Chimeric anti-staphylococcal enterotoxin B antibodies and lovastatin act synergistically to provide in vivo protection against lethal doses of SEB

Staphylococcal enterotoxin B (SEB) is one of a family of toxins secreted by Staphylococcus aureus that act as superantigens, activating a large fraction of the T-cell population and inducing production of high levels of inflammatory cytokines that can cause toxic shock syndrome (TSS) and death. Extracellular engagement of the TCR of T-cells and class II MHC of antigen presenting cells by SEB triggers the activation of many intracellular signaling processes. We engineered chimeric antibodies to block the extracellular engagement of cellular receptors by SEB and used a statin to inhibit intracellular signaling. Chimeric human-mouse antibodies directed against different neutralizing epitopes of SEB synergistically inhibited its activation of human T-cells in vitro. In the in vivo model of lethal toxic shock syndrome (TSS) in HLA-DR3 transgenic mice, two of these antibodies conferred significant partial protection when administered individually, but offered complete protection in a synergistic manner when given together. Similarly, in vivo, lovastatin alone conferred only partial protection from TSS similar to single anti-SEB antibodies. However, used in combination with one chimeric neutralizing anti-SEB antibody, lovastatin provided complete protection against lethal TSS in HLA-DR3 transgenic mice. These experiments demonstrate that in vivo protection against lethal doses of SEB can be achieved by a statin of proven clinical safety and chimeric human-mouse antibodies, agents now widely used and known to be of low immunogenicity in human hosts.     

Potent Neutralization of Staphylococcal Enterotoxin B In Vivo by Antibodies that Block Binding to the T-Cell Receptor

To develop an antibody (Ab) therapeutic against staphylococcal enterotoxin B (SEB), a potential incapacitating bioterrorism agent and a major cause of food poisoning, we developed a “class T" anti-SEB neutralizing Ab (GC132) targeting an epitope on SEB distinct from that of previously developed “class M" Abs. A systematic engineering approach was applied to affinity-mature Ab GC132 to yield an optimized therapeutic candidate (GC132a) with sub-nanomolar binding affinity. Mapping of the binding interface by hydrogen–deuterium exchange coupled to mass spectrometry revealed that the class T epitope on SEB overlapped with the T-cell receptor binding site, whereas other evidence suggested that the class M epitope overlapped with the binding site for the major histocompatibility complex. In the IgG format, GC132a showed ∼ 50-fold more potent toxin-neutralizing efficacy than the best class M Ab in vitro, and fully protected mice from lethal challenge in a toxic shock post-exposure model. We also engineered bispecific Abs (bsAbs) that bound tetravalently by utilizing two class M binding sites and two class T binding sites. The bsAbs displayed enhanced toxin neutralization efficacy compared with the respective monospecific Ab subunits as well as a mixture of the two, indicating that enhanced efficacy was due to heterotypic tetravalent binding to two non-overlapping epitopes on SEB. Together, these results suggest that class T anti-SEB Ab GC132a is an excellent candidate for clinical development and for bsAb engineering.     

Engineered pH-dependent recycling antibodies enhance elimination of Staphylococcal enterotoxin B superantigen in mice

A new modality in antibody engineering has emerged in which the antigen affinity is designed to be pH dependent (PHD). In particular, combining high affinity binding at neutral pH with low affinity binding at acidic pH leads to a novel antibody that can more effectively neutralize the target antigen while avoiding antibody-mediated antigen accumulation. Here, we studied how the in vivo pharmacokinetics of the superantigen, Staphylococcal enterotoxin B (SEB), is affected by an engineered antibody with pH-dependent binding. PHD anti-SEB antibodies were engineered by introducing mutations into a high affinity anti-SEB antibody, 3E2, by rational design and directed evolution. Three antibody mutants engineered in the study have an affinity at pH 6.0 that is up to 68-fold weaker than the control antibody. The pH dependency of each mutant, measured as the pH-dependent affinity ratio (PAR – ratio of affinity at pH 7.4 and pH 6.0), ranged from 6.7–11.5 compared to 1.5 for the control antibody. The antibodies were characterized in mice by measuring their effects on the pharmacodynamics and pharmacokinetics (PK) of SEB after co-administration. All antibodies were effective in neutralizing the toxin and reducing the toxin-induced cytokine production. However, engineered PHD antibodies led to significantly faster elimination of the toxin from the circulation than wild type 3E2. The area under the curve computed from the SEB PK profile correlated well with the PAR value of antibody, indicating the importance of fine tuning the pH dependency of binding. These results suggest that a PHD recycling antibody may be useful to treat intoxication from a bacterial toxin by accelerating its clearance.     

A phage‐displayed single‐chain Fab library optimized for rapid production of single‐chain IgGs

Phage‐displayed synthetic antibody (Ab) repertoires have become a major source of affinity reagents for basic and clinical research. Specific Abs identified from such libraries are often screened as fragments antigen binding (Fabs) produced in bacteria, and those with desired biochemical characteristics are reformatted for production as full‐length immunoglobulin G (IgG) in mammalian cells. The conversion of Fabs to IgGs is a cumbersome and often rate‐limiting step in the development of Abs. Moreover, biochemical properties required for lead IgG development are not always shared by the Fabs, and these issues are not uncovered until a significant effort has been spent on Abs that ultimately will not be useful. Thus, there is a need for simple and rapid techniques to convert phage‐displayed Fabs to IgGs at an early stage of the Ab screening process. We report the generation of a highly diverse phage‐displayed synthetic single‐chain Fab (scFab) library, in which the light and heavy chains were tethered with an optimized linker. Following selection, pools of scFabs were converted to single‐chain IgGs (scIgGs) en masse, enabling facile screening of hundreds of phage‐derived scIgGs. We show that this approach can be used to rapidly screen for and select scIgGs that target cell‐surface receptors, and scIgGs behave the same as conventional IgGs.     

Improved methods for prepurification and detection of staphylococcal enterotoxin B from cell-free culture filtrate

An improved ELISA method for the detection of Staphylococcal Enterotoxin B (SEB) in protein A preparations is presented. Fab fragments were obtained by digestion with papain of anti-SEB IgG bound to SEB immobilized on Sepharose 4B. Anti-SEB and peroxidase-labeled Fab fragments from secondary antibodies were successfully used in a modified ELISA of SEB in protein A preparations. SEB-Sepharose was used repeatedly for the production of anti-SEB Fab fragments by papain digestion without loss of affinity. In addition, for the purification of SEB from crude culture filtrates, an initial step utilizing a combined heat and pH treatment for the removal of significant amounts of contaminating proteins without losses of toxin activity is presented. This pretreatment step yielded positive effects in further downstream processing considering both shortened time and an increase in total recovery.     

Engineering aggregation resistance in IgG by two independent mechanisms: lessons from comparison of Pichia pastoris and mammalian cell expression

Aggregation is an important concern for therapeutic antibodies, since it can lead to reduced bioactivity and increase the risk of immunogenicity. In our analysis of immunoglobulin G (IgG) molecules of identical amino acid sequence but produced either in mammalian cells (HEK293) or in the yeast Pichia pastoris (PP), dramatic differences in their aggregation susceptibilities were encountered. The antibodies produced in Pichia were much more resistant to aggregation under many conditions, a phenomenon found to be mainly caused by two factors. First, the mannose-rich glycan of the IgG from Pichia, while slightly thermally destabilizing the IgG, strongly inhibited its aggregation susceptibility, compared to the complex mammalian glycan. Second, on the Pichia-produced IgGs, amino acids belonging to the α-factor pre-pro sequence were left at the N-termini of both chains. These additional residues proved to considerably increase the temperature of the onset of aggregation and reduced the aggregate formation after extended incubation at elevated temperatures. The attachment of these residues to IgGs produced in cell culture confirmed their beneficial effect on the aggregation resistance. Secretion of IgGs with native N-termini in the yeast system became possible after systematic engineering of the precursor proteins and the processing site. Taken together, the present results will be useful for the successful production of full-length IgGs in Pichia, give indications on how to engineer aggregation-resistant IgGs and shed new light on potential biophysical effects of tag sequences in general.     

Construction of a single-chain variable-fragment antibody against the superantigen Staphylococcal enterotoxin B

Staphylococcal food poisoning (SFP) is one of the most prevalent causes of food-borne illness throughout the world. SFP is caused by 21 different types of staphylococcal enterotoxins produced by Staphylococcus aureus. Among these, staphylococcal enterotoxin B (SEB) is the most potent toxin and is a listed biological warfare (BW) agent. Therefore, development of immunological reagents for detection of SEB is of the utmost importance. High-affinity and specific monoclonal antibodies are being used for detection of SEB, but hybridoma clones tend to lose their antibody-secreting ability over time. This problem can be overcome by the use of recombinant antibodies produced in a bacterial system. In the present investigation, genes from a hybridoma clone encoding monoclonal antibody against SEB were immortalized using antibody phage display technology. A murine phage display library containing single-chain variable-fragment (ScFv) antibody genes was constructed in a pCANTAB 5E phagemid vector. Phage particles displaying ScFv were rescued by reinfection of helper phage followed by four rounds of biopanning for selection of SEB binding ScFv antibody fragments by using phage enzyme-linked immunosorbent assay (ELISA). Soluble SEB-ScFv antibodies were characterized from one of the clones showing high affinity for SEB. The anti-SEB ScFv antibody was highly specific, and its affinity constant was 3.16 nM as determined by surface plasmon resonance (SPR). These results demonstrate that the recombinant antibody constructed by immortalizing the antibody genes from a hybridoma clone is useful for immunodetection of SEB.     

Optimizing Production of Antigens and Fabs in the Context of Generating Recombinant Antibodies to Human Proteins

We developed and optimized a high-throughput project workflow to generate renewable recombinant antibodies to human proteins involved in epigenetic signalling. Three different strategies to produce phage display compatible protein antigens in bacterial systems were compared, and we found that in vivo biotinylation through the use of an Avi tag was the most productive method. Phage display selections were performed on 265 in vivo biotinylated antigen domains. High-affinity Fabs (<20nM) were obtained for 196. We constructed and optimized a new expression vector to produce in vivo biotinylated Fabs in E. coli. This increased average yields up to 10-fold, with an average yield of 4 mg/L. For 118 antigens, we identified Fabs that could immunoprecipitate their full-length endogenous targets from mammalian cell lysates. One Fab for each antigen was converted to a recombinant IgG and produced in mammalian cells, with an average yield of 15 mg/L. In summary, we have optimized each step of the pipeline to produce recombinant antibodies, significantly increasing both efficiency and yield, and also showed that these Fabs and IgGs can be generally useful for chromatin immunoprecipitation (ChIP) protocols.     

bisFabs: Tools for rapidly screening hybridoma IgGs for their activities as bispecific antibodies

Bispecific antibodies are a growing class of therapeutic molecules. Many of the current bispecific formats require DNA engineering to convert the parental monoclonal antibodies into the final bispecific molecules. We describe here a method to generate bispecific molecules from hybridoma IgGs in 3–4 d using chemical conjugation of antigen-binding fragments (Fabs) (bisFabs). Proteolytic digestion conditions for each IgG isotype were analyzed to optimize the yield and quality of the final conjugates. The resulting bisFabs showed no significant amounts of homodimers or aggregates. The predictive value of murine bisFabs was tested by comparing the T-cell redirected cytotoxic activity of a panel of antibodies in either the bisFab or full-length IgG formats. A variety of antigens with different structures and expression levels was used to extend the comparison to a wide range of binding geometries and antigen densities. The activity observed for different murine bisFabs correlated with those observed for the full-length IgG format across multiple different antigen targets, supporting the use of bisFabs as a screening tool. Our method may also be used for the screening of bispecific antibodies with other mechanisms of action, allowing for a more rapid selection of lead therapeutic candidates.     

Production and characterization of anti-peptide monoclonal antibodies with specificity for staphylococcal enterotoxins A and B

A synthetic peptide containing selected epitopes from staphylococcal enterotoxin A (SEA) and enterotoxin B (SEB) was used to produce monoclonal antibodies (Mabs) to respective enterotoxins in a single fusion procedure. The peptide inhibited the reaction of polyclonal anti-SEA or anti-SEB antisera with their homologous enterotoxin, thus showing that the chosen epitopes are part of the antibody-inducing enterotoxin sequences. Two Mabs, Mab-A and Mab-B, reacted with both the peptide and with either SEA or SEB. Used in a double antibody sandwich ELISA, the Mabs were able to quantitate the native SEA or SEB toxins at nanogram levels.     

The use of the in vitro toxin binding inhibition (ToBI) test for the estimation of the potency of tetanus toxoid

The in vitro toxin binding inhibition (ToBI) test was used to determine antitoxin responses in mice immunized with tetanus toxoid. The ToBI test showed good correlation with the in vivo toxin neutralization (TN) test in titration of sera of mice immunized with various doses of DPT-Polio, DT-Polio and a tetanus reference preparation. Estimates of potency of tetanus toxoid obtained in mice by ToBI test correlated significantly with those obtained in mice by the lethal challenge test. In addition, potency values of the European reference preparation, succeedingly estimated by ToBI test and lethal challenge test in a single group of guinea-pigs, showed good correlation. From the study it is concluded that the ToBI test is a promising alternative to the toxic challenge procedure in the potency assay of tetanus toxoid vaccines. A substantial refinement and reduction in the use of animals can be achieved. Additional savings can be made by combining diphtheria and tetanus potency testing.     

The mechanism of superantigen-mediated toxic shock: not a simple Th1 cytokine storm

The profound clinical consequences of Gram-positive toxic shock are hypothesized to stem from excessive Th1 responses to superantigens. We used a new superantigen-sensitive transgenic model to explore the role of TCRalphabeta T cells in responses to staphylococcal enterotoxin B (SEB) in vitro and in two different in vivo models. The proliferative and cytokine responses of HLA-DR1 spleen cells were 100-fold more sensitive than controls and were entirely dependent on TCRalphabeta T cells. HLA-DR1 mice showed greater sensitivity in vivo to two doses of SEB with higher mortality and serum cytokines than controls. When d-galactosamine was used as a sensitizing agent with a single dose of SEB, HLA-DR1 mice died of toxic shock whereas controls did not. In this sensitized model of toxic shock there was a biphasic release of cytokines, including TNF-alpha, at 2 h and before death at 7 h. In both models, mortality and cytokine release at both time points were dependent on TCRalphabeta T cells. Anti-TNF-alpha pretreatment was protective against shock whereas anti-IFN gamma pretreatment and delayed anti-TNF-alpha treatment were not. Importantly, anti-TNF-alpha pretreatment inhibited the early TNF-alpha response but did not inhibit the later TNF-alpha burst, to which mortality has previously been attributed. Splenic T cells were shown definitively to be the major source of TNF-alpha during the acute cytokine response. Our results demonstrate unequivocally that TCRalphabeta T cells are critical for lethality in toxic shock but it is the early TNF-alpha response and not the later cytokine surge that mediates lethal shock.     

Analytical FcRn affinity chromatography for functional characterization of monoclonal antibodies

The neonatal Fc receptor (FcRn) is important for the metabolic fate of IgG antibodies in vivo. Analysis of the interaction between FcRn and IgG in vitro might provide insight into the structural and functional integrity of therapeutic IgG that may affect pharmacokinetics (PK) in vivo. We developed a standardized pH gradient FcRn affinity liquid chromatography method with conditions closely resembling the physiological mechanism of interaction between IgG and FcRn. This method allows the separation of molecular IgG isoforms, degradation products and engineered molecules based on their affinity to FcRn. Human FcRn was immobilized on the column and a linear pH gradient from pH 5.5 to 8.8 was applied. FcRn chromatography was used in comparison to surface plasmon resonance to characterize different monoclonal IgG preparations, e.g., oxidized or aggregated species. Wild-type and engineered IgGs were compared in vitro by FcRn chromatography and in vivo by PK studies in huFcRn transgenic mice. Analytical FcRn chromatography allows differentiation of IgG samples and variants by peak pattern and retention time profile. The method can distinguish: 1) IgGs with different Fabs, 2) oxidized from native IgG, 3) aggregates from monomer and 4) antibodies with mutations in the Fc part from wild-type IgGs. Changes in the FcRn chromatographic behavior of mutant IgGs relative to the wild-type IgG correlate to changes in the PK profile in the FcRn transgenic mice. These results demonstrate that FcRn affinity chromatography is a useful new method for the assessment of IgG integrity.     

Generation, characterization, and epitope mapping of neutralizing and protective monoclonal antibodies against staphylococcal enterotoxin B-induced lethal shock

T-cell stimulating activity of Staphylococcal enterotoxin B (SEB) is an important factor in the pathogenesis of certain staphylococcal diseases including SEB mediated shock. SEB is one of the most potent superantigens known and treatment of SEB induced shock remains a challenge. We generated and characterized murine monoclonal antibodies (mAbs) to SEB in mice. We tested mAbs neutralize mitogenic effects of SEB in vitro and in vivo with T-cell proliferation assays and 2 murine models for SEB induced lethal shock (SEBILS). Epitope mapping suggests that all these mAbs recognize conformational epitopes that are destroyed by deleting the C terminus of the protein. Further site-directed mutagenesis identified potential residues involved in binding to SEB that differ between Methicillin resistant and sensitive Staphylococcus aureus strains. Only mAb 20B1 was effective as a monotherapy in treating SEBILS in HLA DR3 transgenic mice, which exhibit enhanced sensitivity to SEB. It is noteworthy that mAbs, 14G8 and 6D3 were not protective when given alone in the HLA DR3 mice but their efficacy of protection could be greatly enhanced when mAbs were co-administered simultaneously. Our data suggest combinations of defined mAbs may constitute a better treatment strategy and provide a new insight for the development of passive immunotherapy.     

“Inclonals”

Full-length antibodies and antibodies that ferry a cargo to target cells are desired biopharmaceuticals. We describe the production of full-length IgGs and IgG-toxin fusion proteins in E. coli. In the presented examples of anti CD30 and anti EGF-receptor antibodies, the antibody heavy and light chains or toxin fusions thereof were expressed in separate bacterial cultures, where they accumulated as insoluble inclusion bodies. Following refolding and purification, a high yield (up to 50 mg /L of shake flask culture) of highly purified (>90%) full-length antibodies and antibody-toxin fusions were obtained. The bacterially produced antibodies, named "Inclonals," equaled the performance of the same IgGs that were produced using conventional mammalian cell culture in binding properties as well as in cell killing potency. The rapid and cost effective IgG production process and the high quality of the resultant product may make the bacterial production of full-length IgG and IgG-drug fusion proteins an attractive option for antibody production. and a significant contribution to recombinant antibody technology.     

Protective effect of Bacillus anthracis surface protein EA1 against anthrax in mice

Bacillus anthracis spores germinate to vegetative forms in host cells, and produced fatal toxins. A toxin-targeting prophylaxis blocks the effect of toxin, but may allow to grow vegetative cells which create subsequent toxemia. In this study, we examined protective effect of extractable antigen 1 (EA1), a major S-layer component of B. anthracis, against anthrax. Mice were intranasally immunized with recombinant EA1, followed by a lethal challenge of B. anthracis spores. Mucosal immunization with EA1 resulted in a significant level of anti-EA1 antibodies in feces, saliva and serum. It also delayed the onset of anthrax and remarkably decreased the mortality rate. In addition, the combination of EA1 and protective antigen (PA) protected all immunized mice from a lethal challenge with B. anthracis spores. The numbers of bacteria in tissues of EA1-immunized mice were significantly decreased compared to those in the control and PA alone-immunized mice. Immunity to EA1 might contribute to protection at the early phase of infection, i.e., before massive multiplication and toxin production by vegetative cells. These results suggest that EA1 is a novel candidate for anthrax vaccine and provides a more effective protection when used in combination with PA.     

Human antibodies from immunized donors are protective against anthrax toxin in vivo

A panel of Fabs that neutralize anthrax toxin in vitro was selected from libraries generated from human donors vaccinated against anthrax. At least two of these antibodies protect rats from anthrax intoxication in vivo. Fabs 83K7C and 63L1D bind with subnanomolar affinity to protective antigen (PA) 63, and Fab 63L1D neutralizes toxin substoichiometrically, inhibits lethal factor (LF) interaction with PA63 and binds to a conformational epitope formed by PA63.     

Monoclonal antibodies against staphylococcal enterotoxins

MAb anti-Staphylococcal enterotoxins (MAb-SE) were produced in mice with a mixture of reference SE types A, B, C₁ and D at a concentration of 1 g each per mouse; the last booster injection was by intrasplenic route. Nine clones were chosen, two produced anti-SEB and anti-SED, one anti-SEA and anti-SEB, two anti-SED, two anti-SEB and one produced anti-SEC_1. The MAb-SE were partially purified as judged by PAGE–SDS. The partially purified antibodies could demonstrate the presence of SE in milk samples containing 0.5 g of toxin m l^–1.     

Coupling immunomagnetic separation on magnetic beads with matrix-assisted laser desorption ionization-time of flight mass spectrometry for detection of staphylococcal enterotoxin B

The growing importance of mass spectrometry for the identification and characterization of bacterial protein toxins is a consequence of the improved sensitivity and specificity of mass spectrometry-based techniques, especially when these techniques are combined with affinity methods. Here we describe a novel method based on the use of immunoaffinity capture and matrix-assisted laser desorption ionization-time of flight mass spectrometry for selective purification and detection of staphylococcal enterotoxin B (SEB). SEB is a potent bacterial protein toxin responsible for food poisoning, as well as a potential biological warfare agent. Unambiguous detection of SEB at low-nanogram levels in complex matrices is thus an important objective. In this work, an affinity molecular probe was prepared by immobilizing anti-SEB antibody on the surface of para-toluene-sulfonyl-functionalized monodisperse magnetic particles and used to selectively isolate SEB. Immobilization and affinity capture procedures were optimized to maximize the density of anti-SEB immunoglobulin G and the amount of captured SEB, respectively, on the surface of magnetic beads. SEB could be detected directly "on beads" by placing the molecular probe on the matrix-assisted laser desorption ionization target plate or, alternatively, "off beads" after its acidic elution. Application of this method to complex biological matrices was demonstrated by selective detection of SEB present in different matrices, such as cultivation media of Staphylococcus aureus strains and raw milk samples.     

Direct skin test in highly sensitized guinea pigs for rapid and sensitive determination of staphylococcal enterotoxin B

The direct skin test in highly sensitized guinea pigs was developed as a rapid and extremely sensitive assay for detection of staphylococcal enterotoxin B (SEB) in foods. This report details the experimental conditions required to elicit optimal sensitization of guinea pigs to SEB. An intense and persistent immunoglobulin E (IgE) anti-SEB response was established in strain 13 guinea pigs pretreated with cyclophosphamide followed by four sensitizing doses of 10 micrograms of SEB 1 month apart. The conditions, however, optimal for eliciting IgE responses led to a sustained failure to produce antibody of the IgG1 subclass. With the use of highly sensitized guinea pigs, one can achieve a sensitivity ranging from 0.1 to 1.0 pg of purified SEB by the direct skin test for at least 7 months after the last challenge. For analysis of SEB in food extracts, the entire assay can be accomplished within 20 min with a sensitivity of 10 to 100 pg SEB per ml of prepared food samples, and the recovery of enterotoxin from spiked food products ranged between 75 and 89% of the amount added.