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Department of Chemistry and Biochemistry, University of Texas at Austin, Austin, Texas,1 Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas,2 Department of Comparative Medicine, Southwest Foundation for Biomedical Research, San Antonio, Texas,3 Department of Virology and Immunology, Southwest Foundation for Biomedical Research, San Antonio, Texas,4 Department of Chemical Engineering and Biomedical Engineering, University of Texas at Austin, Austin, Texas5
Received 13 July 2005/ Returned for modification 16 August 2005/ Accepted 4 September 2005
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ABSTRACT |
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INTRODUCTION |
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The anthrax attacks of October 2001 heightened awareness concerning the necessity of effective countermeasures for inhalation anthrax exposure. A high anti-PA antibody titer has been correlated with protection in guinea pigs and rabbits immunized with the Anthrax Vaccine Adsorbed vaccine (20, 42). Partial prophylaxis of animals treated with anti-PA rabbit polyclonal antibodies was demonstrated in a guinea pig spore challenge (30). Several murine monoclonal antibodies with moderate affinities towards PA failed to show protection in the same study, although one monoclonal antibody did show a significant increase in the time to death (TTD) of treated animals (30). The detailed mechanism of protection by anti-PA antibodies remains a subject of intense study (51, 52).
Several groups have been pursuing a therapeutic strategy directed against PA (7, 16, 21, 33, 35, 36, 41, 45, 47). We have used the 14B7 antibody (equilibrium dissociation constant [KD], 4.3 nM) (16) as the starting point for a protein-engineering campaign that led to several variants exhibiting 20-fold to 200-fold greater affinity (16, 33). ETI-204, a humanized immunoglobulin G (IgG) derived from an engineered affinity-enhanced 14B7 variant (31), was recently reported to provide excellent protection against inhalation challenge with spores (100x to 300x 50% lethal dose [LD50]) of the Ames strain of B. anthracis in rabbits in both prophylactic and postexposure settings (35). High-affinity anti-PA therapeutic antibodies can serve as effective prophylactics as well as late-stage infection antidotes. Scenarios in which both activities are important can be envisioned, especially in the event of exposure to an antibiotic-resistant anthrax strain (3).
In the adaptive immune response, antibodies are thought to represent the molecular link between recognition of a pathogen and its elimination through phagocytosis (antibody-dependent cytotoxicity) or complement activation (complement-dependent cytotoxicity). In both cases, Fc receptors provide the critical connection between an antibody-antigen binding event and effector functions. Fc receptors recognize and bind either to the hinge region/CH2 domain interface, or, in some cases, to regions near the CH2/CH3 domain interface (8).
The B. anthracis exotoxin is essential for the survival of the organism in the host (13), so it is possible that the neutralization of PA by long-circulating, ultra-high-affinity antibody fragments may be sufficient to confer passive protection against anthrax despite the absence of Fc-mediated immune responses. Benefits of such a strategy could include (i) substantially lower costs of manufacturing antibody fragments, a critical issue since the current prophylactic IgG antibodies require large dosages (4 to 10 mg/kg of body weight) and must be produced by recombinant CHO cells, (ii) elimination of potentially adverse effects associated with Fc, and (iii) rapid production in bacterial cells.
Polyethylene glycol (PEG) has previously been used as a conjugating agent for enhancing pharmacokinetics/bioavailability, improving stability issues, and decreasing immunogenicity among other attributes for therapeutics (22, 28, 29, 48). PEGylation of Fab fragments has provided increased serum half-lives and efficacy for antibody therapy for a variety of applications (23-25). The availability of maleimide-PEG has allowed conjugation to antibodies through reaction with free native or engineered cysteines (12). This chemistry has proven to be suitable not only for Fab but also for single-chain variable fragments (scFvs) (1, 37, 54), immunoliposomes (38), and other conjugates (19).
Here we report the prevention of toxemia from inhalation of B. anthracis spores by the very-high-affinity (KD = 35 pM) anti-PA antibody fragment (M18) conjugated to PEG. The M18 antibody fragment was derived from a library of random mutants of 14B7 scFv (31) screened by anchored periplasmic expression (APEx) (16). Intrinsic to the APEx screening strategy is that isolated antibody fragments are highly expressed in bacteria, and M18 is no exception. The conjugation of an M18 antibody fragment with a 40-kDa PEG polymer increased the serum half-life of the construct beyond that of full-length immunoglobulin. Subcutaneous administration of the conjugate resulted in a significant increase in survival and an improved overall mean TTD in a guinea pig model challenged with a relatively high dose of anthrax spores (Vollum 1B). These results reveal that binding to the toxin impairs the ability of B. anthracis to colonize the host and demonstrate the feasibility of using bacterially expressed antibody fragments as a prophylactic anthrax treatment. PEGylated antibody fragments can be developed and produced much more rapidly than current whole IgG therapeutics and, thus, could constitute a critical technology for rapid response to emerging bacterial and possibly even viral (e.g., influenza H5N1) pathogenic agents.
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MATERIALS AND METHODS |
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scAb expression and purification.
The M18 scFv gene (16) was cloned via terminal SfiI sites into pMoPac16 (18), a modified version of the pAk4000 vector (26) that carries an scFv gene with the human 
light chain to create a single-chain antibody (scAb) (18, 34). The pMoPac16 plasmid also carries a gene for the coexpression of the skp periplasmic chaperone (17, 18). To create a protein suitable for PEG conjugation, the M18 scAb gene was amplified with a C-terminal primer that incorporated a Cys residue downstream of the C-terminal His6 purification tag. This latter gene was then ligated into pMoPac16 via NcoI and AscI restriction sites to create the pMoPac16Cys plasmid that contains the M18 scAb-His6-Cys construct.
Escherichia coli Tuner cells (Novagen, Madison, WI) were transformed with pMoPac16_M18 and pMoPac16Cys_M18 and were grown at 25°C in 2-liter baffled flasks containing 400 ml Terrific Broth medium with 2% glucose and 200 µg/ml ampicillin. Cultures were induced at an optical density at 600 nm of 1.6 with 1 mM IPTG (isopropyl-ß-D-thiogalactopyranoside) (Sigma-Aldrich, St. Louis, MO) for 4 h, and then the cells were pelleted by centrifugation (10 min at 8,000 x g). Osmotic shock was carried out at 0°C as previously described (18). Cells were resuspended in 20 ml 0.75 M sucrose and 100 mM Tris-HCl (pH 8.0), with the addition of 1.0 ml of 10 mg/ml lysozyme in the same buffer. After shaking for 10 min at 0°C, 40 ml of 1 mM EDTA was added dropwise, followed by 15 min of further incubation at 0°C. A total of 3.0 ml of 0.5 M MgCl2 was then added dropwise, followed by an additional 15 min of incubation at 0°C. Spheroplasts were pelleted by centrifugation, and the clarified supernatant was mixed with a 1/10 volume of 10x IMAC buffer (100 mM Tris-HCl, 5 M NaCl, 0.2 M imidazole, pH 8.0) and applied to 1.5 ml Ni-nitrilotriacetic acid agarose resin (QIAGEN, Madison, WI). Following washing with 3 x 10 ml IMAC buffer, scAb protein was eluted with 500 mM imidazole in IMAC buffer and dialyzed against 2 x 2 liters phosphate-buffered saline (PBS) at 4°C overnight. Native M18 scAb was then applied to a Superdex 200 HR10/30 column (Amersham Biosciences, Piscataway, NJ) on an 
kta fast-protein liquid chromatography system (Amersham Pharmacia, Piscataway, NJ). Fractions were isolated and then concentrated with an Amicon ultra centrifugal filter device (molecular weight cutoff, 10,000; Millipore Corp., Bedford, MA). Protein concentrations were quantified using a micro-bicinchoninic acid quantification kit (Pierce, Rockford, IL).
scAb conjugation and purification. The conjugation of M18 scAb with PEG was performed as described previously (1, 37) with the following modifications: a twofold excess of tris(2-carboxyethyl)phosphine hydrochloride (TCEP; Molecular Probes, Eugene, OR) in PBS was added to purified scAb protein (2 mg/ml in PBS) and incubated overnight, with stirring at 4°C. The solution was then brought to room temperature and a twofold excess of maleimide-PEG (20 kDa or 40 kDa) (Nektar Therapeutics, San Carlos, CA) in PBS was added dropwise over 2 to 4 h. The scAb-PEG conjugate was purified by IMAC using Ni-nitrilotriacetic acid agarose. Eluate was then purified by size exclusion chromatography, concentrated, and quantified as mentioned for native scAb.
Biacore analysis. Surface plasmon resonance (SPR) analysis was performed using a Biacore 3000 instrument (Biacore, Piscataway, NJ). Recombinant PA (List Biological Laboratories, Campbell, CA) in 10 mM NaC2H3O2 (pH 5.0) was immobilized on a CM5 chip using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide N-hydroxy-succinimide chemistry. The PA solution was added as a 5 µg/ml solution until an amount equivalent to 500 response units was obtained. scAb and scAb-PEG proteins (2.5 nM to 40 nM) in HBS-N (Biacore) were used at a flow rate of 100 µl/min. A solution of 4 M MgCl2 was used as the regeneration buffer between runs. Data were analyzed using BIAevaluation software (version 3.0). A second flow cell with bovine serum albumin was used for data baseline correction. For some experiments, the scAb with or without PEG protein was immobilized onto the chip at a level corresponding to 120 response units under the same coupling conditions as described above and PA was used as the analyte.
In vitro anthrax toxin challenge. Anthrax LeTx challenges were performed with RAW 264.7 mouse macrophage cells as previously described (33, 50).
Pharmacokinetic studies.
Female Hartley guinea pigs (225 to 305 g) (Charles River Laboratories, Wilmington, MA) were given dorsal, subcutaneous injections of 1.2 ml of M18 scAb-PEG protein (20 or 40 kDa) or 14B7 IgG to a dose of 10 mg/kg or PBS as a control. Following sedation with ketamine (80 mg/kg) and xylazine (10 mg/kg), animals were bled when time reached 0 for 15 min, 1 h, 3 h, 6 h, 12 h, 24 h, 48 h, and 72 h at the femoral artery. The serum was collected and centrifuged as mentioned above. The construct concentrations were determined by enzyme-linked immunosorbent assay as follows: PA (2.5 µg/ml) in PBS was coated onto CoStar 96-well plates (Corning Inc., Corning, NY) and incubated overnight at 4°C. The wells were then blocked with the addition of 2% milk-PBS for 3 h at room temperature, and diluted serum (1:10) was added in 2% milk-PBS. scAb-PEG constructs were detected by using goat anti- light chain horseradish peroxidase (Sigma-Aldrich, St. Louis, MO). 14B7 murine IgG was detected with goat anti-mouse IgG horseradish peroxidase (Bio-Rad, CA). Data were modeled using WinNonlin software (Pharsight, MountainView, CA), formatted to be consistent with a one-compartment, bolus, first-order elimination model.
Inhalation challenge with B. anthracis spores. Animals were housed individually in a One Cage 2100 AllerZone interchangeable microisolator high density housing system (Lab Products Inc., Seaford, DE) in the BSL-4 safety facility at the Southwest Foundation for Biomedical Research (San Antonio, TX). Anthrax spore inocula were prepared on the day of challenge and diluted to the desired concentration in PBS. Female Hartley guinea pigs (225 to 305 g) were sedated with ketamine (80 mg/kg) and xylazine (10 mg/kg) during all bleeds, injections, and inhalation instillations. Animals were injected subcutaneously with 3.0 ml of PBS containing M18 scAb-40-kDa PEG to a dosage of 40 mg/kg or 80 mg/kg of body weight. Control animals were injected with 3.0 ml of either PBS, unconjugated 40-kDa PEG-maleimide (40 mg/kg), or native M18 scAb (40 mg/kg). Four hours later, the animals were challenged with anthrax spores by unilateral instillation of 250x to 625x LD50 (1 x 107 to 2.5 x 107) of B. anthracis Vollum 1B (2) spore inocula (100 µl total volume) in the nares of animals. Animals were monitored for a 2-week period and euthanized by cardiac injection of sodium pentobarbital when considered moribund or at project end.
Lungs and spleens were removed from the guinea pigs after euthanasia. A 100-mg section was excised and homogenized with 0.4 ml of sterile PBS in a sterile tissue grinder. The homogenates were serially diluted in sterile PBS and then plated in duplicate on sheep blood agar plates. After 24 h of incubation at 37°C, the colonies were enumerated to determine CFU/100 mg of tissue.
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RESULTS |
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domain (34), has superior expression, stability, and serum half-lives compared to scFv fragments (33). Expression from a lac promoter in E. coli Tuner cells, followed by purification by IMAC, produced a mixture of a monomer and a disulfide-linked dimer with an average yield of 8 mg/liter (1.3 mg/liter optical density at 600 nm) in shake flask culture. Reduction of the inter-scAb disulfide by using TCEP produced pure monomeric M18 scAb, which was conjugated to either 20-kDa or 40-kDa PEG-maleimide (Fig. 1). Following gel filtration fast-protein liquid chromatography, the purified protein conjugates were found to contain <2 ng lipopolysaccharide/mg of protein by using the QCL-1000 Limulus amoebocyte lysate assay kit (BioWhittaker, MD).
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102 to 105 CFU/100 mg tissue) of anthrax bacteria in the lungs and spleens. Analysis of a guinea pig that succumbed in the 80 mg/kg antibody conjugate group showed a very small amount of hemorrhage and lymph depletion, yet no bacteria could be seen in a microscopic inspection of lung or spleen tissues. |
DISCUSSION |
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Conjugation of PEG to M18 scAb had no effect on the dissociation rate constant, KD, while the association rate constant, KA, was found to be dependent on the Biacore assay format. When the PA was immobilized on the Biacore chip and the scAb constructs were in the mobile phase, a decrease in KA with increasing PEG size was seen. No such trend was observed when the M18 scAb constructs were immobilized on the chip. Although the biologically relevant value of KA is not clear at this point, it should be noted that the therapeutic potency of anti-PA antibody fragments can be influenced by the dissociation rate constant, KD, (33) which, in the case of the M18 scAb, is clearly not affected by PEGylation. Consistent with this assertion, PEGylation did not affect the activity of the antibody in protecting RAW 264.7 macrophages from toxin challenge in vitro. Importantly, the PEG modification significantly increased serum persistence to the point that following subcutaneous administration, the M18 scAb-40-kDa PEG conjugate exhibited a serum half-life beyond that of the 14B7 whole IgG.
Prophylactic administration of the M18 scAb-40-kDa PEG construct resulted in a significant increase in TTD and survival of between 50 to 60% of the animal group for the 14-day duration of the experiments compared to a 3-day mean TTD for the group of animals immunized with PBS alone, unconjugated PEG, or with native scAb. No statistically significant increase in protection was observed with the 80 mg/kg dose of M18 scAb-40-kDa PEG compared to the 40 mg/kg dose, indicating that the latter or perhaps an even smaller dose may be adequate to confer the same level of protection.
In the group that was treated with 40 mg/kg, two of the six animals died on days 12 and 13, raising the possibility that perhaps the percentage survival might have been lower if the experiments had been continued for longer times. This is probably not the case, however, since histological inspection of the surviving animals did not reveal any bacteria in the lungs or the spleens, suggesting that the infection had been eradicated.
It is not yet entirely clear how a high anti-PA antibody serum titer, elicited either through active immunization or through passive immunization with anti-PA antibodies (30, 35), prevents inhaled spores from causing anthrax. The data presented here indicate that an ultra-high-affinity, anti-PA antibody fragment lacking an Fc region is sufficient to confer prophylactic protection against heavy challenge with inhalation anthrax spores. The prolonged TTD and low or nonexistent amount of live bacteria in all treated animals suggests that a higher degree of prophylaxis could be possible upon repeated antibody fragment administration.
To our knowledge, the results presented here represent the first report of immunological protection against pathogen infection by antibody fragments lacking Fc domains and administered directly to the animal prior to challenge. In the absence of Fc, the mechanism of protection is unlikely to involve either antibody-dependent cytotoxicity or complement-dependent cytotoxicity. Thus, it appears that the interaction of the high- affinity antibody fragment with toxin is sufficient to prevent the establishment of infection by preventing spore germination (52), by preventing the dissemination of vegetative bacteria from lymphoid organs, or by perhaps some other mechanism.
Regardless of the mechanism, the extent of protection conferred by M18 scAb-40-kDa PEG is roughly comparable to that observed with guinea pigs treated with rabbit polyclonal anti-PA IgG in a previous study (30). We note, however, in the earlier study by Little et al. (30), the guinea pigs were exposed to a substantially lower dose of spores relative to the present experiments (40x LD50 versus 250x to 600x LD50) and a different strain (Ames versus Vollum 1B), so caution must be used when making any direct comparison. Nevertheless, it is reasonable to suggest that the protection observed with M18 scAb-40-kDa PEG is significantly better than that reported for the parent 14B7 IgG monoclonal antibody (30). A reasonable conclusion is that the very high affinity of the engineered M18 antibody fragment is responsible for the increased protective activity. As support for this hypothesis, we note that the interaction between PA and the CMG2 receptor is relatively strong; KD was 170 pM (53), meaning that M18 (KD = 35 pM) can effectively compete with this interaction, while 14B7 (KD = 4.3 nM) cannot.
Since the anti-PA M18 scAb-40-kDa PEG lacking an Fc region is able to confer prophylactic protection against heavy challenge with inhalation anthrax spores, it is reasonable to assume that other means of inactivating anthrax toxins should also hold promise as potential therapeutics for anthrax. These include multivalent peptides that bind to PA (36), small molecule inhibitors of LF (39, 46) and dominant negative PA mutants (45).
From a therapeutic development perspective, the key advantages of PEGylated antibody fragments include the ease of isolation of scFv or scAb from combinatorial libraries and the facile low-cost production by well-established techniques using E. coli. Combined with the ease of subcutaneous injection, the approach presented here represents a highly practical strategy for a large-scale prophylactic response to anthrax exposure, including antibiotic-resistant strains (3). Studies are under way to determine whether PEGylated, high-affinity antibody fragments can be employed for protection or therapy against other bacterial agents where pathogenicity is intimately associated with toxin production, e.g., Shiga toxin-producing hemolytic E. coli (49), other microbial pathogens, and, finally, viral infections. If that proves to be the case, PEGylated antibodies will likely represent a practical and rapidly deployable therapeutic avenue for combating emerging infections.
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ACKNOWLEDGMENTS |
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This work was supported by the NIH (U01 AI56431) and the DOD-Army (DAAD17-01-D0001).
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FOOTNOTES |
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