Cancer Immunol Immunother (2004) 53: 118–124 DOI 10.1007/s00262-003-0437-0
O R I GI N A L A R T IC L E
Wenxue Ma Æ Hai Yu Æ Qingqing Wang Æ Jianfang Bao Jie Yan Æ Hongchuan Jin
In vitro biological activities of transmembrane superantigen staphylococcal enterotoxin A fusion protein
Received: 20 November 2002 / Accepted: 7 July 2003 / Published online: 22 October 2003 Springer-Verlag 2003
Abstract The bacterial superantigen staphylococcal enterotoxin A (SEA) stimulates T cells bearing certain TCR Vb domains when binding to MHC II molecules, and is a potent inducer of CTL activity and cytokine production. Antibody-targeted SEA such as C215 FabSEA and C242 Fab-SEA has been investigated for cancer therapy in recent years. We have previously reported significant tumor inhibition and prolonged survival time in tumor-bearing mice treated with a combination of both C215Fab-SEA and Ad IL-18 (Wang et al., Gene Therapy 8:542–550, 2001). In order to develop SEA as an universal biological preparation in cancer therapy, we first cloned a SEA gene from S. aureus (ATCC 13565) and a transmembrane (TM) sequence from a c-erb-b2 gene derived from human ovarian cancer cell line HO-8910, then generated a TMSEA fusion gene by using the splice overlap extension method, and constructed the recombinant expression
Drs W. Ma and H. Yu are joint corresponding authors for this article. W. Ma Æ H. Yu Æ H. Jin Cancer Institute, Zhejiang University School of Medicine, 88 Jiefang Road, 310009 Hangzhou, China Q. Wang Institute of Immunology, Zhejiang University, 310006 Hangzhou, China J. Bao Center for Cellular and Molecular Biology, Zhejiang University School of Medicine, 310006 Hangzhou, China J. Yan Department of Microbiology, Zhejiang University School of Medicine, 310006 Hangzhou, China W. Ma (&) Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE 68198-6025, USA E-mail:
[email protected] Tel.: +1-402-5592407 Fax: +1-402-5599543
vector pET-28a-TM-SEA. Fusion protein TM-SEA was expressed in E. coli BL21(DE3)pLysS and purified by using the histidine tag in this vector. Purified TM-SEA spontaneously associated with cell membranes as detected by flow cytometry. TM-SEA stimulated the proliferation of both human PBLs and splenocytes derived from C57BL/6 (H-2b) mice in vitro. This study thus demonstrated a novel strategy for anchoring superantigen SEA onto the surfaces of tumor cells without any genetic manipulation. Keywords Transmembrane Æ Superantigen Æ Cloning Æ Proliferation Æ Anchoring Abbreviations SEA staphylococcal enterotoxin A Æ TM transmembrane Æ NK cell natural killer cell Æ CTL cytotoxic T lymphocyte
Introduction Superantigens (SAgs) include a class of certain bacterial and viral proteins exhibiting highly potent lymphocyte-transforming (mitogenic) activity toward human and or other mammalian T lymphocytes. Unlike conventional antigens, SAgs bind to certain regions of major histocompatibility complex (MHC) class II molecules of antigen-presenting cells (APCs) outside the classical antigen-binding groove and concomitantly bind in their native form to T cells at specific motifs of the variable region of the beta chain (Vb) of the T cell receptor (TCR). This interaction triggers the activation (proliferation) of the targeted T lymphocytes and leads to the in vivo or in vitro release of large amounts of various cytokines and other effectors by immune cells. The biological activities of SAgs make them attractive for use in T-cell activation and elimination of tumor cells expressing major histocompatibility complex (MHC) class II. Staphylococcal enterotoxin A (SEA;
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27.8 kDa) is a bacterial SAg that is produced by Staphylococcus aureus [1]. The SEA gene was cloned and sequenced in 1988 [2]; SEA protein has an extremely potent activity on T lymphocytes presenting MHC class II molecules [3]. The purified toxin has been shown to cause the activation and proliferation of T cells that express a restricted number of TCR Vb domains. SEA stimulates T cells bearing Vb 1, 5.3, 6.3, 6.4, 6.9, 7.4, 9.1, and 23 [4]. The existence of T-cellspecific tumor immunity has been demonstrated in several human malignancies including, melanoma and renal carcinoma [5, 6, 7]; however, MHC class II molecules are also expressed on normal B cells and monocytes. To develop tumor-specific SAgs for cancer therapy, the SEA gene was genetically fused to the Fab region of C215 and C242 monoclonal antibodies (mAbs) specific for human colon carcinoma [8]. The Fab-SEA fusion protein expressed a 100-fold stronger affinity for the tumor antigen than MHC class II molecules [9]. Both in vitro and in vivo studies indicated that Fab-SEA could target cytotoxic T cells against MHC class II) tumor cells bearing the proper tumor antigen without obvious systemic side effects [10, 11, 12, 13, 14]. However, not all the malignancies express C215 or C242 antigen, and therefore, antibodytargeted SEA is limited by its applicability to only certain tumor types. Tumor cells escape protective immune response because they lack or down-regulate stimulatory surface antigens [15]. Introduction of immunostimulatory antigens, for example, MHCI, MHC II, or B7-1 onto the tumor cells can induce antitumor immunity as demonstrated by rejection of parent tumor in vivo [16]. These approaches include transfection of the relevant genes into tumor cells; however, transfection is time consuming, and primary tumor cells often do not grow very well in vitro. Hence, the application of this strategy to cancer therapy might be limited. The transmembrane (TM) protein-encoding sequence corresponding to amino acid residues 644–687 derived from the c-erb-b2 gene [17], has been shown to have hydrophobic characteristics. Increasing the hydrophobicity of this sequence has been shown to change the translocating sequence into a stop anchor sequence [18, 19]. It has been further demonstrated in E. coli that the hydrophobic segment can facilitate its own membrane insertion [20], and the hydrophilic transmembrane segments have considerable freedom to move in relation to the membrane [21]. We therefore hypothesized that bacterial SAg can be introduced as a tumor surface antigen without transfection through fusion of the SAg with a TM protein. In the current study, we have genetically fused the SEA gene to the hydrophobic TM sequence (TM-SEA) and have studied the in vitro biological activities of the TM-SEA fusion protein, including the ability of the fusion protein to anchor onto tumor cells and to stimulate the proliferation of both human PBLs and splenocytes derived from C57BL/6 (H-2b) mice.
Materials and methods Bacterial strain and cell lines S. aureus (ATCC 13565) producing staphylococcal enterotoxin A was purchased from ATCC, and was grown on brain heart infusion (BHI) medium plate supplemented with adenine, guanine, cytosine, and uracil (final concentration of each was 5 lg/ml), and thymine (20 lg/ml). For transformation and protein expression, E. coli BL21(DE3)pLysS (Novagen) and JM109 (Invitrogen) were routinely cultured in LB (Luria-Bertani) medium. Antibiotics were added in the following concentrations: ampicillin (100 lg/ml), kanamycin (50 lg/ml), and chloramphenicol (34 lg/ml). HO-8910 cancer cell line, derived from human ovarian cancer, which express the c-erb-b2 gene [22] and B16 cell line, derived from C57BL/6 mice (H-2b), were maintained in RPMI-1640 medium supplemented with penicillin 100 U/ml, streptomycin 100 lg/ml, and 10% fetal calf serum (FCS). All culture media were purchased from Gibco-BRL (Gaithersburg, MD, USA).
Mice Female wild-type C57BL/6 mice (H-2b), 6–8 weeks old, purchased from joint ventures Sipper BK experimental animal company (Shanghai, China), were housed in a specific pathogen-free condition at the experimental animal center of Zhejiang University. All experiments were conducted in accordance with Zhejiang University Animal Facility guidelines.
Reagents The reagents used were isopropyl-D-thiogalactopyranoside (IPTG), 5-bromo-4-chloro-3-indolyl-b-D-galactoside (X-gal), MTT, antirabbit IgG/HRP, and antirabbit IgG/FITC (Sigma), purified SEA and rabbit anti-SEA IgG (Toxin Technology, Sarasota, FL), restriction endonucleases Nhe I and Hind III, T4 DNA ligase, RNase A and pGEM-T vector (Promega), high fidelity PCR polymerase (Roche), prokaryotic expression vector pET-28a, and the Ni-NTA His.Bind Resin Purification System (Novagen), QIAquick DNA Gel Extract Kit and Mini-preparation of Plasmid Kit (QIAGEN), Limulus amoebocyte lysate (LAL) assay kit (Charles River Endosave, Charleston, SC), ECL detection reagents and Hyperfilm-ECL (Amersham, Arlington Heights, IL).
Construction of the expression vector containing TM-SEA target gene For cloning and subcloning of PCR products, pGEM-T and pET28a vectors were used, respectively. All DNA manipulations and analyses were performed according to standard procedures [23]. Also, the genome DNA from S. aureus (ATCC 13565) was extracted as described [24]. The SEA gene was cloned by using primer 1 and 2; and total RNA from the HO-8910 cell line was isolated using TRIzol reagent according to the manufacturers instructions. The cDNA product of reverse transcription was used as the template for cloning TM fragment by using primer 3 and 4. The amplified SEA gene (796 bp) and TM encoding sequence (155 bp) were purified and used as templates in subsequent PCR. The TMSEA fusion gene was generated using primer 1 and primer 4 by splice overlap extension, and the amplified target fragment (922 bp) was ligated into a predigested pGEM-T vector. Positive recombinant clones were confirmed by restriction endonuclease digestion with Nhe I and Hind III, as well as DNA sequencing, after which the subcloning was completed, and confirmed again by DNA sequencing. The target protein was expressed with the engineered strain BL21(DE3)pLysS-pET-28a-TM-SEA.
120 Polymerase chain reaction and sequence analysis Typical PCR reaction was performed with a Perkin-Elmer 2400 thermocycler in a volume of 100 ll, containing 30 ng denatured DNA, PCR buffer, dNTP (dATP, dGTP, dCTP and dTTP, each at a final concentration of 1.25 mmol/l), 2.5 mmol/l of MgCl2, 100 pmol of each primer, and 0.5 ll of Taq polymerase. Primer 1 was 5-GCCGCTAGCATGAAAAAAAC AGCATTTAC-3 (Forward); primer 2, 5-GCTCTCTGCTCGGCACTTGTATATAAA-3 (Reverse); primer 3, 5-TTTATATACAAGTGCCGAGCAGAGAGC-3 (Forward); and primer 4, 5-AAGCTTCTTACATCGTGTACTTCCG-3 (Reverse). Primer 1 and 4 were designed to include Nhe I and Hind III restriction sites (underlined). PCR was performed for 35 cycles with 3 min of denaturation at 94C, 1 min of an annealing temperature at 56C, and extension for 1 min at 72C. Nucleotide sequencing was finished by the sequencing services of Southern Human Genome Center of China. All the sequences were confirmed in both directions using recombinant pGEM-T and pET-28a vectors containing the TMSEA target gene.
Charleston, SC). A standard curve ranging from 50 to 0.0005 endotoxin units (EU)/ml was prepared in duplicate from E. coli O55:B5 control standard endotoxin supplied by the manufacturer. TM-SEA protein was serially diluted from 50 lg to 5 ng/ ml into microtiter plates using pyrogen-free distilled water. Reconstituted LAL reagent was added to the endotoxin standard, and TM-SEA protein sample. TM-SEA protein was assayed three times, both the standard and sample was analyzed in duplicate, and absorption was measured at 405 nm by using a Dynatech 5000 microplate reader. All glassware used in the endotoxin handling was rendered pyrogen-free through heating at 180C for 6 h.
Flow cytometry B16 cells were harvested after incubating with TM-SEA for 4 h at 37C, rinsed with PBS three times, and then incubated with rabbit anti-SEA IgG and FITC-labeled antirabbit IgG, respectively, before performing detection.
Expression and analysis of TM-SEA fusion protein
Proliferation of lymphocytes
Overnight culture of BL21(DE3)pLysS-pET-28a-TM-SEA was diluted at 1:100 with LB medium (10-g Bacto-Tryptone, 5-g Bacto-Yeast extract, 10-g sodium chloride per liter, with 1% glucose), and supplemented with kanamycin (50 lg/ml) and chloramphenicol (34 lg/ml). The cells were then incubated at 37C until the A600 was 0.8 with continuous shaking before transcription was induced by IPTG with a final concentration of 1.0 mmol. The cells were harvested after inducting for 5 h, the cell pellet was resuspended in 50 ml TN buffer (20-mmol Tris-HCI, 300-mmol NaCI, pH 7.9), and was frozen at )80C. The cell pellets were thawed and sonicated 3 times for 15 s on ice, centrifuged at 17,800 g for 30 min at 4C. The supernatant was collected and the protein concentrations were determined by Bradford assay (Sigma). The recombinant fusion protein was analyzed by Western blotting. Sample proteins and controls were resolved using 12% SDS polyacrylamide gel electrophoresis. Proteins were transferred to a PVDF membrane at 100 V for 1 hr with cooling. The blot was soaked in TBST for two rinses of 15 min each and then blocked with 10% nonfat dried milk (NFDM) freshly made in TBST. The blot was incubated on a rotating shaker for 15 min at room temperature or overnight at 4C. The blot was probed with Anti-SEA IgG (Toxin Technology, Sarasota, FL) in TBST/1% NFDM for 1 hr at room temperature and with antirabbit IgG/HRP (Sigma) in TBST/1% NFDM for 30 min at room temperature, and proteins were visualized using ECL detection reagents (Amersham, Arlington Heights, IL). Blots were then exposed to Hyperfilm-ECL (Amersham) for various times depending on the amount of target protein present.
Proliferation of lymphocytes was followed by MTT assay as previously described [25]. MTT was dissolved in DPBS at a concentration of 10 mg/ml and sterilized by passage through a 0.22 lm filter. This stock solution was added to each well of a 96-well cell culture plate, and the plate was incubated at 37C for 4 h. DMSO was added to each well and mixed thoroughly to dissolve the dark blue crystals. After a few minutes of incubation at room temperature to ensure that all the crystals were dissolved, the plates were read on a microplate reader at a wavelength of 570 nm. The biological activities of TM-SEA in vitro were detected using a lymphocyte proliferation assay. Human peripheral blood lymphocytes (PBLs) from the blood of healthy donor and splenocytes isolated from C57BL/6 mice were aliquoted to 5·105 cells/ well in 96-well plates. Inactivated B16 cells (treated with mitomycin C 100 lg/ml for 1 hr at 37C) were incubated with 10-fold dilutions of TM-SEA for 4 h at 37C, and then added to the PBLs or splenocytes at a ratio of 1:1 after washing completely. After incubation for 44 h (37C, 5% CO2), MTT assay was performed on the lymphocytes. Absorbance value was measured at a wavelength of 570 nm and the proliferation effects was reported as a proliferation index (PI), PI = Abs value in experimental groups / Abs value in control groups.
Statistical analysis Statistical analysis was performed using Students t-test. The difference was considered statistically significant when the p value was less than 0.05.
Purification of the TM-SEA target protein The target protein was purified according to the manufacturers instructions. Cell pellets from the cultures induced with IPTG were disrupted by sonication in TN buffer, and the homogenates were centrifuged at 17,800 g for 30 min at 4C. The supernatant containing the target protein was further purified using Ni-NTA His.Bind Resin (Novagen), and the samples were then analyzed by Western blotting. The purified protein was detected by endotoxin assay, and then used for further studies.
Endotoxin assays Endotoxin activity of purified TM-SEA fusion protein was determined by using commercial LAL reagent containing limulus amoebocyte lysate in its haemolymph (Charles River Endosave,
Results Cloning and sequencing of TM-SEA fusion gene The SEA gene and TM sequence were cloned respectively as described in ‘‘Materials and methods.’’ Templates used were the genome DNA derived from S. aureus and the total RNA derived from human ovarian cancer cell line HO-8910, which express the c-erb-b2 gene. The TM-SEA fusion gene was generated by using the splice overlap extension method [26], which was cloned into the pGEM-T vector and sequenced in both directions. The sequence of the fusion gene TM-SEA
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Fig. 1 a Schematic representation of the expression vector containing TM-SEA. Arrows indicate relative positions of the primers. Figure not drawn to scale. TM-SEA was generated by splice overlap extension PCR, subcloning of TM-SEA fusion gene into pET-28a vector results in a N-terminal histidine sequence. Kan denotes the kanamycin resistance gene, ori origin of DNA replication. b Electrophoresis of PCR product of TM, SEA, TMSEA gene and analysis of the recombinant plasmid digested with Nhe I and Hind III. TM fragment, SEA gene, and TM-SEA fusion gene were amplified by PCR, respectively; the recombinant pGEMT and pET-28a plasmids were digested with Nhe I and Hind III, all the PCR and digested products were analyzed by electrophoresis in a 1.7% agarose gel containing 0.5 lg/ml ethidium bromide. M 100 bp DNA ladder plus, Lane 1 TM PCR product (155 bp), lane 2 SEA PCR product (796 bp), lane 3 TM-SEA PCR product (922 bp), lane 4 empty pGEM-T vector, lane 5 recombinant pGEM-T-TM-SEA vector digested with Nhe I and Hind III (913 bp), lane 6 empty pET-28a vector, lane 7 recombinant pET28a-TM-SEA vector digested with Nhe I and Hind III (913 bp)
was consistent with the corresponding sequence in GenBank, and has 100% homogeneity [2, 27]. For constructing the expression vector containing the TM-SEA fusion gene, the inserts were subsequently excised from the recombinant pGEM-T vector with the restriction sites of Nhe I and Hind III, and then subcloned into the pET-28a expression vector based on its open reading frame (ORF), and confirmed by restriction endonuclease digestion and sequencing, as shown in Fig. 1. Expression of TM-SEA fusion gene in E. coli BL21(DE3)pLysS TM-SEA fusion protein derived from the cell lysate and the purified TM-SEA were analyzed by Western blotting. The results showed that a protein with a size of 32.6 kDa accumulated to a high level, and it was consistent with the expected size of the TM-SEA fusion protein (Fig. 2).
Fig. 2 a Western blotting analysis of the target protein TM-SEA from BL21(DE3)pLysS-pET-28a-TM-SEA strain. PVDF membrane was immunoblotted with antisera raised against the following samples order: Lane 1 purified SEA (Toxin Technology, USA) 30 ng/well as a positive control, lane 2 BL21(DE3)pLysS-pET-28a vector , lane 3 engineering strain BL21(DE3)pLysS-pET-28a-TM-SEA without inducement of IPTG, lanes 4–7 engineering strain BL21(DE3)pLysS-pET-28a-TM-SEA was induced 1 h, 3 h, 5 h, and 7 h, respectively, by IPTG with the final concentration of 1.0 mmol. b Western blotting analysis of the purified TM-SEA fusion protein. The samples were resolved on a 12% polyacrylamide gel in the following order: Lane 1 purified SEA (30 ng loaded) as a positive control, lane 2 supernatant of the lysates of engineering strain BL21(DE3)pLysS-pET-28a-TM-SEA containing the target protein, lane 3 unbound protein, lane 4 purified target TM-SEA protein
3,986 times less endotoxic than the control E. coli O55:B5 LPS standard.
TM-SEA spontaneously anchored onto the tumor cells Results from flow cytometry indicated that 94.9% of B16 tumor cells were positive for TM-SEA fusion protein (Fig. 3b). SEA protein did not bind to B16 cells (Fig. 3a). B16 cells were incubated with TM-SEA fusion protein for 4 h, and then the cells were washed and incubated with medium for 4 h, after that treated with antibodies; 87% of B16 cells were positive to the TM-SEA protein (Fig. 3c).
Endotoxicity of purified TM-SEA protein TM-SEA stimulate the lymphocytes proliferation The endotoxicity of purified TM-SEA fusion protein from E. coli BL21(DE3)pLysS was determined by LAL assay, and its endotoxic activity was compared with that of E. coli O55:B5 LPS. The results showed that purified TM-SEA protein from E. coli BL21(DE3)pLysS did exhibit very minimal endotoxicity: it was
Significant proliferation of human PBLs and the splenocytes from C57BL/6 mice was induced after stimulation with TM-SEA of 0.003 nmol and 0.045 nmol, respectively, when compared with the controls (p
