Potent Neutralization of Staphylococcal Enterotoxin B by Synergistic Action of Chimeric Antibodies

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 T_H1 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).