ABSTRACT
Staphylococcal enterotoxin B (SEB), a shock-inducing exotoxin synthesized by Staphylococcus aureus, is an important cause of food poisoning and is a class B bioterrorism agent. SEB mediates antigen-independent activation of a major subset of the T-cell population by cross-linking T-cell receptors (TCRs) with class II major histocompatibility complex (MHC-II) molecules of antigen-presenting cells, resulting in the induction of antigen independent proliferation and cytokine secretion by a significant fraction of the T-cell population. Neutralizing antibodies inhibit SEB-mediated T-cell activation by blocking the toxin's interaction with the TCR or MHC-II and provide protection against the debilitating effects of this superantigen. We derived and searched a set of monoclonal mouse anti-SEB antibodies to identify neutralizing anti-SEB antibodies that bind to different sites on the toxin. A pair of non-cross-reactive, neutralizing anti-SEB monoclonal antibodies (MAbs) was found, and a combination of these antibodies inhibited SEB-induced T-cell proliferation in a synergistic rather than merely additive manner. In order to engineer antibodies more suitable than mouse MAbs for use in humans, the genes encoding the VL and VH gene segments of a synergistically acting pair of mouse MAbs were grafted, respectively, onto genes encoding the constant regions of human Igκ and human IgG1, transfected into mammalian cells, and used to generate chimeric versions of these antibodies that had affinity and neutralization profiles essentially identical to their mouse counterparts. When tested in cultures of human peripheral blood mononuclear cells or splenocytes derived from HLA-DR3 transgenic mice, the chimeric human-mouse antibodies synergistically neutralized SEB-induced T-cell activation and cytokine production.
Staphylococcal enterotoxin B (SEB) is one of several potent exotoxins secreted by Staphylococcus aureus that cause toxic shock syndrome (TSS) (14, 18, 33, 35, 47). This illness, which is characterized by high fever, erythematous rash, and hypotension, can result in multiorgan failure and death. SEB is also a primary cause of classical food poisoning (4). SEB is a superantigen, a category that includes a large number of proteins that can stimulate a large fraction, up to 20%, of the host's T-cell population (41, 42). Like other superantigens, it binds simultaneously to major histocompatibility complex class II (MHC-II) molecules on antigen-presenting cells (APCs) and to the T-cell receptors (TCRs) that incorporate Vβ chains belonging to particular Vβ families or subfamilies (13, 14, 18, 22, 33). The SEB-induced pathology of TSS results from massive induction of proinflammatory cytokines, which include interleukin-2 (IL-2), gamma interferon (IFN-γ), and tumor necrosis factor beta (TNF-β) derived from TH1 cells (2, 18, 32, 35) and IL-1 and TNF-α from activated APCs (34, 41). Notably, SEB is resistant to denaturation and highly toxic (in humans, the estimated 50% lethal dose is <100 ng/kg of body weight and the 50% effective dose is <1 ng/kg by aerosolized exposure [15, 46]) and can be readily produced by the techniques of recombinant DNA technology. These attributes have led to its classification as a priority B bioterrorism agent.
Blockade of SEB's simultaneous cross-linking of MHC-II on APCs to the TCR on T cells prevents the formation of the MHC-II/SEB/TCR complex and inhibits the action of the toxin. A number of experimental approaches to preventing or disrupting the formation of MHC-II/SAg/TCR complexes have been explored by different laboratories. These include immunization with proteasome-SEB toxoid vaccines (29, 30), inactivated recombinant SEB vaccine (5, 26, 52), and synthetic peptides (53) to induce anti-SEB antibodies, passive immunoprophylaxis and immunotherapy with intravenous immunoglobulin (IVIG) (9, 10, 21, 23), the use of peptide antagonists (1-3), synthetic chimeric mimics of MHC-II/TCR complex (19, 27, 36) or mimics of TCR Vβ (7) engineered to interfere with the binding of SEB to the native forms of these receptors on APCs or T cells. Perhaps the most successful of these approaches have involved TCR Vβ chain mimics that blocked SEB activation in vitro and showed promising results when tested in vivo in a rabbit model (7). However, these TCR mimics reported by Buonpane et al. (7) have a short half-life (325 min) in rabbits and are likely to display short half-lives if deployed in clinical settings. However, rapid in vivo turnover of SEB blocking agents can be avoided by use of antibodies well matched to the host's FcRn, a receptor responsible for protecting IgG from proteolysis and hence endowing it with a long half-life (24). The use of monoclonal antibodies to neutralize the effects of SEB was first demonstrated by the pioneering studies of Hamad et al. (17) and later by the work of Pang et al. (39). Furthermore, using genes encoding the V regions of monoclonal antibodies derived in nonhuman species, it has been possible to engineer a number of useful chimeric antibodies that manifest relatively long half-lives and low immunogenicity in humans (8). Confident that the V regions of neutralizing mouse monoclonal anti-SEB antibodies could be chimerized with human constant regions, we selected a library of neutralizing anti-SEB from a collection of monoclonal antibodies derived by immunization of BALB/c mice with native SEB.
We are also aware that the crystal structures of SEB in complex with MHC-II or TCR reveal that the two binding sites are spatially distinct with the contact areas for each of these different binding sites displaying multiple and potentially immunogenic epitopes against which antibodies can be raised (17). Since multiple epitopes are involved in this interaction, it was possible that our library contained neutralizing antibodies directed against different and spatially distinct epitopes. This suggested that a mixture of anti-SEB antibodies directed against spatially separated neutralizing epitopes would be more effective than an equivalent amount of any component of the mixture used alone. In order to test this hypothesis, it was necessary to identify non-cross-reacting neutralizing antibodies in our library. A pair of non-cross-reactive neutralizing anti-SEB monoclonal mouse antibodies was found and a combination of the two produced a greater degree of neutralization in cultures of mouse splenocytes than equivalent amounts of either member of the pair acting alone. This synergistic action was observed whether the mouse antibodies or chimeric equivalents of the antibody pair were used. However, because it is well established that SEB-mediated effects are seen at much lower toxin concentrations in systems bearing human rather than mouse class II MHC (11, 14), it was important to determine the ability of our pair of chimeric antibodies to neutralize SEB in HLA-DR3 transgenic mice, a more demanding and humanlike model system than conventional mice (11, 44, 45, 50, 51). Both chimeric antibodies effected neutralization in this transgenic model and, when used together, were synergistic in their neutralization of SEB in cultures derived from HLA-DR3 transgenic mice. Similar observations were made in cultures of human peripheral blood mononuclear cells (PBMC).
MATERIALS AND METHODS
Mice.BALB/c (H-2d) mice were used for hybridoma production and as a source of splenocytes for some in vitro neutralization studies. HLA-DR3 transgenic mice expressing functional HLA-DRA1*0101 and HLA-DRB1*0301 transgenes on a completely mouse MHC-II-deficient background (AE0) were generated as described elsewhere (43, 50, 51) and are hereafter referred to as DR3 transgenic mice. All mouse work was conducted in accordance with protocols approved by institutional animal care and use committees.
SEB.SEB was purchased from Toxin Technology (Sarasota, FL), and laboratory stocks were always less than the federally mandated limit of 5 mg. The manufacturer has certified that the SEB batch purchased has >95% purity on the basis of gel analysis.
Immunization and hybridoma production.BALB/c mice were primed by intraperitoneal injection of 10 μg of native SEB (Toxin Technology, Inc., Sarasota, FL) per mouse emulsified in complete Freund's adjuvant and boosted once or twice after intervals of 21 days by intraperitoneal injection of 20 μg of native SEB in 0.5 ml of phosphate-buffered saline (PBS). Immunized mice were sacrificed 3 days after secondary or tertiary immunization, and hybridomas were derived and processed along the outlines of the procedures described elsewhere (16).
Neutralization assays.Splenic mononuclear cells from BALB/c or DR3 transgenic mice were isolated by Ficoll-Paque Plus (GE Healthcare, Uppsala, Sweden) gradient centrifugation. The isolated cells were washed two to three times with PBS and resuspended in growth medium (RDGS) containing 45% Dulbecco modified Eagle medium (DMEM), 45% RPMI 1640, 10% fetal bovine serum (FBS), and 55 μM 2-mercaptoethanol (all from Invitrogen, Carlsbad, CA) and 20 μg of gentamicin (Sigma-Aldrich, St. Louis, MO)/ml. Cell suspensions (5 × 106 cells/ml) were distributed in 100-μl aliquots to the wells of 96-well flat-bottom plates (5 × 105 cells/well) containing 50 μl of various concentrations of SEB and 50 μl of the indicated concentration of anti-SEB antibody. Each condition was tested in triplicate. The cells were incubated (at 37°C in 7.5% CO2) for 48 h and then pulsed with 1 μCi of [3H]thymidine (Amersham/GE Healthcare) per well, incubated an additional 18 h, and then harvested onto glass fiber filter strips with a PhD cell harvester (Brandel, Gaithersburg, MD). The incorporated radioactivity was measured by liquid scintillation counting, and stimulation indices (SI) were calculated as follows: SI = (average net cpm from SEB-treated cultures)/(average net cpm from untreated cultures).
Statistical methods.Mean values and their standard deviations of SI and cytokine levels were compared by using Student's t test.
In vitro metabolic labeling of monoclonal antibodies.Monoclonal anti-SEB antibody secreting hybridoma cells were harvested by centrifugation and washed in 37°C cysteine-methionine (CM)-free DMEM (Invitrogen, Carlsbad, CA), 9% heat-inactivated dialyzed FBS, and 1% undialyzed FBS. Cells were suspended at 5 × 106 cells/ml in CM-free DMEM, and 1-ml aliquots were distributed to the wells of a 24-well cell culture dish. Then, 10 μCi of 35S-labeled CM (Perkin-Elmer Life and Analytical Sciences, Boston, MA) in 0.1 ml of CM-free DMEM was added to each well, and the cultures were incubated for 24 h at 37°C in 7.5% CO2, after which 1 ml of CM-free DMEM was added to each well, and the contents were subsequently quantitatively transferred to centrifuge tubes and spun down at 1,500 rpm (400 × g) for 10 min. The supernatants were then transferred to a fresh tube and spun for an additional 10 min. Harvested supernatants were transferred into Slide-A-Lyzer dialysis cassettes (Pierce, Rockford, IL) and dialyzed overnight at 4°C against two changes of serum-free growth medium.
Competition assay.Subsets of neutralizing anti-SEB antibodies that do not cross-react with the same epitope were identified by using competition assays by incubating equimolar concentration of 35S-labeled 82M.1.2 or 63.1.1 with an unlabeled member of the set and compared to the radioactivity bound during parallel incubations with growth medium (negative control) and with unlabeled 82M.1.2 or 63.1.1 as appropriate. Parallel incubations were performed on SEB-coated and on bovine serum albumin (BSA)-coated 96-well flexible immunoplates. After a 1-h incubation, plates were washed five times with 0.1% BSA-PBS; wells were then cut out of the plates and transferred into scintillation vials, and the bound radioactivity was counted with the aid of a liquid scintillation counter. Subset members that reduced binding of radioactivity by ≥30% are considered to compete with the labeled member for the same epitope or overlapping epitopes. The chances of detecting competition between a more strongly binding labeled antibody and a weaker binding competitor were increased by repeating the competition assay using a 10-fold excess of the unlabeled competitor.
IFN-γ and IL-2 ELISA.Splenic mononuclear cells were isolated from DR3 transgenic mice as described above. Cells (5 × 105/well in 96-well flat bottom plates) were incubated in RDGS medium at 37°C in 7.5% CO2 with the indicated concentrations of SEB or SEB and antibody. After 24 h of incubation, supernatants were collected and IL-2 and IFN-γ were measured by enzyme-linked immunosorbent assay (ELISA) using a sandwich assay involving capture and detecting anti-IL-2 or anti-IFN-γ antibodies (BD Pharmingen, San Diego, CA). The serum levels of IL-2 were measured by ELISA from blood samples collected 6 h after the indicated treatment.
Engineering of neutralizing chimeric human-mouse anti-SEB antibody.Total RNA was made from selected anti-SEB-secreting hybridomas using the Ultraspec RNA isolation system (Biotecx Laboratories, Houston, TX) according to the manufacturer's instructions and used as a template for cDNA synthesis according to standard protocols. The cDNA was amplified by PCR using universal primer sequences designed for the amplification of mouse immunoglobulin VL and VH genes (54); the PCR products were gel purified with a QIAquick gel extraction kit (Qiagen, Valencia, CA) and sequenced, and a second PCR was performed to incorporate cloning sites necessary for insertion into human immunoglobulin VH (BssHII and SalI) and VL (ApaLI and BglII) expression cassettes (40) using primers that contain the desired restriction sites. The PCR products obtained were then cloned into the TOPO TA cloning vector and transformed into TOP10 Escherichia coli by using a TOPO TA cloning kit (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Plasmid DNA was purified from overnight cultures of E. coli using the QIAprep spin mini-prep kit (Qiagen, Valencia, CA), quantified, and digested with the indicated restriction enzymes to confirm the presence of VH and VL inserts. Mouse anti-SEB binding VH and VL fragments were cloned into the corresponding restriction sites of the VHExpress and VκExpress plasmids (40), and the chimeric constructs were transformed into DH5α for amplification. Plasmids were purified and digested with restriction enzymes to confirm the presence of VH and VL inserts. The chimeric constructs were sequenced to confirm appropriate insertion, and in-frame human-mouse chimeric constructs were then transfected into SP2/0 cells by electroporation using a cell line Nucleofector kit (Amaxa, Gaithersburg, MD). Double transfectants were selected with neomycin (for the HC-bearing plasmid) and mycophenolic acid (for the LC-bearing plasmid). Supernatants from clones that survived double selection were tested for the secretion of human anti-SEB antibodies by ELISA.
Affinity measurements.Purified monoclonal antibodies were coupled to the dextran surfaces of CM-5 chips by using standard 1-ethyl-3-(3-dimethylaminopropyl)-1-carbodiimide hydrochloride-N-hydroxysuccinimide coupling chemistry in a BIACore 2000. The amount coupled varied from 700 to 900 resonance units. Graded concentrations of SEB ranging from 1.5 to 500 nM were offered at a flow rate of 10 μl/min in 10 mM HEPES (pH 7) buffer containing 150 mM NaCl, 3 mM EDTA, and 0.005% Tween 20 at 25°C. Background binding to an IgG1 surface was subtracted, and the data were analyzed by using Scrubber 2 by steady-state analysis since the dissociation half times were too long for kinetic assessment.
RESULTS
Generation of hybridomas secreting neutralizing anti-SEB antibodies.Previous studies have demonstrated that the MHC-II and TCR binding sites of SEB are found in regionally distinct sites of SEB (28), and each of these different binding sites display multiple epitopes against which antibodies might be raised (28). Our work required a library of neutralizing antibodies from which non-cross-reacting neutralizing antibodies could be isolated and tested for synergistic inhibition of SEB-mediated T-cell activation. A series of fusions of spleen cells from primed and boosted BALB/c mice allowed us to accumulate a library of hybridoma lines secreting monoclonal anti-SEB antibody. Further analysis identified a subset that had SEB-neutralizing capacity (Table 1). Figure 1 presents data from a representative sample of neutralizing anti-SEB antibodies. Varying in their capacity to inhibit SEB-induced T-cell proliferation, all of the antibodies demonstrate greater degrees of inhibition against lower than against higher concentrations of the toxin. However, inhibition is clear at both concentrations of SEB.
Neutralization data produced by a representative sample of neutralizing anti-SEB monoclonal antibody obtained with 100 ng of SEB/ml (a) or 10 ng of SEB/ml (b). BALB/c splenocytes (5 × 105 cells/well) were incubated at 37°C for 48 h in the presence of medium alone, SEB alone (10 ng/ml or 100 ng/ml), SEB plus anti-SEB monoclonal antibody (2 μg/ml), or SEB plus isotype control [2 μg of mouse IgG1(κ)/ml] as indicated. Cells were then pulsed with [3H]thymidine (1 μCi/well), incubated for an additional 18 h, and harvested, and cell proliferation was determined by measuring [3H]thymidine incorporation. Each bar represents the means ± the standard deviation (SD) of triplicate measurements, and the data shown are representative of two or more independent experiments. Neutralization of SEB-induced T-cell proliferation by anti-SEB antibodies were significant (P < 0.01) compared to results obtained with SEB alone or isotype control.
Library of anti-SEB monoclonal antibodiesa
Identification of non-cross-reacting anti-SEB antibodies that synergistically neutralize SEB.Since multiple epitopes are involved in the interaction of SEB with its ligands, it was possible that our library contained neutralizing antibodies directed against different and spatially distinct epitopes. A selected subset of the library of neutralizing antibodies listed in Table 1 was subjected to competition to determine whether antibodies binding different epitopes could be identified. We identified a pair of non-cross-reacting antibodies, 82M.1.2 and 63.1.1 that reacted with different epitopes. Table 2 demonstrates that these antibodies do not compete with each other for binding to epitopes on SEB. It is also clear that the subset of antibodies tested contains other antibodies that do not compete with 82M.1.2 or 63.1.1 and some that do compete with one or the other of these antibodies. Neutralization assays were conducted using equimolar amounts of 82M.1.2, 63.1.1, and a mixture containing 50% of each. The data in Fig. 2 show that at concentrations of 10 μg of either antibody/ml, SEB-induced cell proliferation was inhibited: 82M.1.2 gave 3-fold inhibition, and 63.1.1 delivered 4-fold inhibition. However, treatment with a combination of 82M.1.2 plus 63.1.1, each present at a concentration of 5 μg/ml, gave an inhibition of 24-fold. This synergistic effect is clearly greater than the additive effect of the antibodies acting alone.
Non-cross-reactive neutralizing antibodies act synergistically. BALB/c splenocytes (5 × 105 cells/well) were incubated at 37°C for 48 h with growth medium (GM), SEB alone (1 μg/ml), SEB plus 10 or 5 μg of α-SEB 82M.1.2/ml, SEB plus 10 or 5 μg of α-SEB 63.1.1/ml, SEB plus the combination of (5 μg of α-SEB 82M.1.2/ml plus 5 μg of α-SEB 63.1.1/ml), or SEB plus 10 μg of mouse IgG1(κ)/ml. Cells were then pulsed with [3H]thymidine (1 μCi/well), incubated for an additional 18 h, and harvested, and cell proliferation was determined by measuring [3H]thymidine incorporation. Each bar represents the means ± the SD of triplicate measurements, and the data shown are representative of two or more independent experiments. The combination of 82M.1.2 and 63.1.1 provided significant neutralization (P < 0.01) compared to each antibody alone.
Competition assay between 35S-labeled and unlabeled anti-SEB monoclonal antibodies
Construction of neutralizing human-mouse chimeric anti-SEB antibodies.To construct and test chimeric versions of the synergistically acting pair of mouse antibodies described above, the mouse VH and VL regions of these antibodies were amplified by reverse transcription-PCR and cloned into VHExpress and VκExpress plasmids, which incorporate sequences that direct the synthesis of the human IgG1 heavy-chain and the human kappa light-chain constant regions (40). Gel characterization and sequencing confirmed the in-frame joining of VH and VL (Fig. 3). SP2/0 cells doubly transfected with the two constructs secreted an antibody that reacted with SEB and, indicative of its chimeric nature, could be detected by a polyclonal anti-human antibody (Fig. 4) or a polyclonal anti-mouse antibody (data not shown).
(A) Results of restriction enzyme digestion of the chimeric human-mouse construct. Plasmids purified from overnight cultures of DH5α cells transformed with the chimeric constructs were digested with BssHII and SalI for the HC construct and with ApaLI and BglII for the LC construct. The expected size of VH (∼400 bp) and VL (∼340 to 350 bp) inserts were present in all clones tested, indicating that cloning of mouse VH and VL chains into human immunoglobulin expression vectors was successful. Lanes: 1, 100-bp ladder; 2 to 7, digested HC (panel 1) and LC (panel 2) constructs; 8, digested VH and Vκ express vectors; 9, undigested HC and LC chimeric constructs; 10, 1-kb DNA ladder. (B) Nucleotide and translated amino acid sequences of the VH and VL Region of Ch 82M (a) and Ch 63 (b). Sequences in boldface correspond to the portion of the sequence from the human IgG1 heavy-chain (VHExpress) and Igκ light-chain (VκExpress) expression vectors.
Transfected SP2/0 cells secrete chimeric human-mouse SEB-specific antibody. Supernatants from untransfected SP2/0 and SP2/0 transfected with chimeric HC and LC plasmids encoding expression of chimeric anti-SEB were added to wells of plates coated with SEB, unlabeled rabbit anti-human (RAH), or 0.1% BSA in PBS. Other wells received the indicated negative controls, polyclonal human immunoglobulin, supernatants from untransfected SP2/0, or growth medium. After incubation and washing, the wells were treated with rabbit anti-human IgG-HRP (H+L) to determine whether the transfected cells generated antibody with human sequences that was reactive with SEB. The absorbance was measured at 405 nm. Each bar represents the means ± the SD of triplicate measurements, and the data shown are representative of two or more independent experiments. Binding of chimeric anti-SEB antibody to SEB-coated plate was significantly higher (P < 0.001) than human IgG control, supernatants from untransfected SP2/0 and growth medium. On the other hand, binding of the chimeric anti-SEB antibody to RAH-coated plate was not significantly different (P > 0.05) from the human IgG control.
The chimeric anti-SEB antibodies and their mouse counterparts have similar affinities.To determine the binding affinities to SEB of the murine antibodies, as well as their chimeric counterparts, we undertook surface plasmon resonance (SPR) binding studies. Side by side SPR determinations of affinity (Fig. 5) show that affinities of the mouse antibodies are 63.1.1 (KD = 449 pM) and 82M.1.2 (KD = 704 pM), while those of the chimeric antibodies are Ch 63 (KD = 437 pM) and Ch 82M (KD = 602 pM). Clearly, all four antibodies bind to SEB with KD values in the subnanomolar range. These data show that replacement of the murine heavy-chain constant domains with human domains yields antibodies with affinities quite similar to the parent mouse antibodies. Furthermore, as demonstrated by their inhibition of SEB-induced T-cell proliferation in splenocyte cultures, both chimeric human-mouse anti-SEB antibodies (Ch 82M and Ch 63) retained neutralization ability (Fig. 6 and 7B).
SPR determinations of the affinity (expressed as KD) of monoclonal mouse anti-SEBs 63 and 82M and of their chimeric counterparts. Antibodies were coupled by standard amine coupling chemistry to the dextran surfaces of a CM-5 chip. Serial 2-fold dilutions of SEB ranging from 62.5 to 1.5 nM were sequentially injected over each surface at a flow rate of 10 μl/min. The zero time point is the start of the SEB injection. Buffer washout was initiated at 240 s. The data were globally fit to a simple monovalent interaction model in BIAeval 3.2 to obtain the indicated KD values. The data points are indicated by dotted lines in shades of gray, and the corresponding curve fits are displayed in black. 63, Mouse anti-SEB 63.1.1; 82M, mouse anti-SEB 82M.1.2.
(A) Chimeric anti-SEB neutralizes SEB-induced T-cell proliferation. BALB/c splenocytes (5 × 105 cells/well) were incubated at 37°C for 48 h with growth medium (GM) alone, with various concentration of SEB alone, SEB plus supernatants from mouse anti-SEB 82M.1.2-secreting cell culture, or SEB plus supernatants from chimeric 82M-secreting cells. Cells were then pulsed with [3H]thymidine (1μCi/well), incubated for an additional 18 h, and harvested, and the cell proliferation was determined by measuring [3H]thymidine incorporation. Each bar represents the means ± the SD of triplicate measurements, and the data shown are representative of two or more independent experiments. Neutralization of SEB-induced T-cell proliferation by mouse and chimeric anti-SEB antibodies were significant (P < 0.01) compared to cells treated with SEB alone. (B) Chimeric anti-SEB neutralizes SEB-induced T-cell activation in cultures of DR3 transgenic mice splenocytes. Splenocytes from DR3 transgenic mice (5 × 105 cells/well) were incubated for 48 h in the presence of medium alone, various concentrations of SEB, SEB plus supernatants from untransfected SP2/0 cells, SEB plus supernatants from mouse anti-SEB 82M.1.2-secreting cell culture, and SEB plus supernatants from chimeric 82M-secreting cell cultures. Cells were then pulsed with [3H]-thymidine (1 μCi/well) and then incubated for an additional 18 h. At the end of the incubation, the cells were harvested, and cell proliferation was determined by measuring the level of [3H]thymidine incorporation by using a liquid scintillation counter. Each bar represents the means ± the SD of triplicate measurements, and the data shown are representative of two or more independent experiments. Neutralization of SEB-induced T-cell proliferation by mouse and chimeric anti-SEB were significant (P < 0.05) compared to cells treated with SEB alone or supernatants from untransfected SP2/0.
(A) Comparison of SEB-induced T-cell proliferation in cultures derived from BALB/c and DR3 transgenic mice. DR3 and BALB/c splenocytes (5 × 105 cells/well) were incubated for 48 h in the presence of medium alone or with various concentrations of SEB. Samples were then pulsed with [3H]thymidine (1 μCi/well) and incubated for an additional 18 h, after which cells were harvested, and cell proliferation was determined by measuring the level of [3H]thymidine incorporation using a liquid scintillation counter. Each point represents the mean of triplicate samples ± the SD, and the data shown are representative of two or more independent experiments. DR3 transgenic mice responded to 1,000-fold lower amounts of SEB than BALB/c mice, indicating the differences in sensitivity of these two animal models to SEB. At 0.001, 0.01, 0.1, 1, and 10 ng/ml, SEB-induced T-cell proliferation levels were significantly higher (P < 0.001) in DR3 than in BALB/c splenocytes. (B) Neutralization and synergy in cultures derived from DR3 transgenic mice. DR3 transgenic mice splenocytes (5 × 105 cells/well) were incubated at 37°C for 48 h with growth medium (GM), SEB (10 or 1 ng/ml as indicated), SEB plus human IgG1(κ) (10 μg/ml); SEB plus chimeric α-SEB 82M (10 μg/ml), SEB plus chimeric α-SEB 63 (10 μg/ml), or SEB plus a combination of 5 μg of Ch 82M/ml plus 5 μg of Ch 63/ml. The cells were pulsed with [3H]thymidine (1 μCi/well), incubated an additional 18 h, and harvested, and the cell proliferation was determined by measuring the [3H]thymidine incorporation. Each determination was run in triplicate, and the results are means with error bars showing the SD; the data shown are representative of two or more independent experiments. Neutralization between the combination of anti-SEBs and either antibody used alone was significant (P < 0.01).
Chimeric anti-SEB antibodies neutralize SEB in splenocytes of HLA-DR3 transgenic mice and in human PBMC.Unlike human class II molecules, mouse class II MHC molecules lack a critical lysine at position 39 of the IEα chain that is necessary for high-affinity binding of SEB and many other bacterial SAgs (25). Consequently, antibody-mediated inhibition of SEB-induced T-cell activation in mouse systems might not be a true predictor of the ability of such an antibody to inhibit SEB activation in human systems. To obviate such differences between in vitro cultures of cells derived from mice versus those from humans, we used DR3 transgenic mice that have been shown to be more sensitive to SEB than cells from mice bearing mouse MHC-II (11, 44).
As shown in Fig. 7 A, spleen cells from DR3 transgenic mice were found to be more than 1,000 times more sensitive to SEB than those from BALB/c mice. Neutralization assays using splenocytes from DR3 transgenic mice indicated that chimeric anti-SEB antibodies neutralized SEB-induced T-cell activation in vitro (Fig. 6B) and, like the mouse anti-SEB 82M.1.2 and 63.1.1 antibodies, the chimeric versions of these two antibodies neutralized SEB-induced T-cell activation synergistically (Fig. 7B).
In addition to determinations of the effects of the chimeric antibodies in cultures of DR3 splenocytes, the antibodies were also tested in cultures of human PBMC and yielded the results shown in Fig. 8A. The data show that SEB-induced activation of human PBMC can be inhibited by either of the chimeric versions of these antibodies and that the chimeric human-mouse antibodies synergistically protect cultures of human PBMC from SEB-induced activation. Since under physiological conditions, SEB is likely to be present at low concentrations, chimeric forms of the antibodies were also tested for their capacity to inhibit low concentrations of SEB. The data in Fig. 8B demonstrate that either antibody Ch 63 or Ch 82M neutralize pictogram concentrations of SEB.
(A) Neutralization and synergy in cultures of human PBMC. Human PBMC (5 × 105 cells/well) were incubated at 37°C for 48 h with growth medium alone (GM), SEB (1 or 0.1 ng/ml as indicated), SEB plus human IgG1(κ) (10 μg/ml), SEB plus chimeric α-SEB 82M (10 μg/ml), SEB plus chimeric α-SEB 63 (10 μg/ml), or SEB plus the combination of 5 μg of Ch 82M/ml plus 5 μg of Ch 63/ml. Cells were pulsed with [3H]thymidine (1 μCi/well), incubated an additional 18 h, and harvested, and the cell proliferation was determined by measuring [3H]thymidine incorporation. Each determination was run in triplicate, and the results are means with error bars showing ± the SD; the data shown are representative of two or more independent experiments. Neutralization between the combination of anti-SEBs and either antibody used alone were significant (P < 0.05). (B) Chimeric anti-SEB 82M and 63 neutralize low concentrations of SEB. Human PBMC (5 × 105 cells/well) were incubated at 37°C for 48 h with growth medium, SEB (various concentrations as indicated), SEB plus chimeric α-SEB 82M (10 μg/ml), or SEB plus chimeric α-SEB 63 (10 μg/ml). Cells were pulsed with [3H]thymidine (1 μCi/well), incubated an additional 18 h, and harvested, and the cell proliferation was determined by measuring [3H]thymidine incorporation. Each determination was run in triplicate, and the results are means with error bars showing the SD; the data shown are representative of two or more independent experiments. At all SEB concentrations, neutralization of SEB-induced T-cell proliferation by Ch 82M or Ch 63 was significant (P < 0.01) compared to that of cells treated with SEB alone.
Anti-SEB antibodies neutralize SEB-induced cytokine production in vitro and in vivo.SEB-mediated T-cell activation induces the synthesis of a number of cytokines. Complementary to the finding of inhibition of T-cell proliferation in DR3 transgenics, measurement of key cytokine synthesis also showed that the chimeric forms of the antibodies significantly inhibited IL-2 and IFN-γ synthesis in cells derived from DR3 transgenic mice. Furthermore, a combination of Ch 82M and Ch 63 demonstrated synergy of inhibition of cytokine synthesis (Fig. 9A). In a pilot in vivo experiment, administration of either antibody to DR3 transgenic mice injected with 50 μg of SEB, significantly (P < 0.05) reduced IL-2 production (Fig. 9B).
(A) Anti-SEB antibodies neutralize SEB-induced cytokine production in cultures of splenocytes from DR3 transgenic mice. Splenocytes from DR3 transgenic mice were incubated for 24 h in the presence of medium alone, SEB alone, SEB plus Ch 82M (10 μg/ml), SEB plus Ch 63 (10 μg/ml), SEB plus Ch 82M plus Ch 63 (5 μg of each/ml), or SEB plus human IgG1(κ) (10 μg/ml). SEB was used at a concentration of 10 ng/ml. Cell-free supernatants were collected and IL-2 and IFN-γ measured by ELISA. Each bar are means ± the SD among triplicate samples, and the data shown are representative of two or more independent experiments. Neutralization between the combination of anti-SEBs and either antibody used alone were significant (P < 0.01). (B) Chimeric anti-SEB 82M and Ch 63 neutralize SEB-induced IL-2 production in vivo. Serum levels of IL-2 were determined at 6 h post-SEB injection in groups of DR3 transgenic mice treated with the following: naive, PBS only; Ch 82M plus SEB, 50 μg of SEB plus 1 mg of Ch 82M; Ch 63 plus SEB, 50 μg of SEB plus 1 mg of Ch 63; and SEB alone, 50 μg of SEB in PBS. Each bar are means ± the SD among triplicate samples. Neutralization of SEB-induced IL-2 production by Ch 82M or Ch 63 was significant (P < 0.05) compared to mice injected with SEB alone.
DISCUSSION
The key step in SEB-mediated T-cell activation is the cross-linking of class II MHC molecules on APCs to the TCRs of T cells by the binding of this toxin, an interaction readily inhibited by appropriate anti-SEB antibodies. By immunizing mice with native toxin, we derived several hybridomas secreting anti-SEB which neutralized SEB-mediated induction of T-cell proliferation or secretion of IL-2 or IFN-γ, two cytokines that typically accompany SEB-mediated T-cell activation.
However, all of our initial tests for neutralization were performed in mouse systems. Since this work was undertaken to identify antibodies that might ultimately find application in the prophylaxis and treatment of SEB intoxication in humans, evaluation in systems bearing a human version of MHC-II were required since SEB has a higher affinity for human class II MHC than for mouse class II (32, 42). Consequently, it is possible that some antibodies that bind neutralizing epitopes of SEB that happen to bind MHC-II with sufficient affinity to prevent their binding to mouse class II may not successfully compete with human MHC-II for these epitopes. Therefore, antibodies identified as particularly interesting in tests using splenocytes from normal mice were also tested in cultures derived from transgenic mice that bear HLA-DR3 and in cultures of human PBMC. With regard to the specificity of the monoclonal anti-SEBs used in the present study, we have checked them for activity against the superantigen toxins, TSST-1 and SEA, and found that they failed to react with either of these. This differs sharply from the behavior of batches of human IVIG, which is polyclonal and contains a mixture of anti-toxin antibodies having various specificities, the result being a preparation that has antibodies against a variety of toxins and therefore can be broadly neutralizing. However, despite the ability of some batches of IVIG to broadly neutralize, batches of IVIG can differ considerably in their titers of neutralizing antibody against various SAg toxins (10, 37, 48).
SEB interacts with the β chain of the TCR, and a distinct but adjacent site interacts with the α chain of the MHC-II molecule (28). This led us to the hypothesis that a combination comprised of a pair of neutralizing antibodies that bound to different and spatially separated epitopes of the toxin would be more effective neutralizers than either member of the pair alone. We used competition assays to determine whether the library contained members that reacted with different epitopes and identified a pair of such antibodies. A combination comprised of such a pair was compared in neutralization assays with equimolar amounts of either member of the pair alone. The combination was significantly more effective and the effect of combining the pair was more than additive, i.e., synergistic. It was clear in in vitro experiments and in a pilot in vivo trial that the use of neutralizing antibodies targeting different and non-cross-reacting epitopes allow greater inhibition with smaller quantities of antibody.
A long-range goal of this work is the generation of antibodies that might be considered for the prophylaxis and treatment of SEB intoxication in humans. However, the immunogenicity of the mouse monoclonal anti-SEBs initially generated in these studies makes them unsuitable for this purpose. Fortunately, a number of examples demonstrate that the creation of chimeric human-mouse antibodies provides a practical method for using mouse monoclonal antibodies with useful binding properties as sources of variable regions for the engineering of antibodies that have low and acceptable levels of immunogenicity in humans. To test the properties of chimeric versions of the synergistically acting pair of mouse anti-SEBs (anti-SEB 82M.1.2 and 63.1.1) identified during the course of this work, we engineered human-mouse chimeric forms of each of the members of this pair (chimeric 82M [Ch 82M] and chimeric 63 [Ch 63]). Studies using SPR comparing each of these chimeric antibodies to their mouse counterparts demonstrated that the chimeric versions had affinities that were indistinguishable from their mouse counterparts. Functional studies showed that each of the chimeric antibodies effectively neutralized SEB in splenocyte cultures from BALB/c, HLA-DR3 transgenic mice and in cultures of human PBMC, demonstrating that chimeric forms of these antibodies are effective and synergistic in systems featuring human class II MHC. This result is fully consonant with the kinetic and equilibrium properties of the mouse and human-mouse chimeric forms of these antibodies, which were shown by SPR to be practically identical.
Because SEB is of clinical and biosecurity interest, many approaches to its prophylaxis and therapy have been explored. Many of these have sought to prevent or disrupt the formation of MHC-II/SAg/TCR complexes. These have ranged from explorations of active immunization with inactivated recombinant SEB vaccines (5, 26, 52), synthetic peptides (53), and proteasome-SEB toxoid combinations (29, 30) to investigations of antibody-based passive immunoprophylaxis/immunotherapy (9, 10, 21, 23, 38, 49), as well as synthetic peptides antagonists (1-3) and receptor mimics such as chimeric mimics of MHC-II-TCR (19, 27, 36) and of the TCR Vβ (7). Although all of these approaches have shown some promise, based on the long history of clinical experience with the use of antibodies, our approach has focused on demonstration of an approach to the derivation of forms of monoclonal anti-SEB antibodies that might be suitable for clinical use. Our pursuit of this approach is encouraged by the knowledge that some batches of IVIG has been shown to have significant titers of neutralizing antibody against SEB. Furthermore, chimeric human-mouse antibodies, such as the anti-CD20, Rituxan, and the anti-TNF-α, Remicade, have been in clinical use for many years (20, 31). We choose to construct chimeric anti-SEBs because years of clinical experience with chimeric human-mouse antibodies has shown that despite the fact that their VH and VL sequences (about one-third of the total amino acid sequence) are of mouse origin, the levels of human anti-chimeric antibody (HACA), though variable, are often low, anaphylactic reactions are uncommon, and true serum sickness is rare (12). Although it remains difficult to predict the immunogenicity of any novel therapeutic protein, including an immunoglobulin, chimeric antibodies have been in clinical use, sometimes chronically, for quite some time (6). Thus, antibodies, even when chimerized, are a class of biologicals that have been clinically demonstrated to display levels of immunogenicity that are often low, generally acceptable, and manageable in humans.
The results reported here make it clear that a diverse collection of neutralizing anti-SEB antibodies can be accessed by immunization of mice with native forms of SEB. In the present study, a search of the library of monoclonal antibodies revealed a pair of antibodies that were more effective neutralizers in combination than when used separately. The ability of combinations of these antibodies and their chimeric counterparts to deliver inhibition of SEB action at lower concentrations is a significant finding. It suggests that significantly smaller amounts of the combination would be needed for prophylaxis or therapy than would be required if a single monoclonal antibody comparable to a member of the set was used alone. This conclusion has clear implications for cost and side effects. If smaller amounts of the antibody combination are required for therapeutic potency than either used alone, costs will be less and side effects should be fewer and less severe.
ACKNOWLEDGMENTS
We thank members of the Goldsby and Osborne labs for many helpful discussions. We also thank Katherine L. Knight for suggesting the VκExpress and VHExpress plasmids as vectors for the expression of chimeric antibodies. We are also very grateful for the helpful advice and generous support of Chella S. David.
This study was supported by National Institutes of Health grants AI057652, AI076944, and AI68741.
FOOTNOTES
- Received 5 October 2009.
- Returned for modification 28 October 2009.
- Accepted 8 March 2010.
- Copyright © 2010 American Society for Microbiology
