Evaluation of plant-produced humanized
anti-ricin antibody
Wei-Gang Hu
Sarah Hayward
DRDC – Suffield Research Centre
Junfei Yin
Canada West Biosciences Inc.
Defence Research and Development Canada
Scientific Report
DRDC-RDDC-2016-R235
November 2016
IMPORTANT INFORMATIVE STATEMENTS
This research has been done in collaboration between Defence Research and Development Canada and
Canada West Biosciences Inc.
In conducting the research described in this report, the investigators adhered to the ‘Guide to the Care and
Use of Experimental Animals, Vol. I, 2nd Ed.’ published by the Canadian Council on Animal Care.
Template in use: (2010) SR Advanced Template_EN (051115).dotm
© Her Majesty the Queen in Right of Canada, as represented by the Minister of National Defence, 2016
© Sa Majesté la Reine (en droit du Canada), telle que représentée par le ministre de la Défense nationale,
2016
Abstract
Therapeutic antibodies can provide specific, instant, and consistent protection against infectious
agents/toxins and therefore offer great value for the Canadian Armed Forces as an effective
medical countermeasure (MedCM) against biological warfare agents. Unfortunately, therapeutic
antibodies are among the most expensive drugs with an average cost of $300/gram when
produced in mammalian cell lines. This production cost significantly impacts the development of
antibodies as therapeutics. DRDC Suffield Research Centre has developed a highly effective
humanized anti-ricin antibody, hD9. To reduce the manufacturing cost, the production of hD9
was explored in plants and purified hD9 was produced in the genetically engineered tobacco-like
plants by PlantForm Corporation. In this report, plant-produced hD9 (PhD9) was compared to its
mammalian cell-produced counterpart, hD9 to investigate whether the plant manufacturing
process would affect the antibody’s ricin-binding affinity and anti-ricin potency. In a
ricin-binding affinity assay, a cell-based ricin neutralization assay and in a systemic ricin
intoxication mouse model, PhD9 demonstrated its ricin-binding affinity and anti-ricin potency to
be comparable to hD9, indicating that plants can be used as bioreactors for cost-effective and
large-scale production of functional PhD9. Additionally, PhD9 was further compared both
in vitro and in vivo with the UK Defence Science and Technology Laboratory (Dstl)-developed
sheep anti-ricin polyclonal antibodies, DV057/29-2 (IgG) and P37501A [F(ab´)2]. PhD9 was not
as effective as the Dstl polyclonal antibodies in a cell based ricin neutralization assay; however,
in terms of therapeutic potency against ricin in an intranasal ricin intoxication mouse model,
PhD9 outperformed the Dstl polyclonal antibodies by about 100 times. Overall, PhD9 has great
potential to be developed as a potent MedCM against ricin intoxication.
Significance to defence and security
Ricin is a deadly toxin, which in 2014 was ranked by the North Atlantic Treaty Organization
Biological Medical Advisory Council as the top future biothreat on a list of 32 potential biothreat
agents due to its high morbidity and mortality, ease of large-scale production and storage, aerosol
stability, ease of dispersal, etc. Ricin has the potential to pose a severe threat to public health and
safety and a ricin crisis could occur at any time throughout the world. Unfortunately, there are
currently no approved anti-ricin therapeutic medical countermeasures (MedCMs) available. The
development of potent MedCMs against ricin intoxication will provide important protection in the
event of attack for both biodefence and public health.
DRDC-RDDC-2016-R235
i
Résumé
Les anticorps thérapeutiques peuvent conférer une protection spécifique, immédiate et uniforme
contre les agents infectieux et les toxines. De ce fait, ils présentent un intérêt majeur pour les
Forces armées canadiennes en tant que contre-mesure médicale efficace contre les agents de
guerre biologique. Malheureusement, les anticorps thérapeutiques figurent parmi les médicaments
les plus chers : leur coût atteint, en moyenne, 300 $ par gramme lorsqu’ils sont produits au moyen
de lignées cellulaires de mammifères. Ce coût de production influence considérablement
l’élaboration d’anticorps à des fins thérapeutiques. Le Centre de recherches de Suffield de
Recherche et développement pour la défense Canada (RDDC) a mis au point un anticorps
anti-ricine humanisé très efficace, le hD9. Afin de réduire le coût de fabrication, on a étudié la
production d’anticorps hD9 à l’aide de végétaux Ainsi, le hD9 purifié a été produit à partir de
plantes proches du tabac modifiées génétiquement par PlantForm Corporation. Dans ce rapport,
on a comparé l’anticorps hD9 d’origine végétale (PhD9) à son équivalent produit au moyen de
cellules de mammifères afin de déterminer si le procédé de fabrication grâce aux plantes influait
sur l’affinité de l’anticorps avec la ricine et sur son efficacité contre la ricine. Lors d’un test
d’affinité, d’une réaction cellulaire de neutralisation et de l’utilisation d’un modèle d’intoxication
générale à la ricine chez la souris, le PhD9 a présenté une affinité et une efficacité comparables à
celles du hD9, ce qui indique que les plantes étudiées peuvent servir de bioréacteurs en vue d’une
production rentable et à grande échelle de PhD9 fonctionnel. En outre, on a également fait la
comparaison in vitro et in vivo du PhD9 avec les anticorps polyclonaux de moutons mis au point
par le Defence Science and Technology Laboratory (Dstl) au Royaume-Uni, les DV057/29-2
(IgG) et P37501A [F(ab´)2]. Le PhD9 ne s’est pas révélé aussi efficace que les anticorps
polyclonaux du Dstl lors d’une réaction cellulaire de neutralisation de la ricine. Cependant, en ce
qui concerne l’efficacité thérapeutique selon un modèle d’intoxication intranasale chez la
souris, le PhD9 a surpassé les anticorps polyclonaux du Dstl par un facteur d’environ 100.
Globalement, le PhD9 a un potentiel considérable pour la mise au point d’une contre-mesure
médicale efficace contre l’intoxication à la ricine.
Importance pour la défense et la sécurité
La ricine est une toxine mortelle. En 2014, le Comité consultatif de l’OTAN sur la défense
biomédicale considérait que la ricine était la menace biologique future figurant en tête de liste
parmi 32 agents potentiels, notamment en raison de ses taux élevés de morbidité et de mortalité,
de sa facilité de production et d’entreposage à grande échelle, de la stabilité des aérosols et de sa
facilité de diffusion. La ricine peut représenter une grave menace pour la santé et la sécurité
publiques, et son utilisation pourrait entraîner une crise à n’importe quel moment, n’importe où
dans le monde. Malheureusement, il n’existe actuellement aucune contre-mesure médicale
approuvée pour le traitement de l’intoxication à la ricine. La mise au point d’une contre-mesure
médicale efficace contre l’intoxication à la ricine offrira une protection importante en cas
d’attaque, tant pour la défense biochimique que la santé publique.
ii
DRDC-RDDC-2016-R235
Table of contents
Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i
Significance to defence and security . . . . . . . . . . . . . . . . . . . . . . i
Résumé . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
ii
Importance pour la défense et la sécurité . . . . . . . . . . . . . . . . . . . .
Table of contents . . . . . . . . . . . . . . . . . . . . . . . . . . . .
ii
iii
List of figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
iv
List of tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
v
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . .
vi
1
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1
2
Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . .
2.1 Preparation of ricin stock . . . . . . . . . . . . . . . . . . . . . .
2.2 Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3 Affinity analysis . . . . . . . . . . . . . . . . . . . . . . . . .
2.4 In vitro neutralization assay . . . . . . . . . . . . . . . . . . . . .
2.5 In vivo protection assay . . . . . . . . . . . . . . . . . . . . . .
Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1 Ricin-binding affinity and anti-ricin potency comparison between PhD9 and hD9 .
3.1.1 Evaluation in vitro . . . . . . . . . . . . . . . . . . . . .
3.1.2 Evaluation in vivo . . . . . . . . . . . . . . . . . . . . . .
3.2 Ricin-binding affinity and anti-ricin potency comparison between PhD9 and Dstl
polyclonal anti-ricin antibodies . . . . . . . . . . . . . . . . . . .
3.2.1 Evaluation in vitro . . . . . . . . . . . . . . . . . . . . .
3.2.2 Evaluation in vivo . . . . . . . . . . . . . . . . . . . . . .
Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2
2
2
2
3
3
4
4
4
5
6
6
7
10
5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12
13
3
4
DRDC-RDDC-2016-R235
iii
List of figures
Figure 1:
Kinetic constants of PhD9 and hD9 binding to ricin. . . . . . . . . . . .
4
Figure 2:
In vitro neutralization assay. . . . . . . . . . . . . . . . . . . . . .
5
Figure 3:
In vivo protection assay. . . . . . . . . . . . . . . . . . . . . . .
5
Figure 4:
Kinetic constants of Dstl polyclonal antibodies binding to ricin. . . . . . .
6
Figure 5:
Anti-ricin neutralization potency comparison in vitro. . . . . . . . . . .
7
Figure 6:
In vivo protection assay. . . . . . . . . . . . . . . . . . . . . . .
9
iv
DRDC-RDDC-2016-R235
List of tables
Table 1:
Anti-ricin potency comparison between PhD9 and Dstl polyclonal anti-ricin
antibodies, Dstl IgG, and Dstl F(ab´)2 in vivo. . . . . . . . . . . . . .
DRDC-RDDC-2016-R235
8
v
Acknowledgements
The authors wish to acknowledge the retired Defence Scientist, Dr. John Cherwonogrodzky for
his endeavors in initial setup of intranasal ricin intoxication mouse model. The technical
assistance of the animal care support group at DRDC – Suffield Research Centre to the project is
also greatly appreciated.
vi
DRDC-RDDC-2016-R235
1
Introduction
Ricin is a 60–65 kDa glycoprotein derived from beans of the castor plant [1]. It consists of a ricin
toxin A (RTA) protein and a ricin toxin B (RTB) protein linked by a disulfide bond. RTB binds to
galactose residues on the mammalian cell surfaces to trigger cellular uptake of ricin. RTA
enzymatically cleaves ribosomal RNA to stop protein synthesis [2]. Ricin can be lethal when
injected, inhaled, or ingested and is therefore regarded as a high terrorist risk for the public [3, 4].
Currently, there are no vaccines or antidotes available against ricin.
Therapeutic antibodies have the capacity to play a critical role in the emergency management of
deadly infectious diseases/intoxications. For example, the use of antibodies was proven effective
in both of the 2010 and 2012 outbreaks of Hendra virus in Australia [5] and in the 2014 largest
recorded outbreak of Ebola virus in Africa [6]. Therefore, therapeutic antibodies can offer great
value for the Canadian Armed Forces (CAF) as an effective medical countermeasure (MedCM)
against biothreat agents and are in line with Canadian Forces Health Services’ (CFHS) demands.
With the support of the Canadian Safety and Security Program (CSSP), a highly potent anti-ricin
humanized antibody, hD9 has been developed at Defence Research and Development Canada
(DRDC) Suffield Research Centre [7–9]. To advance this antibody to the preclinical stage of
development, large-scale production of this antibody was required. Therapeutic antibodies are
among the most expensive drugs to produce, with an average cost of $300/gram when produced
in mammalian cell lines [10]; this production cost significantly impacts the development of
antibodies as therapeutics.
The manufacturing cost can potentially be reduced by novel technologies such as plant-based
antibody production. Plants can be used as bioreactors for large-scale production of therapeutic
antibodies at a low cost [11]. The lowest reported cost for the production and purification of
antibodies in a transgenic crop is estimated at $12–15/gram [12], although there is no approved
antibody product to validate this estimate. To reduce the manufacturing cost, the production of
hD9 was explored in plants by a Canadian industrial contract with PlantForm Corporation,
supported by the CFHS Biological Warfare Threat Medical Countermeasure (BWTMCM)
project. In the first production run, a total of 350 mg of purified hD9 was produced in genetically
engineered tobacco-like plants by PlantForm Corporation.
In this report, the study was designed to investigate whether the plant manufacturing process
would affect the ricin-binding and anti-ricin potency of the hD9. Additionally, plant-produced
hD9 (PhD9) was further evaluated in terms of ricin-binding affinity and anti-ricin potency in
comparison to the UK Defence Science and Technology Laboratory (Dstl)-developed sheep
anti-ricin polyclonal antibodies, IgG and F(ab´)2 under the Memorandum of Understanding
Concerning the Research, Development, and Acquisition of Chemical, Biological and
Radiological Defense Material (CBR MOU) MedCM Consortium Working Group (MCMC) Task
6-Ricin MedCMs.
DRDC-RDDC-2016-R235
1
2
2.1
Materials and methods
Preparation of ricin stock
Ricin was prepared from castor beans at DRDC Suffield Research Centre [8] and the toxicity of
this ricin stock was also determined. For the systemic ricin intoxication mouse model via the
intraperitoneal (i.p.) route, 0.215 µg of ricin was equivalent to 1×LD50 and with 5×LD50 (1.09 µg)
all mice died within two days. For the intranasal (i.n.) instillation ricin intoxication mouse model,
0.09 µg of ricin was equivalent to 1×LD50; with 2.2×LD50 (0.2 µg) the mean time to death was
7 days. All research with ricin was conducted in a secure Containment Level 2 area and under the
purview of Organisation for the Prohibition of Chemical Weapons.
2.2
Antibodies
HD9 was produced in HEK 293 mammalian cells (Thermo Fisher Scientific, Burlington, ON)
transfected with a recombinant adenovirus expressing hD9. The expressed recombinant hD9 was
purified using ImmunoPure Protein (L) agarose gel (Pierce, Brockville, ON) [7]. PhD9 was
produced by PlantForm Corporation [13, 14]. Briefly, hD9 gene was transiently expressed, along
with human galactosyl transferase, in a line of Nicotiana benthamiana (ΔFX) with suppressed
expression of the plant-specific glycosyltransferases (xylosyl-fucosyl-transferases) to mimic
human glycosylation patterns on the produced protein. The PhD9 was successfully purified and
polished and the formulated product was 97% pure and contained ≤1 endotoxin unit per mg of
PhD9. The UK Dstl polyclonal anti-ricin antibodies, DV057/29-2 (IgG) and P37501A [F(ab´)2]
were raised in sheep following a series of immunizations with ricin toxoid plus incomplete
Freund’s adjuvants; they were prepared and provided by Dstl [15].
2.3
Affinity analysis
The binding affinity of PhD9, hD9, and Dstl polyclonal antibodies (DV057/29-2, P37501A) for ricin was
determined using a Surface Plasmon Resonance (SPR) biosensor, SensiQ Pioneer (ICx Technologies,
Oklahoma, OK). Briefly, ricin (10 μg/mL) diluted in 10 mM acetate buffer pH 4.5 was first immobilized
onto the COOH1 chip following the standard 1-ethyl-3-(3-dimethylpropyl)-carbodiimide (EDC) plus
N-hydroxysuccinimide (NHS) (Sigma-Aldrich, Oakville, ON) coupling chemistry and
350 response units (RU) of ricin were immobilized. The system was operated at 25 °C. Kinetic
measurements were made using a two minute injection at a flow rate of 25 μL/min of serial
dilutions of antibodies in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES,
Sigma-Aldrich)-buffered saline containing 3 mM ethylenediaminetetraacetic acid (EDTA,
Sigma-Aldrich), 150 mM NaCl and 0.005% Tween-20 followed by a dissociation for ten minutes.
The ricin-immobilized chip surface was regenerated by injection of 10 mM phosphoric acid for
120 seconds after each cycle. The data of dissociation (koff) and association (kon) rate constants
were obtained with the SensiQ Qdat software, corrected by subtraction of the zero antibody
concentration flow cell as well as zero ricin flow cell; values for the apparent equilibrium
dissociation constant (Kd) were calculated from the ratio of koff and kon.
2
DRDC-RDDC-2016-R235
2.4
In vitro neutralization assay
A Vero cell-based (ATCC, Burlington, ON) ricin toxicity neutralization assay was performed
utilizing Alamar blue (TREK Diagnostic System, Cleveland, OH) as a cell viability indicator [8].
Ricin was incubated with a serial dilution of anti-ricin antibodies for two hours at 37 °C in
96-well plates. Ten thousand Vero cells cultured in 50 µL of DMEM medium (Thermo Fisher
Scientific) with 10% fetal bovine serum (Hyclone, Fisher Canada) were added into the mixture.
The final volume of the cell mixture was 200 µL with the ricin concentration of 7.5 ng/mL and
antibody concentrations from 0.14 to 33.33 nM, or 0.26 to 33.33 nM. After incubation at 37 °C,
5% CO2 in a humidified incubator for two days, 20 µL of Alamar blue was added to each well
and the plate was then incubated for 6–7 hours. On a plate reader (Molecular Devices, Sunnyvale,
CA), the plate was read at an absorbance of 570 nm with 600 nm as a reference wavelength.
Readings were normalized by subtracting the absorbance reading of wells without cells. Cell
viability is expressed as cell survival rate relative to the control without ricin (Vero cells plus
antibodies).
2.5
In vivo protection assay
Female Balb/c mice (18–20 g) were obtained from the pathogen-free mouse-breeding colony
from Charles River Canada (St Constant, QC). All animal experiments were performed in strict
accordance with the guidelines set out by the Canadian Council on Animal Care. The animal care
protocol was reviewed and approved by the Committee on the Ethics of Animal Experiments of
DRDC, Suffield Research Centre. All efforts were made to minimize suffering.
For post-exposure therapeutic potency study in a systemic ricin intoxication mouse model, groups
of eight mice were given 5×LD50 of ricin i.p. per mouse and then 5 μg per mouse of either the
antibody (PhD9 or hD9) or Hank’s Balanced Salt Solution (HBSS) (Thermo Fisher Scientific)
was administered i.p. at 1, 2, 4, or 6 hours after ricin exposure. The mice were observed for
morbidity and mortality over one week.
For the post-exposure therapeutic potency study using an i.n. instillation ricin intoxication mouse
model, groups of four or twelve mice were anesthetized with metophane in a sealed chamber until
the mice were unconscious. The mice were then given 0.2 µg/50 µL of ricin per mouse by i.n.
instillation and a series of doses of antibodies were administered by either the intramuscular (i.m.)
or intravenous (i.v.) route at different hours (therapeutic windows) following ricin exposure. The
mice were observed for morbidity and mortality over 14 days.
DRDC-RDDC-2016-R235
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Cell survival rate (%)
100
Dstl IgG
Dstl F(ab')2
PhD9
Control
80
60
40
20
0
33.33
16.67
8.33
4.17
2.08
1.04
0.52
0.26
Antibody concentrations (nM)
Figure 5: Anti-ricin neutralization potency comparison in vitro. Ricin (7.5 ng/mL) was
pre-incubated with a serial dilution of different antibodies and then exposed to 104 Vero
cells/well for two days before evaluation of cell viability using Alamar blue staining. The cell
viability is expressed as cell survival rate relative to the control without ricin.
3.2.2
Evaluation in vivo
An i.n. ricin intoxication mouse model was used in order to compare the protective potency
between PhD9 and the Dstl polyclonal antibodies. Mice were challenged i.n. with ricin at a dose
of 2.2×LD50. A series of doses of different antibodies were then administered by either i.m. or i.v.
route to the mice at different hours (therapeutic windows) after ricin challenge. The results are
shown in Table 1.
The data for a dose of 1 mg of Dstl IgG or F(ab´)2, or 100 µg of PhD9 administered by the i.m.
route at 16 hours post-exposure are plotted in Figure 6. PhD9 was able to rescue 100% of the
mice, while the Dstl IgG and F(ab´)2 polyclonal antibodies were able to rescue only 33% and 67%
of the animals.
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7
Table 1: Anti-ricin potency comparison between PhD9 and Dstl polyclonal anti-ricin
antibodies, Dstl IgG, and Dstl F(ab´)2 in vivo.
Abs
Therapeutic Window
(hours post-exposure)
Route
i.v.
16
i.m.
PhD9
i.m.
20
i.v.
24
Dstl IgG
16
20
Dstl F(ab´)2
16
20
i.v.
i.m.
i.m.
i.v.
i.m.
i.m.
i.v.
i.m.
Dose
(µg)
Survival mice/
total mice
% Survival
30
50
100
10
30
6/8
6/8
4/4
13/16
8/8
75%
75%
100%
81%
100%
100
12/12
100%
100
150
150
200
100
200
1,000
1,000
2,500
1,000
1,000
1,000
2,500
1,000
3/4
3/4
3/4
8/8
3/4
7/8
4/12
4/8
8/8
2/4
8/12
6/8
8/8
2/4
75%
75%
75%
100%
75%
88%
33%
50%
100%
50%
67%
75%
100%
50%
Notes: Ricin was given at the dose of 2.2×LD50 to mice by the i.n. route. A series of doses of different
antibodies were administered by either i.m. or i.v. route to the mice at different hours (therapeutic
windows) after ricin exposure. The mean time to death of the control mice was 7 days post-exposure.
8
DRDC-RDDC-2016-R235
Survival rate (%)
Days
Figure 6: In vivo protection assay. Groups of twelve mice were challenged with 2.2×LD50 ricin
via the i.n. route. PhD9 (100 µg), Dstl IgG (1 mg), Dstl F(ab´)2 (1 mg), or HBSS (control) were
then administered to the animals at 16 hour post-exposure via the i.m. route. The mice were
observed for morbidity and mortality over a two week period.
DRDC-RDDC-2016-R235
9
4
Discussion
Therapeutic antibodies are among the most expensive drugs. The current production of
therapeutic antibodies involves the use of very large cultures of mammalian cells followed by
extensive purification steps, under good manufacturing practice conditions, leading to extremely
high production costs [10]. The high cost has dramatically affected the development of antibodies
as therapeutics. The cost could potentially be reduced by certain novel technologies, such as
plant-based antibody production. As such, hD9 was produced in plants. In this study, the PhD9
was evaluated against its mammalian cell-produced counterpart, hD9. The results demonstrated
that PhD9 was comparable to hD9 in terms of ricin-binding affinity and anti-ricin potency both in
vitro and in vivo, indicating plants can be used as bioreactors for large-scale production of
therapeutics at reduced cost. Meanwhile, the plant production system is fast, efficient, highly
versatile (for new product development), easily scalable for production, and free from
contamination by mammalian pathogens. Furthermore, development of this production platform
would provide DRDC the capability for cost-effective and large-scale production of any
protein-based MedCM against biothreat agents in the future. Currently there are only a few Food
and Drug Administration-approved plant-produced biologics on the market and none of these are
antibody-related. Before the plant-based therapeutic antibodies come to the market, some major
challenges still remain to be addressed, including non-mammalian glycosylation, genetic
instability, few available contract manufacturing organizations, and the rigorous regulatory
requirements and standards for plant-based pharmaceutical products [16, 17].
There are many anti-ricin neutralizing antibodies being developed worldwide. Some of these
anti-ricin antibodies have been evaluated in either inhalation or intranasal instillation ricin
intoxication mouse models, since aerosol is the most likely route of dissemination of biological
select agents and toxins in a bioterrorist attack, regardless of the natural route of exposure to the
agent [18]. Ricin acts rapidly and therefore leaves a very short therapeutic window (effective
timing of administration of therapeutic antibodies) for post-exposure MedCMs. For post-exposure
therapeutic potency study in inhalation/intranasal instillation ricin intoxication mouse models,
currently the best result reported in the literature has been the Dstl polyclonal antibodies, which
demonstrated that the therapeutic window could be pushed up to 16 hours post exposure when
given an i.v. injection of 2.5 mg IgG and F(ab´)2 in an inhalational mouse model with intoxication
of 3 ×LD50 ricin [15].
In this report, PhD9 was compared to the Dstl polyclonal antibodies. In a ricin-binding affinity
assay, the affinity of PhD9 binding to ricin was around nine times higher than Dstl polyclonal
antibodies. The result is understandable since polyclonal antibodies consist of various monoclonal
antibodies, which have different antigen-binding affinities. As such, the Kd of a polyclonal
antibody is an average of various monoclonal antibody Kds. Therefore, this result only indicates
that the monoclonal PhD9 ricin-binding affinity is around nine times higher than the average.
PhD9 was further compared with Dstl polyclonal antibodies in a Vero cell-based anti-ricin
neutralization assay. When the antibody molar concentrations were ≥1.04 nM, PhD9 anti-ricin
potency was not as high as Dstl polyclonal antibodies, but when the antibody molar
concentrations were <1.04 nM, PhD9 anti-ricin potency was better than Dstl polyclonal
antibodies, indicating the anti-ricin neutralization mechanism might be different between PhD9
and Dstl polyclonal antibodies in vitro, or Dstl polyclonal antibodies might contain a subset of
high potency neutralizing antibodies that could neutralize ricin more efficiently than PhD9 at high
10
DRDC-RDDC-2016-R235
concentrations, but at low concentrations, the number of the high potency neutralizing antibodies
might not be enough to be effective to neutralize ricin. When PhD9 was compared with the Dstl
polyclonal antibodies in an i.n. instillation mouse model with intoxication of 2.2×LD50 ricin,
PhD9 outperformed the Dstl polyclonal antibodies when antibodies were administered by an i.m.
route in which PhD9 (30 µg per mouse) was able to rescue 100% of the mice, while Dstl IgG and
F(ab´)2 polyclonal antibodies were able to rescue only 33% and 67% of the mice at an even
higher dose of 1 mg per mouse. Since the molecular weight of F(ab´)2 is around two thirds of
IgG, the anti-ricin protective potency of PhD9 at molar basis is around 100 times better than Dstl
anti-ricin IgG and F(ab´)2. Taken together, although PhD9 was not as efficient as Dstl polyclonal
antibodies in the in vitro ricin neutralization assay, PhD9 outperformed the polyclonal antibodies
in the in vivo rescue study using a ricin-intoxicated mouse model. The antibody functions against
pathogens or toxins involve a series of mechanisms that include direct binding to specific
epitopes to neutralize toxins or inhibit microbial attachment [19] and indirect antibody-mediated
events primarily consisting of antibody-dependent cellular cytotoxicity, antibody-dependent cell
phagocytosis, and complement-dependent cytotoxicity [20]. Unlike in vivo studies, an in vitro
neutralization test is only able to examine the antibody direct function and unable to examine the
antibody indirect functions. Our results indicate that the indirect functions of PhD9 might also
play a pivotal role against ricin intoxication in vivo. Furthermore, PhD9, as a humanized
monoclonal antibody, does not have the animal polyclonal antibody’s drawbacks, such as serious
side effects in humans mainly due to foreignness to humans [21] (serum sickness, low efficacy
resulting from rapid clearance of antibodies from humans, and potentially lethal allergic
response), batch-to-batch variation, uncertain dosing, and a risk of infection from animal serum.
DRDC-RDDC-2016-R235
11
5
Conclusion
The ricin-binding affinity and anti-ricin potency of PhD9 were confirmed to be comparable to
those of hD9 both in vitro and in vivo, indicating that plants can be used as bioreactors for
cost-effective and large-scale production of functional hD9.
In addition, the in vivo anti-ricin efficacy study also demonstrated that PhD9 performed
substantially better than the Dstl polyclonal anti-ricin antibodies, the most efficacious antidote
against ricin intoxication in mouse models reported in the literature. Overall, PhD9 has great
potential to be developed as a potent MedCM against ricin intoxication.
12
DRDC-RDDC-2016-R235
References
[1] Montanaro L, Sperti S, Stirpe F: Inhibition by ricin of protein synthesis in vitro. Ribosomes
as the target of the toxin. Biochem J 1973, 136(3):677–683.
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[3] Audi J, Belson M, Patel M, Schier J, Osterloh J: Ricin poisoning: a comprehensive review.
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[13] Hall JC, Meyers A: Plant-Produced Humanized Antibodies DRDC Contract Report 2016,
Defence Research and Development Canada, DRDC-RDDC-2016-C022.
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Evaluation of plant-produced humanized anti-ricin antibody
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Hu, W.-G.; Hayward, S.; Yin, J.
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Therapeutic antibodies can provide specific, instant, and consistent protection against infectious
agents/toxins and therefore offer great value for the Canadian Armed Forces as an effective
medical countermeasure (MedCM) against biological warfare agents. Unfortunately, therapeutic
antibodies are among the most expensive drugs with an average cost of $300/gram when
produced in mammalian cell lines. This production cost significantly impacts the development
of antibodies as therapeutics. DRDC Suffield Research Centre has developed a highly effective
humanized anti-ricin antibody, hD9. To reduce the manufacturing cost, the production of hD9
was explored in plants and purified hD9 was produced in the genetically engineered
tobacco-like plants by PlantForm Corporation. In this report, plant-produced hD9 (PhD9) was
compared to its mammalian cell-produced counterpart, hD9 to investigate whether the plant
manufacturing process would affect the antibody’s ricin-binding affinity and anti-ricin potency.
In a ricin-binding affinity assay, a cell-based ricin neutralization assay and in a systemic ricin
intoxication mouse model, PhD9 demonstrated its ricin-binding affinity and anti-ricin potency
to be comparable to hD9, indicating that plants can be used as bioreactors for cost-effective and
large-scale production of functional PhD9. Additionally, PhD9 was further compared both
in vitro and in vivo with the UK Defence Science and Technology Laboratory (Dstl)-developed
sheep anti-ricin polyclonal antibodies, DV057/29-2 (IgG) and P37501A [F(ab´)2]. PhD9 was not
as effective as the Dstl polyclonal antibodies in a cell based ricin neutralization assay; however,
in terms of therapeutic potency against ricin in an intranasal ricin intoxication mouse model,
PhD9 outperformed the Dstl polyclonal antibodies by about 100 times. Overall, PhD9 has great
potential to be developed as a potent MedCM against ricin intoxication.
--------------------------------------------------------------------------------------------------------------Les anticorps thérapeutiques peuvent conférer une protection spécifique, immédiate et uniforme
contre les agents infectieux et les toxines. De ce fait, ils présentent un intérêt majeur pour les
Forces armées canadiennes en tant que contre-mesure médicale efficace contre les agents de
guerre biologique. Malheureusement, les anticorps thérapeutiques figurent parmi les
médicaments les plus chers : leur coût atteint, en moyenne, 300 $ par gramme lorsqu’ils sont
produits au moyen de lignées cellulaires de mammifères. Ce coût de production influence
considérablement l’élaboration d’anticorps à des fins thérapeutiques. Le Centre de recherches de
Suffield de Recherche et développement pour la défense Canada (RDDC) a mis au point un
anticorps anti-ricine humanisé très efficace, le hD9. Afin de réduire le coût de fabrication, on a
étudié la production d’anticorps hD9 à l’aide de végétaux Ainsi, le hD9 purifié a été produit à
partir de plantes proches du tabac modifiées génétiquement par PlantForm Corporation. Dans ce
rapport, on a comparé l’anticorps hD9 d’origine végétale (PhD9) à son équivalent produit au
moyen de cellules de mammifères afin de déterminer si le procédé de fabrication grâce aux
plantes influait sur l’affinité de l’anticorps avec la ricine et sur son efficacité contre la ricine.
Lors d’un test d’affinité, d’une réaction cellulaire de neutralisation et de l’utilisation d’un
modèle d’intoxication générale à la ricine chez la souris, le PhD9 a présenté une affinité et une
efficacité comparables à celles du hD9, ce qui indique que les plantes étudiées peuvent servir de
bioréacteurs en vue d’une production rentable et à grande échelle de PhD9 fonctionnel. En
outre, on a également fait la comparaison in vitro et in vivo du PhD9 avec les anticorps
polyclonaux de moutons mis au point par le Defence Science and Technology Laboratory (Dstl)
au Royaume-Uni, les DV057/29-2 (IgG) et P37501A [F(ab´)2]. Le PhD9 ne s’est pas révélé
aussi efficace que les anticorps polyclonaux du Dstl lors d’une réaction cellulaire de
neutralisation de la ricine. Cependant, en ce qui concerne l’efficacité thérapeutique selon un
modèle d’intoxication intranasale chez la souris, le PhD9 a surpassé les anticorps polyclonaux
du Dstl par un facteur d’environ 100. Globalement, le PhD9 a un potentiel considérable pour la
mise au point d’une contre-mesure médicale efficace contre l’intoxication à la ricine.
14. KEYWORDS, DESCRIPTORS or IDENTIFIERS (Technically meaningful terms or short phrases that characterize a document and could be helpful
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Unclassified, the classification of each should be indicated as with the title.)
therapeutic antibodies; anti-ricin; plant-produced; efficacy