Potent Neutralization of Anthrax Edema Toxin by a Humanized Monoclonal Antibody That Competes with Calmodulin for Edema Factor Binding

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Potent neutralization of anthrax edema toxin by a humanized monoclonal antibody that competes with calmodulin for edema factor binding

Zhaochun Chena,1,2, Mahtab Moayerib,1, Huaying Zhaoc, Devorah Crownb, Stephen H. Lepplab, and Robert H. Purcella,2

aLaboratory of Infectious Diseases and bLaboratory of Bacterial Diseases, National Institute of Allergy and Infectious Diseases, and cNational Institute for Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD 20892

Contributed by Robert H. Purcell, June 15, 2009 (sent for review May 5, 2009) This study describes the isolation and characterization of a neu- The purpose of this study was to determine (i) if anti-EF tralizing monoclonal antibody (mAb) against anthrax edema fac- neutralizing mAbs could be isolated; (ii) the effectiveness of such tor, EF13D. EF13D neutralized edema toxin (ET)-mediated cyclic antibodies against anthrax ET effects; and (iii) the neutralization AMP (cAMP) responses in cells and protected mice from both mechanism of these antibodies. We have made a Fab combina- ET-induced footpad edema and systemic ET-mediated lethality. The torial phage display library from chimpanzees that were immu- antibody epitope was mapped to domain IV of EF. The mAb was nized with anthrax toxins (17). From the library, 4 EF-specific able to compete with calmodulin (CaM) for EF binding and dis- Fabs were recovered, and 1 of them had potent neutralizing placed CaM from EF-CaM complexes. EF-mAb binding afﬁnity activity independent of the homologous PA-binding N-terminal (0.05–0.12 nM) was 50- to 130-fold higher than that reported for (1–254) domain of LF. In this report, we describe the detailed EF-CaM. This anti-EF neutralizing mAb could potentially be used characterization of these anti-EF clones. alone or with an anti-PA mAb in the emergency prophylaxis and treatment of anthrax infection. Results Isolation and Characterization of EF-Specific Fabs. A phage library nfection by inhalational anthrax is often fatal if treatment is expressing chimpanzee ␥1/␬ antibody genes was constructed Idelayed. Anthrax bacteria can be killed by vigorous treatment after immunization of chimpanzees with full-length EF. After 3 MEDICAL SCIENCES with antibiotics, but patients may still die because the lethality of rounds of panning against EF, 96 individual EF-specific clones anthrax is largely because of the action of toxins (1). Anti-toxin were identified by phage ELISA. Sequence analysis identified 4 neutralizing monoclonal antibodies (mAbs) are the only viable unique Fab clones with distinct VH and VK sequences. These choice for immediate neutralization of toxin and they could were designated EF12A, EF13D, EF14H, and EF15A. The augment the effectiveness of antibiotics. closest human V-gene germline origins of the 4 clones were Anthrax bacteria produce 3 toxin components: Protective assigned by conducting sequence similarity searches of all of the antigen (PA), lethal factor (LF), and edema factor (EF) (2, 3). known human Ig genes. The findings are shown in Table 1. PA binds to cellular receptors and acts as a vehicle to deliver LF The Fabs were converted to full-length chimeric IgG mole- or EF into the cytosol where they exert their enzymatic activities cules with human ␥1 heavy chain constant regions, and the (4–8). LF is a zinc-dependent protease that cleaves mitogen- binding specificity of all 4 clones for EF was verified by ELISA activated protein kinase kinases and causes lysis of macrophages (Fig. 1A). No binding was observed for negative controls LF, PA, (9, 10). EF is a calcium-calmodulin (CaM)-dependent adenylate BSA, thyroglobulin, lysozyme, or phosphorylase b. cyclase and causes local inflammation and edema (11). The combination of PA with LF results in lethal toxin (LT). LT can mAb EF13D Potently Neutralizes ET In Vitro and In Vivo. Of all of the replicate symptoms of anthrax disease when injected into ani- anti-EF mAbs tested for neutralization of ET activity, only a mals (12). PA together with EF forms edema toxin (ET) and ET single antibody, EF13D, was able to inhibit ET-mediated cyclic can produce a range of toxic effects in the host (11, 13). AMP (cAMP) production in RAW264.7 cells and did so with an PA has been regarded as the most important target for EC50 of 10–118 ng/mL. Thus, the potency of EF13D was prophylaxis and therapy of anthrax, because PA is common to comparable to the potent anti-PA mAb 14B7 in a similar assay both LTs and ETs, initiates the toxic process via receptor (29), but lower than that of anti-PA mAb W1 (17) (Fig. 1B). We binding, and is highly immunogenic. In fact, PA is the major tested the in vivo efficacy of mAb EF13D in 2 different models. component in the current anthrax vaccine and the target for most The antibody significantly inhibited ET-mediated edema in the of the available human or human-like neutralizing mAbs that mouse footpad model when premixed with toxin before admin- have been shown to be very effective in protection against istration in the footpad (Fig. 2 A–C) as well as when given anthrax toxin or spore challenge (14–19). However, there is systemically (IV) before footpad administration of toxin (Fig. evidence that LF and EF may play important roles in providing 2D). The antibody was able to prevent ET-induced footpad protective immunity (20–22). Furthermore, concerns that PA edema almost completely when compared with untreated or could potentially be manipulated, such that it would no longer PBS-treated mice after administration of 3 different doses of be neutralized by current anti-PA neutralizing mAbs have led to toxin. More significantly, systemic preadministration of mAb interest in therapeutics against the other 2 toxin components. A EF13D (50 ␮g per mouse) also provided significant protection mixture of mAbs that recognize distinct epitopes on multiple toxin components (PA, LF, or EF) would not only enhance the

protective efficacy but also broaden the spectrum of protection. Author contributions: Z.C., M.M., H.Z., S.H.L., and R.H.P. designed research; Z.C., M.M., H.Z., Thus, in recent years, several anti-LF mAbs have also been and D.C. performed research; Z.C., M.M., H.Z., S.H.L., and R.H.P. analyzed data; and Z.C., reported (23–27). However, no anti-EF neutralizing mAbs have M.M., H.Z., S.H.L., and R.H.P. wrote the paper. been reported to date. A previous report had indicated that The authors declare no conﬂicts of interest. immunization with the PA-binding N-terminal domain of EF 1Z.C. and M.M. contributed equally to this work. (amino acids 1–254) resulted in polyclonal sera containing both 2To whom correspondence may be addressed. E-mail: [email protected] or EF and LF neutralizing activities (28). [email protected].

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0906581106 PNAS Early Edition ͉ 1of6 Downloaded by guest on September 26, 2021 could interrupt this process and thereby block the toxin’s enzymatic activity.

mAb EF13D Prevents CaM Binding to EF and Can Also Displace Prebound CaM from EF. To determine if the mAb competes with CaM for binding to EF, we performed native gel electrophoresis and analytical ultracentrifugation (AUC) assays. Because the mobility of proteins or protein complexes on native gel is dependent on their size, conformation, and charge, and the Phast native gel system allows visualization of sharp bands, we assumed that the mobility of a complex made of 3 components (Fab⅐EF⅐CaM) would be easily distinguished from that of the complex made of 2 components (Fab⅐EF or EF⅐CaM). When EF was mixed with 4-fold molar excess of Fab, the EF⅐Fab complex and free Fab were seen as distinct bands on the native gel (Fig. 5A, lane 3). Interestingly, the mixture of 3 components (EF, Fab, and CaM) also always resulted in the same shifted band as the binary complex (EF⅐Fab), in a manner independent of whether CaM was prebound to EF (EF-CaM) or added after EF-Fab complexes were formed (Fig. 5A, lane 4 and 5). Therefore, it appeared that CaM could not bind or remain bound to EF when Fab was present. We hypothesized that despite the clear charge and conformation-based shift difference CaM induced in EF mobility after binding to EF (causing the EF⅐CaM band to migrate much faster than EF) (Fig. 5A, lane 2), perhaps the ternary complex Fab⅐EF⅐CaM was simply indistinguishable from the Fab⅐EF complex because of CaM-mediated conformational changes. Thus, to make sure that the CaM was not present in the shifted complexes formed from ternary reaction of EF, Fab, and Fig. 1. Antibody binding and in vitro neutralization assays. (A) ELISA titra- CaM, Phast-Western blots with anti-CaM antibody were per- tion of 4 anti-EF mAbs: Recombinant EF was used to coat the wells of an ELISA formed. While reactivity with the free CaM and the EF⅐CaM plate. Wells were then incubated with various dilutions of mAbs, and bound bands were strong, CaM was not detected in the expected ternary IgGs were detected by the addition of peroxidase-conjugated anti-human Fc complexes. Next, to rule out the possibility that the inability to antibody followed by TMB substrate. (B) EF13D inhibits ET-mediated cAMP detect CaM by Western blot in the complex was because of production in cells. ET was ﬁrst incubated with serial dilutions of antibody. The ET-mAb mixture was then incubated with RAW264.7 macrophage cells as insufficient transfer of the large complex (158 kDa) to nitrocel- described in the Methods. The cAMP production levels were assessed with the lulose membranes or other epitope masking issues, we labeled BioTRAK cAMP enzyme immunoassay from Amersham Pharmacia Biotech CaM with an IR-dye. This allowed direct detection of CaM on according to the manufacturer’s protocol. Results were plotted and analyzed gels with high sensitivity in any complex in which it was present. with Prism software (version 5, Graphpad Software). Although labeling resulted in only partial conversion of EF to EF⅐CaM, it was clear that the trace amounts of Fab⅐EF⅐CaM complex (green bands) was formed, which migrated faster than against ET-mediated death in the mouse model after challenge the Fab⅐EF complex (red bands) (Fig. 5B). Therefore, in all of with a lethal dose of ET in C57BL/6J mice (Fig. 3). the ternary mixtures (different premix configurations), Fab⅐EF complex was the predominant species, although with high sen- mAb EF13D Recognizes EF Domain IV. We next wanted to establish sitivity labeling, trace amounts of the ternary complex can be the mechanism for the potent neutralizing activity of EF13D. detected. The same results were obtained in 8 experiments Full-length mature EF protein consists of 776 aa and is struc- performed with both IgG and Fab. Thus, it appeared that turally organized into 4 domains (Fig. 4A). Domain I (amino acid anti-EF neutralizing antibody was not only able to prevent CaM 1–259) binds to PA, the interface of domain II and III (amino from binding to EF but could even displace CaM that was acid 260–588) forms the catalytic center, and domain IV (amino prebound to EF. acid 589–766) is connected to domain IIb by a linker (30, 31). We next confirmed the results from the native gel analyses by Binding of CaM induces a large reorganization of the structure sedimentation velocity analytical ultracentrifugation (SV-AUC), that creates the catalytic center. We initially tested whether taking advantage of the size-dependent migration difference in EF13D could recognize LF or FP119 (a fusion protein of EF the centrifugal field. Sedimentation coefficient distributions domain I and diphtheria toxin) in Western blots, as domain I were calculated from the measured concentration profiles (Fig. 5C). For solutions of single-protein components, the sedimen- (amino acid 1–259) of EF has been previously shown to contain tation data yielded major peaks for CaM, Fab, and EF with PA-binding epitopes shared by EF and LF (28). We found that sedimentation coefficients of 1.87 S, 3.62 S, and 4.92 S, respec- EF13D did not react with LF, PA, or FP119 in Western blots, tively. Because of the high sensitivity of SV-AUC for trace suggesting the epitope was not in the N terminus. Next we populations, minor peaks from low level impurities and aggre- constructed and purified truncated EF peptides representing gates were also observed. The binary mixtures showed close to different domains of the toxin (Fig. 4B). The binding assay stoichiometric binding, with only minor populations of unreacted (ELISA) showed that, of all of the anti-EF antibodies, only free species visible at the s-values of the individual components. EF13D IgG bound to domain IV (Fig. 4 C and D). This suggested For the mixture of CaM and EF, as indicated in Fig. 5C, the that the difference between neutralizing and nonneutralizing complex sedimented as a major peak at 5.46 S, whereas for the mAbs we isolated could be because of their different binding Fab and EF mixture, a major peak at 6.16 S confirmed formation sites. As domain IV is a helical domain that facilitates CaM- of Fab⅐EF. For the ternary mixture, the major peak appeared at mediated activation of EF, binding to this region by antibody an s-value of 6.17 S, mutually identical to the value for the binary

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Fig. 2. Anti-EF mAb EF13D protects mice against ET-mediated footpad edema. (A) Comparison of left footpad treated with 0.5 ␮gET(Top), 0.5 ␮g ET premixed with 5 ␮g anti-EF mAb EF13D (Middle), and ET premixed with 5 ␮g anti-PA mAb W1 (Lower). (B and C) mAb EF13D (5 ␮g) and 5 ␮g W1 mediated inhibition of footpad edema induced by ET (panel B, 0.5 ␮g; panel C, 1.0 ␮g). Averages and standard errors presented are based on n ϭ 3 mice per treatment. (D) mAb EF13D (50 and 100 ␮g) injected IV 1 h before toxin administration inhibited footpad edema induced by 0.25 ␮g ET. Footpad experiment results shown are for the Balb/cJ strain.

complex Fab⅐EF. A peak corresponding to CaM⅐EF was not ported independently by the analysis of the signal amplitudes detected. This suggested that a ternary complex was not present. from the absorbance relative to the interference data, taking Theoretically, assuming the common (2/3)-power scaling law of advantage of the much lower extinction coefficient of CaM sedimentation coefficients relative to molar masses of globular compared with EF and Fab . proteins, one would expect a ternary complex to sediment with These results unambiguously show that under the SV-AUC s-values between 7.2–7.7 S. There is only a minor peak at ϳ7.1 conditions, Fab and CaM bind with high negative cooperativity S, which we believe to represent low level aggregates in this to EF. In a separate SV-AUC experiment, we observed the same experiment. In fact, in a separate experiment at higher (equimo- Fab⅐EF complex formation when Fab was added to a CaM-EF lar) protein concentrations, this peak could not be detected. That mixture preincubated for 2 h, confirming that Fab can displace ⅐ the major species in the ternary mixture is the binary Fab EF CaM from EF. complex is supported by the fact that, in this mixture, the peak of free CaM reappears with an area quantitatively corresponding mAb EF13D Binds to EF with High Affinity. Our results that the to the loading concentration. Further, the virtually exclusive ⅐ antibody can displace prebound CaM from EF suggest that presence of Fab EF complexes in the ternary mixture is sup- EF13D binds to EF more tightly than CaM does. Surface plasmon resonance (SPR) analyses showed that the dissociation of EF from the Fab⅐EF complex was extremely slow. Using the binding affinity distribution model, the kon was determined as 5 Ϫ1 Ϫ1 10 M s . Because of the extremely slow dissociation, Kd and koff could not be directly measured. However, the lower and upper limit of koff can be estimated. Consequently, based on the relationship between Kd, koff, and kon (Kd ϭ koff /kon), the upper Ϫ5 Ϫ1 limits of koff and Kd are 1.2 ϫ 10 s and 0.12 nM, respectively. Ϫ6 Ϫ1 For the lower limit of koff at 10 s , the estimated Kd from the distribution analysis is 0.05 nM. Thus, the affinity for Fab-EF interaction is 50- to 130-fold higher than that of EF-CaM (32).

Discussion

Fig. 3. Anti-EF mAb EF13D protects mice against ET-mediated death. We report here the isolation and characterization not only of an C57BL/6J mice received a single IV dose of 50 ␮g mAb EF13D or PBS 1 h before EF neutralizing mAb, but one with high binding affinity (Kd of a lethal dose of ET (25 ␮g, IV), and survival was monitored for 100 h. 0.12 to 0.05 nM). The neutralizing mAb, EF13D, was as potent

Chen et al. PNAS Early Edition ͉ 3of6 Downloaded by guest on September 26, 2021 Fig. 5. Analysis of complex formation among CaM, EF, and Fab on the native gel and by AUC. (A) Phast-gel electrophoresis of single, binary, and ternary complex of EF, Fab, and CaM followed by Coomasie blue staining. Lane 1, EF; lane 2, EF ϩ CaM; lane 3, EF ϩ Fab; lane 4, EF-CaM ϩ Fab; lane 5, EF-Fab ϩ CaM. (B) Phast-gel electrophoresis of protein complex with IR dye-labeled CaM. Gels were scanned at 800 nm for labeled CaM (green) and at 700 nm for Coomasie blue-stained proteins (red). Lane 1, EF; lane 2, EF ϩ CaM; lane 3, EF ϩ Fab; lane Fig. 4. mAb EF13D recognizes EF domain IV. (A) Schematic diagram of 4, EF-CaM ϩ Fab; lane 5, EF-Fab ϩ CaM; lane 6, Fab; lane 7, CaM. The shifted full-length EF showing the domain structure. Numbers refer to the amino acid bands of Fab⅐EF binary complex were indicated by arrows (A and B). (C) The residues at the beginning of the each domain or the beginning and end of the sedimentation coefficient distributions c(P)(s) obtained from the Bayesian protein itself. (B) A diagram of 7 constructs generated in this study. Numbers analysis of sedimentation velocity data of individual proteins, binary mixture refer to the amino acid residues at the beginning and end of each peptide. (C) and ternary mixture. The sedimentation profile of the ternary mixture is Reactivity of various peptides with anti-EF mAb EF13D or anti-His mAb. (D) shown in the Inset. c(P)(s) curves: CaM (blue), Fab (yellow), EF (green), mixture Reactivity of full-length EF (amino acid 1–766) and truncated EF (amino acid of CaM and EF (cyan), mixture of Fab and EF (red), ternary mixture of 3 proteins 589–766) with 4 EF-specific Fabs. Expressed peptides were fused to his tag and (black). were detected by 1 ␮g/mL anti-EF mAbs or anti-His mAb (1:3,000). Each reaction was done in triplicate. The average binding signal with standard deviation for each reaction upon binding by anti-EF or anti-His was plotted. gel assay when CaM was labeled with IR-dye. This is somewhat different from results obtained by AUC-SV; the latter did not reveal any presence of the ternary complex. The discrepancy as anti-PA mAb 14B7 in preventing cAMP induction by ET in might be because of variation in the experimental conditions vitro. More impressively, this antibody protected mice from (such as salt, pH, protein concentration, difference in reactivity ET-mediated edema formation and death. The differential between labeled and unlabeled CaM, and sensitivity of detection binding to truncated EF peptides identified domain IV of EF as of the complex). However, if there was a ternary complex of the target of the antibody. Crystallographic analysis has dem- Fab⅐EF⅐CaM formed, the amount was very small. onstrated that domain IV is not part of the catalytic core, but The binding of CaM is essential for the activity of EF, because contains the major sites for CaM binding and undergoes dra- the EF⅐CaM complex is 1,000-fold more active than is EF alone matic conformational changes upon CaM binding (30). Native ⅐ gel and AUC analyses indicated that EF13D was not only able (30, 33). Thus, inhibition of the formation of the EF CaM to prevent CaM from binding to EF, but also able to displace complex could be an important goal of passive immunoprophy- CaM from EF. This suggested to us that the antibody had a laxis and reversal of complex formation an important goal of higher binding affinity for EF than CaM did. Indeed, SPR antibody therapy. Indeed, a search for small molecules that can experiments yielded an estimate for the K of 0.12 to 0.05 nM for inhibit interaction between EF and CaM has been pursued, and d ␮ Fab-EF interaction, compared with 6.7 nM reported for EF- a molecule with a low inhibitory activity (IC50 of 10–15 M) has CaM (32). Of course, the binding affinity of EF-CaM is highly been found (34). Here, we report a naturally occurred EF- dependent on the concentration of calcium (32), but the binding neutralizing mAb that is not only able to inhibit the interaction affinity of EF13D for EF appears to be at least 50-fold higher between CaM and EF, but able to displace prebound CaM from than that of CaM for the toxin even under the highest affinity the EF⅐CaM complex. While the ability of EF13D to displace EF-CaM interaction conditions. The mAb epitope appears to be CaM from EF is certainly fascinating and could be 1 of the conformational because the antibody could not interact with the neutralizing mechanisms, it is unlikely to account for all of the denatured form of EF on Western blots. potency with which this mAb neutralizes EF. Because antibody The presence of trace amounts of the ternary complex, which is not expected to enter the cytoplasm with EF, where CaM is ran faster than the binary complex of Fab⅐EF, was detected in the readily available, it is far more likely that this antibody blocks

4of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0906581106 Chen et al. Downloaded by guest on September 26, 2021 unfolding and translocation of EF through the PA heptamer Proteins were expressed from E. coli BL21(DE3)pLysS (Promega) and purified channel in a manner similar to what has been described for through ProBond nickel-chelating affinity column (Invitrogen). antibodies that block botulinum toxin action (35). The use of this The purity of the peptides was determined by SDS/PAGE and the identity by mAb alone or in combination with anti-PA mAbs may not only Western blots probed with anti-His antibody (Sigma). Each peptide was tested for its binding to mAb EF13D as well as to anti-His by ELISA. An anti-His improve the efficacy, but also broaden the spectrum of protec- reaction was used to quantify each coated peptide. In brief, 5–10 ␮g/mL of tion against anthrax infection. each peptide were coated onto the wells of a 96-well plate. The plate was then incubated with 1 ␮g/mL EF13D IgG or anti-His-HRP conjugate (1:3,000) for 2 h Methods at room temperature (RT). Wells containing EF13D IgG were next incubated Toxins. PA was made from Bacillus anthracis in our laboratory as previously with anti-human IgG Fc-HRP conjugate (1:5,000; Jackson ImmunoResearch) described (36). EF and FP119 (a fusion protein of the N terminus of EF and for 1 h. HRP activity was assessed by adding tetramethylbenzidine substrate diphtheria toxin) were made from Escherichia coli as described previously (37). (TMB; Sigma) and measured at OD450. Each reaction was performed in triplicate. The average binding signal with standard deviation for each reaction Construction and Selection of Phage Library. The combinatorial cDNA library of with anti-EF or anti-His was plotted. chimpanzee ␥1/␬ antibody genes was constructed by inserting the heavy and A similar ELISA was used to determine if the other nonneutralizing anti-EF light chains at XhoI/SpeI and SacI/XbaI sites, respectively, in pComb3H vector Fabs bound to the shortest peptide (amino acid 589–766, EF589–766) that was as described previously (38). The library was panned against recombinant EF still reactive with EF13D. protein immobilized on ELISA wells for 3 rounds, and EF-specific clones were selected by 96-well phage ELISA (39). Native Gel Analysis and Western Blotting. Human, recombinant CaM was from BioMol. A portion was labeled with IRDye 800CW using a kit (Li-Cor Biosciences) Production and Purification of Fab and IgG. Removal of the phage coat protein per manufacturer’s protocol. EF was prebound to CaM or IR-dye-labeled CaM by III-encoding region from phagemid DNA by digestion with NheI/SpeI and mixing 3 ␮M EF and 40 ␮MCaMfor1hatRT.EF13D was then added to EF-CaM religation resulted in phagemid encoding soluble Fab. Soluble Fab was ex- or EF (EF and antibody at final concentrations of 1.7 and 7.5 ␮M), and the mixtures pressed and purified on a nickel-charged column as described (38). The were incubated for 1 h before running on native Phast gels (Amersham Pharma- conversion of Fab to IgG and the expression of IgG were carried out as cia Biotech). In other experiments, 3 ␮M EF was prebound to 12 ␮M EF13D before ␮ described previously (38). The purity of Fab and IgG was determined by SDS the addition of 40 M CaM. All binding experiments were performed with CaCl2 polyacrylamide gel electrophoresis (NuPAGE MOP; Invitrogen). Protein con- present (0.35–0.45 ␮M). Phast Gel were scanned in both 800 and 700 nm channels centrations were determined both by BCA assay (Pierce) and by measurement using the Odyssey Infrared Imaging System (Li-Cor Biosciences), before and subsequent to Coomasie blue staining. For Western blotting, gels were removed of OD280, assuming that 1.35 OD280 is equivalent to 1 mg/mL. from plastic backing and transferred to nitrocellulose according to protocols

Determination of Binding Specificity by ELISA. EF, LF, PA at 5 ␮g/mL and recommended by the manufacturer (Amersham Pharmacia Biotech) for the Phast MEDICAL SCIENCES unrelated proteins (BSA, thyroglobulin, lysozyme, and phosphorylase b; Gel System. The blot was incubated with anti-CaM mAb (1:1,000; Upstate). In Sigma) at 10 ␮g/mL in carbonate buffer (pH 9.5) were coated on ELISA epitope mapping experiments, PA, LF, EF, or FP119 (4–10 ng of each) were subjected to western blotting after conventional SDS/PAGE on 4%–20% Tris- microtiter plates. ELISAs were performed as described previously (17). Glycine gels (Invitrogen), using EF mAbs (1:500) as primary antibody and IR-dye- conjugated secondary antibodies. Blots were scanned using the Odyssey Infrared Nucleic Acid Sequence Analysis of EF-Specific Fab Clones. The genes coding for Imaging System. the variable region of heavy (VH) and light (VL) chains of EF-specific clones were sequenced, and their corresponding amino acid sequences were deter- AUC Sedimentation Velocity. Samples in 400 ␮Lat1␮M each in working buffer mined. The presumed family usage and germ-line origin were determined for (5 mM HEPES, 50 mM NaCl, 100 ␮M CaCl , pH 7.5 at 20 °C) were loaded into each VH and VL gene by search of V-Base, which is a compilation of all of the 2 12-mm path length double sector cells and were centrifuged at 50,000 rpm at available human variable region IG germ line sequences (40). 20 °C in a ProteomeLab-XL-I analytical ultracentrifuge (Beckman Coulter) after the standard protocol (42). The evolution of the resulting concentration Affinity Measurement by Biosensor Analysis. A SPR biosensor (BIAcore 3000; GE gradient was monitored by the interference optics and absorbance optics. Healthcare) was used to measure the binding kinetics and affinity of the Sedimentation coefficients for the protein constructs were obtained from interaction between EF and EF13D Fab. The experiments were performed at the analysis of the sedimentation velocity data using the software SEDFIT. 25 °C in the running buffer HBS (10 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM Twenty-three scans were loaded and modeled with a continuous c(s) distri- EDTA, 0.005% P20) using a CM3 sensor chip (GE Healthcare) with a carboxym- bution model using a resolution of 200 s-values between 0.1 to 15 S and ethylated dextran matrix. The purified Fab was covalently attached to the chip maximum entropy regularization with a 68% confidence interval. It should be surface by amine coupling. An immobilization level of 900 RU was reached noted that the width of the distribution is governed by the regularization and ␮ after applying 10 g/mL Fab in NaOAc buffer, pH 5.5, on the surface. Binding the noise in the data acquisition and that the area under the peaks, not the experiments were conducted by injecting 3-fold serial dilutions of recombi- peak heights, corresponds to the species concentrations. The s-value of the nant EF ranging from 0.1 to 300 nM to the chip surface. After each round of protein species was determined as the weight-averaged sedimentation coef- ␮ injection, the bound EF was completely removed by 2 passages of 10 L2M ficient of the peak. After the analysis of the single protein samples, the ␮ NaCl, 0.01% Triton X-100 followed by 10 L 0.1% SDS. The kinetic traces were obtained sedimentation coefficients for the 3 molecules of interest were used globally fitted with a model for continuous affinity and rate constant distri- as a priori information for the Bayesian analysis (43) of the data of the mixture. butions (41). Animal Studies. Chimpanzees 1603 and 1609 were immunized with recombinant ϭ ϩ ET In Vitro Neutralization. ET (100 ng/mL 100 ng/mL PA 100 ng/mL EF) was PA, LF, and EF, and bone marrow aspirates were used for library construction as prepared in DMEM in a 96-well plate. Antibodies were diluted serially directly described previously (17). Female BALB/cJ or C57BL/6J mice were purchased from into the toxin mixture and incubated for1hat37°C.Anti-PA antibody 14B7 Jackson Laboratories and used at 8–12 weeks of age. For the footpad edema (29) was used on each plate as a positive control. The ET-mAb mixtures were model, BALB/cJ mice were injected in 1 foot pad with 0.5 or 1.0 ␮g ET premixed then transferred to RAW264.7 macrophage cells grown to 80–90% confluence with anti-EF mAb EF13D, anti-PA mAb W1 (17) or PBS. Antibodies were used at 5 in 96-well plates. Cells were incubated for 1 h and total cAMP levels were ␮g. Footpad injection volumes were 20 ␮L. Footpad edema was monitored at 20 assessed using the BioTRAK cAMP enzyme immunoassay from Amersham and 46 h after injection by measuring dorsal/planar and medial/lateral sizes using Pharmacia Biotech according to the manufacturer’s protocol. digital calipers. In alternative experiments, 50 or 100 ␮g per mouse mAb EF13D were preadministered to mice via i.v. (IV) injection 1 h before 0.25 ␮g ET injection Epitope Mapping. Genes coding for full length mature EF (amino acids 1 to into footpads, and footpad size was monitored. To test for efficacy against ET 766), domain I (amino acids 1 to 259), domain II to domain IV (amino acids 260 lethality, C57BL/6J mice received a single mAb EF13D injection (50 ␮g, IV) 1 h to 766), domain IIb, III and IV (amino acids 316 to 766), domain IIb and IV before administration of a lethal dose of ET (25 ␮g ET, IV), and survival was (amino acids 456 to 766), domain II and III (amino acids 260 to 588), and domain monitored for 100 h. All experiments involving animals were performed under IV (amino acids 589 to 766) were amplified by PCR and cloned into pET-31b protocols approved by the respective Institutes as well as by the Animal Care and (Novagen) at NdeI and XhoI sites. The peptides were in frame at the C terminus Use Committee of the National Institute of Allergy and Infectious Diseases, with 6 histidines in the vector. Constructs were confirmed by DNA sequencing. National Institutes of Health.

Chen et al. PNAS Early Edition ͉ 5of6 Downloaded by guest on September 26, 2021 ACKNOWLEDGMENTS. We thank Rasem Fattah for producing the PA, EF, was supported by the Intramural Research Program of the National FP119, and other toxin proteins, Michelle Makayi for producing EF13D Fab, Institutes of Health, National Institute of Allergy and Infectious and Drs. Susan Emerson and Peter Shuck for helpful discussion. This work Diseases.

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