepatobiliary system, but no radioactivity was found projecting onto secondary lymphoid organs. Similarly, SC-injected DCs were not detectable in lymphoid tissues at any of the time points; instead, most of the DCs remained at the site of injection even after 24 hours (Fig. 2BGo). However, IP-injected DCs were detected in the mediastinal lymph nodes 39 minutes after injection (Fig. 2CGo) and in the spleen 143 minutes after injection (Fig. 2DGo). Thus, mature DCs migrate to secondary lymphoid organs after IP injection, and we chose this route of injection as the standard for the subsequent vaccination studies.


Figure 2
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  		FIG. 2. Intraperitoneally injected mature mouse dendritic cells (DCs) migrate to secondary lymphoid organs: copper-64–pyruvaldehyde-bis(N4-methylthiosemicarbazone)–labeled DCs were injected intravenously (i.v.) (A), subcutaneously (s.c.) (B), or intra-peritoneally (i.p.) (C and D) into C57BL/6 mice, and small-animal positron emission tomography was performed at different time points after injection. Shown are representative coronal images for each injection modality at the specified time points. LN, lymph node.

 
Immunization With Tat-ECD–Transduced DCs Significantly Delays Development of Palpable Tumors
In a tumor-prevention model, 10 mice per group received either 2 immunizations or 1 immunization with Tat-ECD–transduced DCs at weekly intervals (2xand 1x DC-Tat-ECD, respectively); one injection of DCs transduced with an irrelevant Tat fusion protein (1x DC-Tat-empty) or no DCs. One week after the last injection, all mice were challenged with neu+ syngeneic breast cancer cells, NT5, injected SC in the left hind limb. Animals were monitored twice weekly for tumor development, and the first appearance of palpable tumor was registered and used to generate a Kaplan-Meier curve (Fig. 3AGo). Twice-immunized mice developed palpable tumors significantly later than mice injected with Tat-empty–transduced DCs (P =.001) or mice that received no DCs at all (P =.001). Likewise, once-immunized mice developed palpable tumors significantly later than the two control groups (P <.05) but slightly earlier than twice-immunized mice.


Figure 3
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  		FIG. 3. Immunization with Tat-extracellular domain (ECD)–transduced dendritic cells (DCs) significantly delayed the development of palpable tumors (A) and resulted in significantly smaller tumors (B). After either 2 immunizations or 1 immunization with Tat-ECD–transduced DCs (2x and 1x DC-Tat-ECD, respectively), 1 injection of DCs transduced with an empty Tat protein (1x DC-Tat-empty), or no DCs, 10 mice per group were challenged with neu+ syngeneic breast cancer cells, NT5. The first appearance of a palpable tumor was considered an event in the Kaplan-Meier curve (A). Ten weeks after tumor challenge, the tumors were explanted for accurate caliper measurement. Shown is the average tumor volume per group (error bar indicates SEM) (B).

 
Immunization With Tat-ECD–Transduced DCs Results in Significantly Smaller Tumors
Ten weeks after tumor challenge, several mice in the control groups had reached tumor diameters of 2 cm, the maximal tolerable size. At this time point, all mice were killed; the tumors were explanted for precise measurement, and the tumor volume was calculated as described in Methods (Fig. 3BGo). Tumor volumes ranged from 40.8 to 163.4 mm3 in the 2x DC-Tat-ECD group, from 50.1 to 120.8 mm3 in the 1x DC-Tat-ECD group, from 80.9 to 189.5 mm3 in the 1x DC Tat-empty group, and from 115.5 to 208.9 mm3 in the no-DC group. The average tumor volume of immunized mice was significantly lower than the tumor volume of mice that received no treatment (P <.001 for 2x DC-Tat-ECD and for 1x DC-Tat-ECD) or of mice once injected with DC-Tat-empty (P <.05 for 2x DC-Tat-ECD and P <.01 for 1x DC-Tat-ECD).

Immunization With Tat-ECD–Transduced DCs Induces the Generation of Tumor-Specific T Cells
To evaluate the potential contribution of tumor-specific T cells to slower tumor growth and smaller tumors in immunized mice, we studied their prevalence in immunized and unimmunized mice. Five mice per group received either two immunizations with Tat-ECD–transduced DCs (2x DC-Tat-ECD) or no DCs, followed by a tumor challenge with neu+ syngeneic breast cancer cells, NT5, as described for the previous experiments. Two weeks after tumor challenge, CD4+ and CD8+ T cells were isolated from spleens and TDLN by antibody-coated magnetic beads. Tumor-specific IFN-{gamma}release of CD8+ T cells was assessed by an overnight IFN-{gamma}ELISPOT by using the neu+ haplotype-matched NT5 cell line as a stimulator and the neu– haplotype-matched NIH-3T3 and the neu+ haplotype-mismatched third ESO cell line as controls (Fig. 4AGo). The number of NT5-specific CD8+ T cells was significantly increased in the spleen of immunized mice as compared with nonimmunized mice (P <.001). NT5 was significantly better recognized by CD8+ T cells than the control cell lines (P <.001 and P <.05 for spleen and for TDLN of immunized mice, respectively).


Figure 4
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  		FIG. 4. Immunization with Tat-extracellular domain (ECD)–transduced dendritic cells (DCs) induced the generation of tumor-specific T cells. Two weeks after the tumor challenge with the neu+ breast cancer line NT5, CD4+ and CD8+ T cells were isolated from spleens and tumor-draining lymph nodes (TDLNs) of immunized (2x DC-Tat-ECD) or unimmunized (no DCs) mice. Tumor-specific interferon (IFN)-{gamma} release of CD8+ T cells was assessed by IFN-{gamma} enzyme-linked immunosorbent spot assay (A) by using NT5 as a stimulator and the neu– NIH-3T3 and the haplotype-mismatched third ESO cell lines as controls. Shown is the average number of spot-forming cells (SFCs) per 100,000 CD8+ T cells. The number of tumor-specific CD8+ T cells was significantly increased in the spleen of immunized mice as compared with nonimmunized mice (P <.001). Additionally, a T-helper type 1 CD4+ T-cell response was induced by Tat-ECD immunization (B). CD4+ T cells were cocultured with Tat-ECD–transduced DCs, with DCs transduced with an irrelevant Tat fusion protein (DC-Tat-empty), or with unpulsed DCs for 24 hours. Supernatants were analyzed for the presence of IFN-{gamma} by enzyme-linked immunosorbent assay.

 
CD4+ T cells were cocultured with Tat-ECD–transduced autologous DCs, with DCs transduced with an irrelevant Tat fusion protein (DC-Tat-empty), or with unpulsed DCs for 24 hours. Supernatants were harvested and analyzed for the presence of IFN-{gamma} by enzyme-linked immunosorbent assay (Fig. 4BGo). Splenic CD4+ T cells from immunized mice secreted high levels of IFN-{gamma} upon recognition of Tat fusion protein–transduced DCs, but not when cocultured with unpulsed DCs or with a medium control only, suggesting that a T-helper type 1 CD4+ T-cell response was induced by Tat-ECD immunization in addition to the CD8+-mediated cytotoxic T lymphocyte response.


   DISCUSSION
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 ABSTRACT
 INTRODUCTION
 METHODS
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REFERENCES
 
Our current data show that Tat fusion protein–transduced DCs induce tumor-specific T cells that significantly reduce tumor growth in vivo. As such, these results extend our earlier data4 showing that Tat-ECD–transduced DCs induce Her2/neu-specific CD8+ T cells in vitro. These CD8+ T cells recognized and lysed Her2/neu+ breast cancer cells.

Protein transduction of DCs is a relatively novel application of a technology based on findings by Frankel and Pabo34 and by Green and Loewenstein35 in 1988. Both groups independently discovered that the HIV Tat protein is able to cross cell membranes. In 1994, Fawell et al.8 demonstrated Tat-mediated delivery of heterologous protein into a variety of cells. Subsequently, Kim et al.9 showed that ovalbumin conjugated to Tat is introduced into the MHC class I pathway. In the following years, Nagahara et al.26 generated a bacterial expression vector and a protocol for expression and purification of Tat fusion proteins.26 In addition, they established a method for protein transduction of full-length proteins into mammalian cells.5–7 Since then, various groups have applied protein transduction to DCs as a means of stimulating antigen-specific T cells.4,11–13,36,37 Whereas some groups use recombinant fusion proteins, others use a synthetic polypeptide that contains a protein transduction domain.

The main advantage of protein transduction of DCs is the strong promotion of cross-priming and, consequently, the stimulation of CD8+ T cells. However, antigen-uptake mechanisms such as endocytosis are also functional, so that fusion protein is processed through the MHC class I and class II pathways. When full-length proteins are used, this increases the likelihood that multiple epitopes for CD4 and CD8 are encoded and presented. Proof of principle was demonstrated by Shibagaki and Udey,11 who used the model antigen ovalbumin. In addition, Lu et al.37 recently reported that a synthetic polypeptide containing the Tat protein transduction domain and several cytotoxic T lymphocyte and T-helper epitopes induced peptide-specific CD4+ and CD8+ T cells. Our own observations with a Tat fusion protein containing the breast cancer antigen, mammaglobin, also support this idea.14 We found that Tat-mammaglobin–transduced DCs induced CD4+ and CD8+ mammaglobin-specific T cells. Our current studies using a Tat-Her2/neu fusion protein containing most of the ECD of Her2/neu further support the idea that protein-transduced DCs stimulate both CD4+ and CD8+ T cells (Fig. 4Go). Mice immunized twice with Tat-ECD–transduced DCs before tumor challenge grew significantly smaller tumors than control mice, as evidenced by measurements of explanted tumors 10 weeks after tumor challenge. Significant differences were observed even after a single injection (Fig. 3BGo). This observation may be explained by a delay in tumor implantation, by a continuous suppression of tumor growth, or by a combination of both effects. Because immunized mice showed a significant delay in the development of palpable tumors (Fig. 3AGo), immunization with Tat-ECD–transduced DCs seems to delay the implantation of tumor cells. However, because no volumetric measurements of tumor growth in vivo were performed over time, our data do not support (or disprove) the hypothesis of continuous suppression of tumor growth.

Protein-transduced DCs were injected IP after studies with radiolabeled DCs suggested that IP injection was superior to IV or SC administration with regard to migration of DCs to lymph nodes (Fig. 2Go). However, caution should be observed in generalizing these findings. To some extent, controversy still exists on which method to use for optimal migration of DCs to lymph nodes and optimal T-cell stimulation in the lymph nodes. Work by Banchereau and Steinman33 and others has demonstrated that immature DCs are better equipped for capturing antigen and migrating to lymph nodes than mature DCs. However, mature DCs are superior in stimulating T cells compared with immature DCs. In keeping with our in vitro studies,4 we decided to transduce mature DCs rather than immature DCs to minimize endocytosis relative to cross-priming. Consequently, in vivo migration of DCs to secondary lymphoid organs may have been suboptimal. Nonetheless, DCs were found to migrate to lymph nodes and to the spleen and subsequently to induce neu-specific T cells. The greater magnitude of the specific CD8+ T-cell response compared with the CD4 response may also be related to the use of mature DCs instead of immature DCs, because antigen uptake via endocytosis by mature DCs is poor. Alternative strategies in which immature DCs are transduced and administered together with a maturation-inducing agent, such as CpG, are currently under consideration.

Although two immunizations seem more effective than one, more than two immunizations have not yet been tested. Additionally, strategies to prolong T-cell survival through, for example, blocking of cytotoxic T cell-associated protein-4, combining vaccination with adjuvants such as CpG37 or depleting CD4+CD25+ regulatory T cells (Treg)38 have not been incorporated yet. Similar vaccination studies with plasmid DNA encoding either full-length Her2/neu39 or Her2/neu fragments40,41 followed protocols of three to four vaccinations given at 2-week intervals. The growth of transplantable tumor was significantly delayed by neu-specific T cells39,40 or prevented by a combination of specific T cells and neu-specific antibodies.41 When applied to neu-transgenic mice that have demonstrated tolerance to neu, the vaccination regimen is even more stringent.24,42 Up to five vaccinations with either (1) plasmid DNA encoding secreted Her2/neu and granulocyte-macrophage colony-stimulating factor42 or (2) neu-encoding vaccinia virus24 were required to observe a significant delay of spontaneous tumor formation. As such, our studies represent proof-of-principle experiments that illustrate the potential of vaccines that consist of Tat fusion protein–transduced DCs.

Clinically, Her2/neu is targeted through adoptive immunotherapy with trastuzumab, a humanized recombinant antibody that has shown beneficial effects when given as a single agent43 or in combination with chemotherapy44 to patients with advanced Her2/neu-overexpressing tumors. In addition, active specific immunotherapy using Her2/neu peptides45 or DCs pulsed with peptide3 has been pursued. However, even though it has been clearly demonstrated that peptide-based immunizations can be successful and that clinical responses can be induced, especially in patients with malignant melanoma,46 alternative strategies are needed to improve clinical response rates.

In conclusion, these findings demonstrate the potential of Tat fusion protein–transduced DCs in inducing tumor-specific T-cell responses. The main characteristic—that multiple T-cell epitopes can be presented simultaneously by professional antigen-presenting cells—may offer unique advantages for use in tumor vaccines and for other diseases.


   ACKNOWLEDGMENTS
 
The authors thank Dr. S. F. Dowdy, San Diego, CA, for providing the Tat-HA vector; Dr. E. M. Jaffee, Baltimore, MD, for the NT5 cell line; Dr. R. J. Battafarano, St. Louis, MO, for the third ESO cell line; and Todd T. Moore for invaluable technical assistance. Supported in part by grants from the Swiss National Science Foundation (81BE-067988) and the Regional Cancer League of Basel, Switzerland (to C.T.V.). PET imaging was supported by a National Institutes of Health/National Cancer Institute Small Animal Imaging Research Program (SAIRP) grant (R24 CA083060), with additional support from the Small Animal Imaging Core (SAIC) of the Alvin J. Siteman Cancer Center at Washington University School of Medicine and Barnes-Jewish Hospital, St. Louis, MO. The SAIC is supported by National Cancer Institute Cancer Center Support Grant 1 P30 CA091842.


   FOOTNOTES
 
Presented at the 57th Annual Cancer Symposium of the Society of Surgical Oncology, New York, New York, March 18–21, 2004.

Received for publication June 18, 2004. Accepted for publication February 3, 2005.


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 ABSTRACT
 INTRODUCTION
 METHODS
 Statistics
 RESULTS
 DISCUSSION
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