Dr. Ma and Dr. Yi contributed equally to this article.
Preparation of murine B7.1-glycosylphosphatidylinositol and transmembrane-anchored staphylococcal enterotoxin
A dual-anchored tumor cell vaccine and its antitumor effect
Article first published online: 28 FEB 2005
Copyright © 2005 American Cancer Society
Volume 103, Issue 7, pages 1519–1528, 1 April 2005
How to Cite
Yi, P., Yu, H., Ma, W., Wang, Q. and Minev, B. R. (2005), Preparation of murine B7.1-glycosylphosphatidylinositol and transmembrane-anchored staphylococcal enterotoxin. Cancer, 103: 1519–1528. doi: 10.1002/cncr.20943
- Issue published online: 18 MAR 2005
- Article first published online: 28 FEB 2005
- Manuscript Accepted: 3 DEC 2004
- Manuscript Revised: 30 NOV 2004
- Manuscript Received: 1 SEP 2004
- National Natural Science Foundation of China. Grant Number: 39770837
- Zhejiang Provincial Natural Science Foundation. Grant Numbers: 399131, 301580
- tumor vaccine;
- murine B7.1;
- staphylococcal enterotoxin A;
- protein transfer
The authors have previously reported a tumor cell vaccine modified with superantigen staphylococcal enterotoxin A (SEA) and its antitumor effect. The tumor cell vaccines modified with multiple immune activators frequently elicited stronger immune responses against established tumors than single-modified vaccines.
The authors explored the effectiveness of a tumor cell vaccine transduced with immune activators, dual-modified using the protein transfer technique. First, a glycosylphosphatidylinositol (GPI)-anchored murine B7.1 (mB7.1-GPI) and a transmembrane-anchored SEA (TM-SEA) were genetically generated. Then, the murine lymphoma EL4 cells were dual modified with the incorporation of mB7.1-GPI and TM-SEA onto the cell surface. Flow cytometry and laser confocal microscopy showed that the incorporation of B7.1 and SEA molecules onto EL4 cells was quite stable.
The dual-modified tumor cell vaccine EL4/mB7.1-GPI + TM-SEA elicited significantly stronger antitumor immune responses both in vitro and in vivo when compared with the single-modified tumor cell vaccines EL4/mB7.1-GPI and EL4/TM-SEA.
The results of the current study validated the novel approach for preparing tumor cell vaccines modified with dual immune active molecules using the protein transfer technique, and supported the feasibility and effectiveness of the dual-modified tumor cell vaccine. Cancer 2005. © 2005 American Cancer Society.
The mechanisms for tumor cells to escape immune surveillance include tumor antigen-specific tolerance,1 local immune dysfunction in tumor-infiltrating mononuclear cells,2 defective antigen presentation processes,3, 4 down-regulated surface major histocompatibity complex (MHC) molecules, and lack of costimulatory molecules.5, 6 Introduction of immunostimulatory molecules such as MHC Class I, MHC Class II, and B7.1 to the tumor cells and use of these surface-modified tumor cells as vaccines can induce antitumor immunity, as demonstrated by tumor rejection.7, 8 These approaches frequently involve gene transfection of target cells, which is time consuming. In addition, the primary tumor cells often do not grow very well in vitro, and the gene expression is unstable.9 Therefore, the practical application of this strategy to induce antitumor immunity is limited.
Alternatively, surface molecules can be introduced to tumor cells through the so-called “protein transfer” approach.10, 11 One of these approaches is the glycosylphosphatidylinositol (GPI)-anchoring technique. Using this technique, the DNA sequence of the target protein is fused with a GPI signal sequence, and the purified, recombinant GPI-linked fusion proteins are incubated with tumor cells. With this approach, the target protein can be anchored to the extracellular membranes of the tumor cells. An in vitro study has demonstrated that GPI-B7.1 can be anchored to the tumor cells and used to stimulate lymphocyte proliferation.12
Bacterial superantigens (SAgs), including staphylococcal enterotoxin A (SEA), staphylococcal enterotoxin B, and toxic shock syndrome toxin-1, are well known as very potent activators of T cells that can elicit strong immune responses both in vitro and in vivo. However, SAgs, as prokaryotic proteins, are not well suited to a GPI-signal-sequence-fusion strategy for cell membrane anchoring, because GPI linkage requires eukaryotic processing. We had developed a strategy for passively attaching SEA to tumor cells, in which the SEA gene was fused with the transmembrane (TM) region sequence of the protooncogene c-erb-B2. The purified TM-SEA protein could be effectively anchored to tumor cells.13 The vaccine derived from these tumor cells can stimulate proliferation of lymphocytes in vitro and induce a systemic antitumor immunity in vivo.
The escape of tumor cells from immune surveillance can involve several mechanisms, which indicates that a single immune activator may be less effective than multiple activators at inducing sufficient immune response against tumor cell growth. Several studies have shown that tumor cell vaccines created by multiple-gene transfection can induce much stronger antitumor immunity than those made by single-gene transfection.14–16 However, multiple-gene transfection is difficult technically and less practicable.
We sought to develop a new approach, other than gene transfection, to prepare surface-modified tumor vaccines in which two different immune-active molecules are anchored to the tumor cell membrane. In the current study, the tumor cells were incubated with fusion protein GPI-linked mouse B7.1 (mB7.1-GPI) and TM-SEA. Thus, those two molecules were passively attached to tumor cells by the protein transfer technique, and the dual-modified tumor cell vaccine was prepared. We investigated the feasibility of the approach for preparing a dual-anchored tumor cell vaccine, and the effectiveness of the tumor cell vaccine made by this novel approach in an animal model.
MATERIALS AND METHODS
The EL4 (T-cell lymphoma) cell line, derived from the C57BL/6 mouse strain, YAC-1, a natural killer cell (NK)-sensitive lymphoma cell line of A/S (H-2a) origin, and the Chinese hamster ovary (CHO) cell line were all obtained from the American Type Culture Collection (ATCC; Rockville, MD). HO-8910 (human ovarian carcinoma) cell line was provided by the Institute for Cancer Research of Zhejiang Province, China. All these cell lines were cultured in complete RPMI-1640 medium supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% fetal calf serum (FCS). All culture media were purchased from Gibco-BRL (Gaithersburg, MD) and FCS was provided by the Shanghai Institute of Biological Products (Shanghai, China).
Female C57BL/6 mice, 6–8 weeks old, were obtained from Sipper-BK Experimental Animal Company (Shanghai, China) and housed in specific pathogen-free conditions at the animal center of Zhejiang University School of Medicine in accordance with institutional guidelines. Mice were housed 5 per cage in a 12-hour light/dark cycle at an ambient temperature of 22 ± 2 °C and humidity of 50 ± 10% with food and water ad libitum. Cages were changed twice weekly to ensure hygienic conditions. The animals were allowed to acclimate to the facility for 2 weeks before randomization into different experimental groups.
Tumor Model and Experimental Design
To establish the tumor model, EL4 cells cultured for 3 days were harvested and washed twice with phosphate-buffered saline (PBS). 1 × 106 EL4 cells in 100 μL of PBS were subcutaneously injected in the right rear flank of each C57BL/6 mouse. Tumor growth was monitored by measuring tumors in 2 dimensions using a digital caliper on every other day, beginning 7 days after inoculation. Tumor size was calculated using the formula, 1/2 (length + width).17
To test the effectiveness of the dual-modified EL4/mB7.1-GPI + TM-SEA vaccine, the tumor-bearing mice were randomized on the third day of posttumor cell inoculation to the following groups with each group consisting of 8 animals. On the seventh day after tumor cell inoculation, the tumor-bearing mice in the groups were injected with 100 μL PBS, EL4 tumor cell vaccine, EL4/mB7.1-GPI tumor cell vaccine, EL4/TM-SEA tumor cell vaccine, or EL4/mB7.1-GPI + TM-SEA tumor cell vaccine, each vaccine suspension containing 1 × 106 cells in 100 μL PBS.
The tumor sizes were measured on Day 25 after tumor cell inoculation (before any deaths occurred) to ensure inclusion of the data from all the mice.
Anti-mouse B7.1 (rat immunoglobulin G 2 alpha [IgG2α], Pharmingen, San Diego, CA), rabbit anti-SEA (Toxin Technology, Sarasota, FL), goat anti-rabbit IgG/TRITC, goat anti-rat IgG/horseradish peroxidase (HRP), and goat anti-rat IgG/fluorescein isothiocyanate (KpL, Gaithersburg, MD) were used.
Construction, Expression, and Purification of Murine B7.1 Glycosylphosphatidylinositol and Transmembrane-Anchored Staphylococcal Enterotoxin A
The DNA sequence encoding the first 247 amino acids of mB7.118 was amplified by polymerase chain reaction (PCR) from pLNSX-mB7.1, a gift provided by Dr. L. Chen (Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, WA).5 5′-GGAATTCGCTATGGCTTGCAATTGTCAG-3′ with an EcoR I restriction site (underlined) and 5′-CGCAGGCGGTGTATGTGTTCTTGCTATCAGG-3′ were the sense and antisense primers, respectively, for amplifying the mB7.1 sequence. The DNA sequence encoding the signal for GPI anchor attachment from human placental alkaline phosphatase (hPLAP)-119 was amplified using DNA extracted from human placental tissue. 5′-GATAGCAAGAACACATACACCGCCTGCGACCT-3′ and 5′-ATCTCGAGTCAGGGAGCAGTGGCCGTCT-3′ with an Xho I restriction site (underlined) were the sense and antisense primers, respectively, for amplifying GPI. The 2 amplified gene sequences were annealed to form a chimeric GPI-anchored mB7.1 molecule by the SOE method.20 The resulting chimera was cloned in the pGEM-T vector (Promega, Madison, WI), subcloned in the neomycin-resistant plasmid pcDNA3.1 (+) (Invitrogen, Carlsbad, CA), and confirmed by sequencing analysis. The recombinant plasmid was transfected into CHO cells by using Lipofectamine 2000 reagent (Invitrogen), and transfectants were selected with G418 (Gibco/BRL) at a concentration of 600 μg/mL. Expression of the mB7.1-GPI fusion protein on the surface of CHO cells was confirmed by flow cytometry, phosphatidylinositol-specific phospholipase C treatment, and confocal microscopy. The mB7.1-GPI fusion protein was purified by anti-mB7.1-monoclonal antibody-CNBr-Sepharose 4B (Pharmacia, Piscataway, NJ) affinity chromatography, then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting.
The SEA gene21 was cloned from Staphylococcus aureus (ATCC 13565) strain. The primers 5′-GCCGCTAGCATGAAAAAAACAGCATTTAC-3′ with a Nhe I restriction site (underlined) and 5′-GCTCTCTGCTCGGCACTTGTATATAAA-3′ were the sense and antisense primers for amplifying the SEA gene13. The sequence encoding the TM region of the c-erb-B2 gene22 was amplified by reverse transcription-PCR from the human ovarian carcinoma cell line HO-8910.13 5′-TTTATATACAAGTGCCGAGCAGAGAGC-3′ and 5′-AAGCTTCTTACATCGTGTACTTCCG-3′ with a Hind III restriction site (underlined) were the sense and antisense primers, respectively, for amplifying the TM sequence. The two amplified gene sequences were annealed to form a chimeric TM-SEA molecule by the SOE method. The resulting chimera was cloned in pGEM-T vector (Promega) and then subcloned into the pET-28a vector (EMD Biosciences, San Diego, CA). The recombinant pET-28a-TM-SEA13 was transfected into the host strain BL21(DE3)pLysS for expression of the TM-SEA fusion protein. Expression of the target protein was induced by incubation at 30 °C and addition of IPTG to a final concentration of 1 mM. Cells were harvested after 5 hours of induction, the cell pellet was resuspended in 50 mL sonication buffer (300 mM NaCl/50 mM sodium phosphate, pH 8/1%Triton X-100), and the suspension was frozen at −80 °C. The cell pellets were thawed and sonicated 3 times for 15 seconds on ice, centrifuged at 17,800 ×g for 30 minutes at 4 °C, and the supernatant was collected for target protein purification by using the Ni-NTA His.Bind resin purification system (EMD Biosciences).
Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis and Western Blot Analysis
Whole cell extracts were obtained by resuspending the PBS-washed cellular pellets in 1% SDS. The lysates were boiled for 5 minutes, and protein amounts were quantified with protein assay reagent (Bio-Rad, Hercules, CA) using a bovine serum albumin standard solution as reference. Protein extracts were separated on 10% SDS-PAGE gels and visualized by silver staining, which was performed according to the manufacturer's instructions (Pharmacia Biotech AB). Then, they were transferred to an Immobilon P membrane (polyvinylidene difluoride; Millipore, Bedford, MA). The membranes were blocked in TBST containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.05% Tween 20 plus 5% nonfat dry milk at room temperature for 30 minutes, incubated with the primary antibodies in a cool room (4 °C) overnight after washing the membrane, and incubated with the second HRP-conjugated antibodies (dilution, from 1:2000 to 5000) at room temperature for 30 minutes after washing. Proteins were visualized by the ECL detection reagents (ECL kit; Amersham Pharmacia Biotech, Piscataway, NJ) after washing five times in TBST.
The following antibodies were used for Western blot analysis: primary antibodies of anti-mouse B7.1 (rat IgG2α, Pharmingen) and rabbit anti-SEA (Toxin Technology). Second antibodies of goat anti-mouse IgG/HRP and goat anti-rat IgG/HRP were obtained from KpL.
Incorporation of Murine B7.1-Glycosylphosphatidylinositol and/or Transmembrane-Anchored Staphylococcal Enterotoxin A into Cell Membranes
EL4 cells were washed 3 times with PBS/5 mM ethylenediaminetetraacetic acid, resuspended (5 × 106 cells/mL), and incubated with 10 μg/mL purified mB7.1-GPI and/or 20 μg/mL purified TM-SEA protein for 4 hours at 37 °C with gently shaking. The incubated cells were washed three times, and analyzed by flow cytometric assay and confocal microscopy. EL4 cells, EL4 cells anchored with mB7.1-GPI protein, EL4 cells anchored with TM-SEA protein, and EL4 cells anchored with both TM-SEA and mB7.1-GPI protein were irradiated (50 gray) for use as tumor cell vaccines.
Flow Cytometric Assay
Tumor cells (1 × 106) were washed once with 2% fetal bovine serum FBS in PBS and resuspended in 5 μL wash buffer containing the primary antibody, incubated at 4 °C for 45 minutes, then washed twice with wash buffer, resuspended in wash buffer containing a fluorescein-labeled secondary antibody, and incubated at 4 °C for 45 minutes. After 2 washes with wash buffer, the cells were resuspended in 500 μL PBS for flow cytometric analysis in a fluorescein-activated cell sorter (Beckman Coulter, Hialeah, FL).
Confocal Microscopic Analysis
Cells were stained at 4 °C and fixed using 2% paraformaldehyde in PBS for 30 minutes, then washed twice in PBS buffer. Five microliters of the cell pellet was mixed with an equal volume of SlowFade (Molecular Probes, Eugene, OR) and placed on a glass slide. Then, the coverslip was sealed with nail polish. Fluorescence distribution was analyzed using a laser confocal scanning microscope.
Measurement of Lymphocyte Proliferation
The proliferation assay used, based on the conversion of the tetrazolium salt 3, [4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to purple formazan crystals by metabolically active cells,23 provides a quantitative determination of viable cells. Splenocytes were derived from C57BL/6 mice and cocultured with irradiated tumor cells at a ratio of 5:1 for 24 hours. Cells were seeded in a 96-well plate, incubated at 37 °C in 5% CO2 for 4 hours, MTT (5 mg/mL) was added to each well of cells, and the plate was incubated for 4 hours at 37 °C. The medium was removed, and the MTT crystals were solubilized in 0.1 mL dimethylsulfoxide and subjected to centrifugation to pellet the cellular debris. Spectrophotometric absorbance of each sample was measured at 570 nm using a Spectra Microplate Reader (Bio-Tek model EXL 800, Winooski, VT).
Cytokine Release Assay
The nonadherent splenocytes derived from C57BL/6 mice, at the concentration of 1 × 107 cells/mL, were stimulated with 5 × 104 tumor vaccine cells. The supernatant was collected for interleukin-2 (IL-2) assay after 24 hours, and for the interferon-gamma (IFN-γ) assay after 48 hours. The concentrations of IL-2 and IFN-γ were determined using a sandwich enzyme-linked immunosorbent assay kit (Eudogen, Woburn, MA).
Immunotherapy with the Inactivated Tumor Cell Vaccines
C57BL/6 mice were inoculated subcutaneously with 1 × 106 EL4 cells in the right rear flank to establish the murine lymphoma model. Three days after inoculation, the tumor-bearing C57BL/6 mice were divided into 5 groups (each group containing 8 mice), and were injected in the left rear flank using 1 of the following preparations: PBS, EL4 vaccine, EL4/mB7.1-GPI vaccine, EL4/TM-SEA vaccine, or EL4/mB7.1-GPI + TM-SEA vaccine. The mice were given a total of 3 injections, at 2-day intervals of tumor cell vaccine (1 × 106 cells) suspended in 100 μL PBS. Tumor sizes were expressed as the mean diameter of the longest and the shortest diameter measured with a digital caliper every other day. Seven days after the last vaccine dose, three mice in each group were killed and splenic lymphocytes were isolated for NK and CTL activity assays. The other 5 mice in each group were monitored for survival (for ≤ 90 days).
Cytotoxic Assays of CTL and Natural Killer Cell Activity
Splenocytes were isolated from the mice that had been killed. The erythrocytes were depleted with 0.83% ammonium chloride and macrophages were removed by adherence of splenocytes on plastic plates for 2 hours. The nonadherent lymphocytes were directly used as NK effector cells. The lymphocytes were cocultured with inactivated EL4 for 7 days in the presence of recombinant murine IL-2 (20 U/mL) and then collected as CTL effector cells. B16 cells were used as the control target cells, and the NK and CTL activity was determined by using the CytoTox 96 nonradioactive cytotoxicity assay kit according to instructions from the manufacturer (Promega). The percentage of specific lysis was determined according to the following formula: 100 × (experimental release − spontaneous release)/(maximum release − spontaneous release).
All experiments were run in triplicate and the results are means ± the standard deviation of triplicate determinations or representative data from one or two independent experiments. Statistical analyses were performed using the Student's t test and log-rank test (for survival analysis). Differences were statistically significant at P < 0.05.
Lymphocyte Proliferation and Cytokine Production
The authors of the current study first tested whether EL4 cells coated with mB7.1-GPI and/or TM-SEA protein could stimulate lymphocyte proliferation and release of cytokines in vitro. The results showed that EL4/mB7.1-GPI or EL4/TM-SEA cells were superior at stimulating lymphocyte proliferation, relative to EL4 (Table 1). Furthermore, the production of IL-2 and IFN-γ cytokines in response toEL4/mB7.1-GPI or EL4/TM-SEA was much higher than in response to EL4 (Table 1, P < 0.05). The EL4/TM-SEA + mB7.1-GPI cells exhibited a greater ability than the EL4/mB7.1-GPI and EL4/TM-SEA vaccines to stimulate lymphocyte proliferation and induce production of IL-2 and IFN-γ cytokines in vitro (P < 0.05, Table 1).
|A||EL4||22.9 ± 1.4||41 ± 3.2||1.98 ± 0.7|
|B||EL4/mB7.1-GPI||508.8 ± 22.1b||805 ± 13.5b||3.43 ± 0.6b|
|C||EL4/TM-SEA||771.3 ± 11.4c||1225 ± 10.9c||4.87 ± 0.3c|
|D||EL4/mB7.1-GPI + TM-SEAd||1563.6 ± 49.4d||2450 ± 188d||7.17 ± 0.6d|
Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis and Western Blot Analysis of Purified Protein
Figure 1A shows the results from SDS-PAGE of the purified recombinant mB7.1-GPI fusion protein, the cell lysate of CHO cells transfected with pcDNA3.1 (+)-mB7.1-GPI, the controls of CHO cell lysates, and the CHO cells transfected with the pcDNA 3.1 (+) empty vector. Figure 1B shows the Western blot results from the purified mB7.1-GPI protein, with the expected size of 62 kilodalton (kD). Figure 1C shows the Western blot results for TM-SEA fusion protein expressed under the induction of IPTG at different time points. Figure 1D shows the Western blot results for the purified TM-SEA protein, with the expected size of 32.6 kD.
Incorporation of Purified Murine B7.1-Glycosylphosphatidylinositol and/or Transmembrane-Anchored Staphylococcal Enterotoxin A Protein into the EL4 Cell Membrane
The mouse lymphoma cell line EL4 was incubated with purified mB7.1-GPI and/or TM-SEA protein. Flow cytometric analysis showed that mB7.1-GPI and/or TM-SEA protein was expressed on the surface of coated EL4 cells. After the incubation of EL4 cells with mB7.1-GPI protein, 96.5% of the cells expressed mB7.1 with a mean fluorescence intensity of 13.2. Similarly, after incubation of EL4 cells with TM-SEA protein, the positive rate of SEA detection was 95.4%, and the mean fluorescence intensity was 13.3. For EL4 cells incubated with TM-SEA and mB7.1-GPI protein, the SEA and mB7.1 were both detected on 94% of the cells. Eight hours after incubation with mB7.1-GPI and/or TM-SEA protein, flow cytometry showed that the proteins were maintained on approximately 85% of the EL4 cells (data not shown). To determine the distribution of mB7.1-GPI and/or TM-SEA protein on the coated cells and the cell integrity after incubation, the cells images were visualized by laser confocal microscopy (Fig. 2).
Enhanced Antitumor Effects of the Dual-Anchored EL4/Transmembrane-Anchored Staphylococcal Enterotoxin A + Murine B7.1- Glycosylphosphatidylinositol Vaccine
Tumor growth in mice treated with the EL4/mB7.1-GPI, EL4/TM-SEA, or dual-anchored EL4/TM-SEA + mB7.1-GPI vaccines was markedly inhibited relative to those treated with PBS or EL4 vaccine controls (Fig. 3A). The antitumor effect of the dual-anchored EL4/TM-SEA + mB7.1-GPI vaccine was significantly stronger than that of the EL4/mB7.1-GPI or EL4/TM-SEA vaccine (P < 0.05). There were no significant differences in tumor growth inhibition between the groups receiving the EL4/mB7.1-GPI and EL4/TM-SEA vaccines.
Five tumor-bearing mice in each group were monitored for their survival period. The results in Figure 3B show that lymphoma-bearing C57BL/6 mice treated with dual-anchored EL4/TM-SEA + mB7.1-GPI vaccine survived longer time than those treated with the EL4/mB7.1-GPI vaccine (P < 0.05), the EL4/TM-SEA vaccine (P < 0.05), and the PBS or EL4 vaccine controls (P < 0.01), but there was no significant difference in the survival period between the mice treated with the EL4/mB7.1-GPI and EL4/TM-SEA vaccines (P > 0.05).
Antitumor Activity Induced by Murine B7.1-Glycosylphosphatidylinositol and Transmembrane-Anchored Staphylococcal Enterotoxin A Tumor Cell Vaccine is Mediated by CTLs and Natural Killer Cells
Splenocytes were isolated from the vaccinated mice (in triplicate) 7 days after the last therapy, cocultured with inactivated EL4 cells (treated with mitomycin C, 100 μg/mL at 37 °C for 1 hour) for 7 days in the presence of recombinant mouse IL-2 (20 U/mL, Sigma, St. Louis, MO), collected as CTL effector cells, and tested against wild-type EL4 cells as target cells, whereas B16 cells were used as control target cells. CTL activity was determined at effector:target (E:T) ratios of 12.5:1, 25:1, and 50:1 by a standard CytoTox 96 nonradioactive cytotoxicity assay. Splenocytes derived from 3 dead mice were also used in an NK cell activity assay (with YAC-1 cells used as the target cells) at the E:T ratios of 25:1, 50:1, and 100:1. As shown in Figure 4, lymphocytes derived from the mice treated with the EL4/TM-SEA + mB7.1-GPI vaccine showed the highest CTL and NK activity compared with lymphocytes derived from all other groups. The CTL and NK activity of the mice in the EL4/mB7.1-GPI and EL4/TM-SEA vaccine groups was also much higher than that in the EL4 vaccine group (P < 0.05).
SAgs derived from bacterial or viral products are known as potent activators of T lymphocytes and efficient inducers of cytokine production. This property of SAgs has been used in cancer immunotherapy,24, 25 including applications in which the SAgs were ligated to tumor-specific monoclonal, antiidiotypic, or bifunctional antibodies for tumor-targeted T-cell activation.26, 27 SAgs have also been genetically transferred to tumor cells, and SAgs-expressing tumor cells have been administered as a tumor vaccine in vivo.28, 29 In addition, genetically engineered TM-SEA could anchor on the surface of tumor cells and was capable of eliciting systemic antitumor immunity without any measurable toxicity.17 In the current study, we showed that the tumor vaccine EL4/TM-SEA stimulated lymphocyte proliferation and caused the considerable release of IL-2 and IFN-γ. The tumor growth was significantly inhibited in tumor-bearing mice treated with the EL4/TM-SEA vaccine, and the survival period was also much longer than the controls. However, the antitumor effect of SAg alone is less satisfactory, especially in preestablished tumors.29 A phase of immune exhaustion, including suboptimal production of IFN-γ and IL-2 and failure to mediate CTL activity, can be induced by repeated administration of SAg, which may be due to the anergy and deletion of the responding T cells.30 Our previous study showed that the antitumor effect by fusion protein C215Fab-SEA was insufficient to cure tumor-bearing animals, because complete tumor remission occurred in only 20% of the treated mice.31
Other studies have shown that SAg, in combination with B7 costimulation, induced a strong lymphocyte proliferation response, accompanied by the release of high concentrations of IL-2 and IFN-γ.32, 33 According to the “two signals” theory, T-cell activation requires at least two distinct signals. The first is from peptide-binding MHC molecules on antigen-presenting cells and the second is from the interaction of CD28 and B7.18 Absence of the second signal may result in T-cell clonal anergy. It was demonstrated that tumors lacking B7.1 were poorly immunogenic, and therefore they failed to initiate an appropriate immune response.34 Introducing B7.1 to the tumor cell surface by gene transfection may improve the immunogenicity of tumor cells and result in the rejection of parental tumors in animals.8, 34 However, gene transfection is time consuming, and not all cells are easily receptive to foreign DNA, especially primary tumor cells.
One class of proteins that is well anchored on the surface of eukaryotic cells comprises GPI-modified polypeptides. Unlike polypeptides with hydrophobic transmembrane anchors, GPI-modified polypeptides are directly anchored on the cell surface. Recently, the protein transfer technique of artificial GPI-modified proteins has been used in experimental cancer immunotherapy by McHugh et al.12 These investigators showed that human B7.1-GPI was able to incorporate into many different tumor cell lines such as K1735, Jurkat, and T47D, and that tumor cell vaccines coated with hB7.1-GPI stimulated an antitumor immune response and protected mice from challenge with live wild-type tumor cells. Consistent with their findings, our study showed that the modified tumor cell vaccine EL4/mB7.1-GPI had exhibited T-cell stimulation and specific antitumor effects in vivo.
To enhance the immunogenicity of tumor cell vaccines, we modified the whole tumor cell vaccine with two distinct immunoactive molecules—SAgs SEA and costimulator B7.1. With the protein transfer technique, the genetically engineered mB7.1-GPI and TM-SEA fusion proteins were stably anchored on the surface of EL4 tumor cells simultaneously. The tumor vaccine prepared using EL4 cells coated with both TM-SEA and mB7.1-GPI fusion protein elicited a significantly stronger antitumor immune response than the EL4/mB7.1-GPI vaccine or EL4/TM-SEA vaccine alone, indicating that mB7.1 and SEA may be able to stimulate antitumor immune responses synergistically.
Protein transfer can be applied to both soluble and membrane-associating proteins and has a number of potential advantages compared with gene transfer. First, protein transfer is not dependent on cellular proliferative potential or transfectability. Second, transfection procedures are rather cumbersome, and hence coordinately expressing multiple genes in the same cell is still, in most instances, quite challenging. In contrast, protein transfer permits the simultaneous delivery of an essentially limitless number of proteins to cells. Third, protein transfer, unlike gene transfer, is a rapid procedure and is thus particularly well suited for therapeutic applications.
In summary, the current study investigated a new approach to prepare a dual immune active molecule-anchored tumor cell vaccine with the protein transfer technique, and the results provided experimental evidence that supports the feasibility and effectiveness of this novel approach in cancer immunotherapy.
The authors thank Dr. Joyce Solheim (Eppley Institute for Cancer Research, University of Nebraska Medical Center, Omaha, NE) for her assistance in the preparation of the current article.
- 14Cooperativity of Staphylococcal aureus enterotoxin B superantigen, major histocompatibility complex class II, and CD80 for immunotherapy of advanced spontaneous metastases in a clinically relevant postoperative mouse breast cancer model. Cancer Res. 2000; 60: 2710–2715., , , et al.