Vaccine 23 (2005) 2082–2086

Human-derived, plant-produced monoclonal antibody for thetreatment of anthrax

Anna K. Hulla, Carolyn J. Criscuoloa, Vadim Metta, Herman Groenb, Wilma Steemanb,Hans Westrab, Gail Chapmanc, Bart Legutkic, Les Bailliec,d, Vidadi Yusibova,∗

a Fraunhofer USA Center for Molecular Biotechnology, 9 Innovation Way Suite 200, Newark, Delaware, DE 19711, USAb IQ Corporation, Rozenburglaan 13a, 9727 DL Groningen, The Netherlands

c Biological Defense Research Directorate, Naval Medical Research Center, 12300 Washington Avenue, Rockville, MD 20852, USAd Medical Biotechnology Center, University of Maryland Biotechnology Institute, 725 West Lombard Street, Baltimore, MD 21201, USA

Available online 13 January 2005

Abstract

The unpredictable nature of bio-terrorism compels us to develop medical countermeasures that will enable authorities to treat individualsexposed to agents such as anthrax. We report the feasibility of producing a protective, human-derived, monoclonal antibody directed againsttb en for itsh©

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he protective antigen (PA) ofBacillus anthracisin plants. This was achieved by transient expression using agroinfiltration ofNicotianaenthamianaplants. The resulting antibody was able to neutralize toxin activity in vitro and in vivo at a comparable level to that seybridoma-produced counterpart.2005 Elsevier Ltd. All rights reserved.

eywords:Anthrax; Antibody; Plant

. Introduction

In a bio-terrorism attack the aggressor is likely to striket a time and place calculated to induce maximum terror

hrough mass casualties. The absence of specific intelligencend integrated real-time detection systems coupled with thenpredictable nature of such events necessitates the devel-pment of medical countermeasures that will enable author-

ties to rapidly treat exposed individuals. The credentials ofacillus anthracisas a bioterror agent were confirmed by

he 2001 postal attacks in the USA. Following inhalation,nthrax spores are phagocytosed by alveolar macrophagesnd transported to hilar and tracheobronchial lymph nodeshere the spores germinate, and multiplication of vegeta-

ive bacilli occurs[1,2]. Fatal bacteraemia and toxaemiahen ensue, with a mortality rate in untreated individualsf >80%[3].

∗ Corresponding author. Tel.: +1 302 369 3766; fax: +1 302 369 8955.E-mail address:vyusibov@fraunhofer.org (V. Yusibov).

Early treatment is essential. Animal studies suggest ththe disease progresses due to the accumulation of a lethaof toxin antibiotics become ineffective[4]. B. anthracispro-duces a tripartite toxin consisting of two enzymatically acsubunits, lethal factor (LF) and edema factor (EF) that cbine with protective antigen (PA), the non-toxic cell-bindcomponent, to form lethal toxin (LT) and edema toxin, restively [5]. Given the central role of anthrax toxin in patholoof the disease, the ability to neutralize its effects would bimmense value at all stages of the infection.

Several animal studies have demonstrated that antoxin-specific antibodies from vaccinated animals cansively protect recipients[6–8]. Horse serum has also beused to treat people exposed to anthrax[9]. However, thedrawbacks inherent in using animal-derived sera (serumness) have prompted the adoption of alternative approsuch as the use of anthrax immunoglobulin derived frommunized donors[10]. While this approach offers a numbof advantages such as reduced adverse reactions, proserum half life, and the targeting of multiple epitopes, it dsuffer from the costly need to maintain, and constantly

264-410X/$ – see front matter © 2005 Elsevier Ltd. All rights reserved.oi:10.1016/j.vaccine.2005.01.013

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A.K. Hull et al. / Vaccine 23 (2005) 2082–2086 2083

new adequate stocks of quality-controlled and safety-testedantibodies with sufficiently high toxin neutralization titer toprotect a large numbers of individuals. An alternative ap-proach would be to develop toxin-neutralizing human mon-oclonal antibodies (mAbs) with specificity for either PA orLF, or both[11]. In conjunction, improved mAb productionsystems will be needed that are capable of generating safe,efficacious antibodies at a scale and cost which will supportlarge scale stockpiling necessary for rapid response to anattack.

Plant-based antibody production systems have the capac-ity to meet this need as they offer specific advantages suchas product safety, low cost and ease of scale-up compared toconventional fermentation methods[12]. Plants have provento be effective systems for producing functional therapeuticmAbs capable of conferring passive protection against bac-terial and viral pathogens in animals[13].

We report here the production and evaluation of a plant-produced PA-specific toxin-neutralizing mAb (pp-mAbPA)derived from a human immunized with a licensed anthraxvaccine. The resulting antibody was found to be as efficaciousas its hybridoma-produced counterpart. This approach offersthe potential to develop urgently needed medical countermea-sures at a fraction of the cost of traditional cell culture-basedapproaches.

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2.2. Transient gene expression

Agrobacterium tumefaciensstrain GLA 4404 (Invitrogen,Carlsbad, CA) was transformed with pBISfi-IQ6E4G andpBISfi-IQ6E4K.Agrobacteriumcultures were grown and in-duced as described[14]. Equal volumes of the induced sus-pensions (each at OD6002.4) were mixed and used in agroin-filtration ofNicotiana benthamianaleaves. The leaves wereharvested 2.5 days post-infiltration and stored at−80◦C.

2.3. Antibody purification

The plant tissue was homogenized in extraction buffer(50 mM Tris–HCl, pH 7.5, 10 mM sodium diethyldithio-carbamate and Complete Mini protease inhibitors (RocheApplied Science, Indianapolis, IN)) with 1% PVPP(polyvinylpolypyrrolidone). The extract was incubated for1 h at 4◦C with 0.1% Tween®20. Plant debris was removedby filtration through Miracloth (Calbiochem, San Diego,CA) followed by centrifugation. The antibody was recov-ered using ammonium sulfate precipitation and HiTrap pro-tein A column chromatography (Amersham, Piscataway, NJ).The pp-mAbPA was further purified by thiophilic adsorp-tion chromatography using T-gel adsorbent (Pierce, Rock-ford, IL). Hybridoma-produced monoclonal antibody (hp-m PA elc

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. Materials and methods

.1. DNA constructs

The SfiI site at position 11031 of pBI121 (Accessiumber AF485783) was mutagenized followed by introd

ion of newSfiI sites in positions 5825 and 7711 to creBISfi.

Total RNA was isolated from the anti-PA mAb-expressybridoma line IQ6E4 (IQ Corporation, The Netherlansing RNeasy Mini Kit (Qiagen, Valencia, CA). Heavy a

ight chain coding sequences were amplified by RT-Psing Superscript One-Step RT-PCR kit with PlatinumNA polymerase (Invitrogen, Carlsbad, CA). The hehain (HC) was amplified using the degenerate 5′-primer, 5′-TCGCGGCCCAGCCGGCCATGGACTGSAYCTGGAG′, in combination with the constant region 3′-primer 5′-CT-GCGGCCTCCGAGGCCTCATTTACCCKGAGACAGG′. The light chain (LC) was amplified using a mixtuf three 5′ primers 5′-CTCGCGGCCCAGCCGGCCATGACATGAGGGTCCYCGC-3′, 5′-CTCGCGGCCCAGCGGCCATGAGGSTCCYTGCTCAGCT-3′, 5′-CTCGC-GCCCAGCCGGCCATGGAARCCCCAGCGCAGCT-′

n combination with the constant region 3′-primer 5′-CTC-CGGCCTCCGAGGCCCTAACACTCTCCCCTGTTGA

′. Resulting HC and LC fragments were cloned in pBISreate pBISfi- IQ6E4G (encoding HC) and pBISfi- IQ6Eencoding HC).

Ab ), was purified similarly using protein A and T-ghromatography.

.4. ELISA and Western blot analysis

Antibody concentrations were estimated by ELISA usunc Maxisorb plates (Fisher Scientific, Hampton, NH).AbPA and hp-mAbPA were detected with horseradish pxidase (HRP)-conjugated rabbit anti-human IgG (Jac

mmunoresearch Laboratories, West Grove, PA). Wholean IgG (Jackson Immunoresearch Laboratories) wass a standard. Specificity of pp-mAbPA was determined bLISA and Western blotting. For ELISA, plates were coaith 0.5�g of PA/well (Alpha Diagnostic International, Sntonio, TX). In Western blot analysis and ELISA pp-mAbPA

ound to PA was detected by HRP-conjugated rabbituman IgG.

.5. Toxin neutralization assay

Toxin-neutralizing activity was determined using a muacrophage/monocyte (RAW 264.7) cell line culture anST-1-based cell viability quantitation assay (Roche Mo

lar Biochemicals, Indianapolis, IN). Serial dilutions of Ppecific antibodies were assayed against standard corations of LF and PA. RAW 264.7 cells (TIB-71, Amean Type Culture Collection, Manassas, VA) were plate× 104 cells/well in a microtiter plate in D-MEM supplented with 2 mMl-glutamine (Gibco BRL, GaithersburD), 10% heat-inactivated fetal bovine serum (FBS,

lone, Logan, UT), 10 mM HEPES buffer solution (Gib

2084 A.K. Hull et al. / Vaccine 23 (2005) 2082–2086

Fig. 1. Purification and characterization of pp-mAbPA. (A) Coomassie staining of purified pp-mAbPA. (B) Binding of pp-mAbPA to PA (→) in Western blot.(C) Binding of pp-mAbPA (�) and hp-mAbPA (�) to PA in ELISA. MWM indicate molecular weight markers.

BRL), Gentamicin (25�g/ml), and 1 mM sodium pyruvate(Gibco BRL) approximately 4 h prior to the assay. All incu-bations were at 37◦C and 5% CO2 atmosphere, 95% relativehumidity. Test antibodies were prepared in a separate 96-wellmicrotiter plate as two-fold dilutions in four or five replicasand incubated with 100 ng/ml PA at 37◦C for 30 min. PA (pre-incubated with or without antibody) and LF were added to thecells (respective concentrations 100 ng/ml and 50 ng/ml) andincubated for 12–15 h. WST-1 was added and cell viabilitymeasured using an ELISA reader (OD450).

2.6. Animal protection studies

The ability of the pp-mAbPA and hp-mAbPA to protect A/Jmice against challenge with spores of the Sterne strain ofB.anthraciswas determined according to the method of Beed-ham and colleagues[8]. A group of five mice were given180�g of pp-mAbPA by the intra-peritoneal route in PBS.Control mice received either the equivalent concentration ofthe hp-mAbPA or PBS. 2.5 h after passive immunization an-imals received spores ofB. anthracis, at a dose of 1× 104

spores in 0.1 ml of PBS (approx 30 median lethal doses).Following challenge, animals were monitored daily for 14days for evidence of morbidity or mortality.

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sorbent chromatography. SDS-PAGE analysis demonstratedhigh purity and integrity of the pp-mAbPA (Fig. 1A). Theyield of purified antibody was approximately 1 mg/kg planttissue.

The hybridoma line producing hp-mAbPA was selectedbased on its ability to bind PA and to neutralize LT. Thespecificity of pp-mAbPA for PA was confirmed by Westernblotting (Fig. 1B) and in ELISA (Fig. 1C). The pp-mAbPA

efficiently bound to PA as determined by Western blotting andwas indistinguishable from hp-mAbPA in its performance inELISA. This result indicates that production in plants did notimpair the specificity of antibody for PA.

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. Results

.1. Characterization of antibody expression

cDNAs for HC and LC of hp-mAbPA were cloned from thQ6E4 hybridoma cell line and transiently expressed fromauliflowerMosaicVirus35S RNA promoter by agroinfiltra

ion inN.benthamianaplants. The pp-mAbPA was purified byrotein A affinity chromatography followed by thiophilic a

ig. 2. Percentage of relative inhibition of cell death as result of toxinralization by pp-mAbPA or hp-mAbPA. mAbs were assessed at differoncentrations for the ability to neutralize the lethal toxin and preveneath. No toxin is a control representing RAW 264.7 cells grown witny toxin or mAbs. Toxin indicates RAW 264.7 cells grown in the presf PA and LF. The data points represent the average from five indepeeplicates.

A.K. Hull et al. / Vaccine 23 (2005) 2082–2086 2085

Fig. 3. Animal protection studies. 2.5 h after receiving a single dose (180�g)of pp-mAbPA A/J mice were exposed to lethal dose of anthrax spores. Controlmice received either equivalent quantity of hp-mAbPA or PBS. Animalsreceiving PBS developed clinical signs of infection and succumbed to disease3 days after challenge. Animals receiving pp-mAbPA or hp-mAbPA did notdevelop disease symptoms and survived the challenge.

3.2. Neutralization of lethal toxin in vitro

The ability of hp-mAbPA and pp-mAbPA to neutralizetoxin activity in vitro was examined. pp-mAbPA, when mixedwith PA prior to exposure to murine macrophages, was ableto neutralize toxin activity in a similar and dose-dependentmanner (Fig. 2) to hp-mAbPA. Cell viability studies indicatedthat at 50 ng/ml pp-mAbPA could efficiently neutralize PA re-sulting in 100% cell survival.

3.3. Animal protection studies

The ability of the pp-mAbPA to protect A/J mice against aninjected lethal challenge ofB. anthracisspores was examinedand compared with hp-mAbPA. While all the control mice(n= 8) died within 3 days of challenge, the pp-mAbPA (n= 5)and hp-mAbPA (n= 8) treatment groups remained healthy un-til the end of the experiment (Fig. 3).

4. Discussion

There is substantial data in the literature to show thatanthrax toxin-neutralizing antibodies can confer protectionacross a range of animal species, including primates, againsta -ti vertb inm ap-p led ndi-v gesi ck tot s hasb bod-i longh cale.T pting

develop humanized or fully human PA-specific toxin neutral-izing monoclonal antibodies[15,16].

Our studies show that plant-produced mAb against PAhad both in vitro and in vivo toxin-neutralizing activity. Infact, the pp-mAbPA neutralized anthrax toxin with the sameefficacy as the hp-mAbPA both in cell assays and in protec-tion assays in mice. The results of this study suggest that ahuman monoclonal antibody produced in plants with speci-ficity for PA can be successfully employed for prophylacticuse.

In summary, the results of this study demonstrate thatfully active human monoclonal antibodies with specificityto PA can be successfully produced in plants and canbe employed for prophylactic use. The study also con-firms that the plant-based approach is a viable alternativefor manufacturing large quantities of biologically activemolecules.

Acknowledgments

The authors thank Dr. Shailaja Rabindran for criticalreading of the manuscript. The authors also thank Mar-garet Schillingford, Christopher Hull, Alison Freeman, PattyWilkins, Tom Taylor, Christine Cool and Kunja Slopsemaf ndedb

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erosol challenge with spores ofB. anthraciswhen adminisered prior to or immediately post challenge[6–8]. The abil-ty to treat exposed individuals several days after a coio-terror attack would result in a significant reductionortality. Given that the half-life of human antibodies isroximately 20 days[10], it raises the possibility that a singose of antibody may be sufficient to treat an infected iidual, a property offering considerable logistic advantan a mass casualty setting. In the past, the major roadblohe adoption of this approach for the treatment of humaneen access to high affinity, anthrax toxin-specific anti

es that are non-toxic to humans, possess a significantlyalf-life, and that can be produced on a commercial so address these issues, researchers have been attem

to

or excellent technical assistance. This project was fuy grants from Fraunhofer USA and from the US Navy.

eferences

[1] Ross JM. The pathogenesis of anthrax following the administrof spores by the respiratory route. J Clin Pathol 1957;73:485–

[2] Guidi-Rontani C, Weber-Levy M, Labruyere E, Mock M. Germition of Bacillus anthracisspores within alveolar macrophages. MMicrobiol 1999;31(1):9–17.

[3] Dixon TC, Meselson M, Guillemin J, Hanna PC. Anthrax. N EJ Med 1999;341(11):815–26.

[4] Smith H, Keppie J, Stanley JL, Harris-Smith PW. The chemical bof the virulence ofBacillus anthracis. IV. Secondary shock as tmajor factor in death of guinea-pigs from anthrax. Br J Exp Pa1955;36(3):323–35.

[5] Baillie L, Read TD.Bacillus anthracis, a bug with attitude! CurOpin Microbioldkjdot 2001;4(1):78–81.

[6] Henderson DW, Peacock S, Belton FC. Observations on thephylaxis of experimental pulmonary anthrax in the monkey. J(Lond) 1956;54(1):28–36.

[7] Pitt ML, Little SF, Ivins BE, et al. In vitro correlate of immunity inrabbit model of inhalational anthrax. Vaccine 2001;19(32):4768

[8] Beedham RJ, Turnbull PC, Williamson EN. Passive transfer oftection againstBacillus anthracisinfection in a murine model. Vacine 2001;19(31):4409–16.

[9] Gold H. Treatment of anthrax. Fed Proc 1967;26(5):1563–8.10] Casadevall A. Antibodies for defense against biological attack

Biotechnol 2002;20(2):114.11] Baillie LW, Williamson D, Quinn C, Groen H. Human anthrax to

neutralizing monoclonal antibodies for the treatment of anthraxPoster 166, ASM Biodefense Meeting, 2004.

12] Schillberg S, Fischer R, Emans N. Molecular farming of recombiantibodies in plants. Cell Mol Life Sci 2003;60(3):433–45.

2086 A.K. Hull et al. / Vaccine 23 (2005) 2082–2086

[13] Peeters K, De Wilde C, De Jaeger G, Angenon G, Depicker A.Production of antibodies and antibody fragments in plants. Vaccine2001;19(17-19):2756–61.

[14] Kapila J, De Rycke R, Van Montagu M, Angenon G. AnAgrobacterium-mediated transient gene expression system for intactleaves. Plant Sci 1997;122:101–8.

[15] Wild MA, Xin H, Maruyama T, et al. Human antibodies from im-munized donors are protective against anthrax toxin in vivo. NatBiotechnol 2003;21(11):1305–6.

[16] Maynard JA, Maassen CB, Leppla SH, et al. Protection against an-thrax toxin by recombinant antibody fragments correlates with anti-gen affinity. Nat Biotechnol 2002;20(6):597–601.