Production and Characterisation of a Neutralising Chimeric Antibody against Botulinum Neurotoxin A
et al. (2010) Production and Characterisation of a Neutralising Chimeric Antibody against
Botulinum Neurotoxin A. PLoS ONE 5(10): e13245. doi:10.1371/journal.pone.0013245
Production and Characterisation of a Neutralising Chimeric Antibody against Botulinum Neurotoxin A
Edgardo Moreno, Universidad Nacional, Costa Rica
0 1 CEA, iBiTec-S, Service de Pharmacologie et d'Immunoanalyse, CEA Saclay, Gif sur Yvette, France, 2 Unite des Toxines et des Bacte ries Anae robies, Institut Pasteur , Paris , France
Botulinum neurotoxins, produced by Clostridium botulinum bacteria, are the causative agent of botulism. This disease only affects a few hundred people each year, thus ranking it among the orphan diseases. However, botulinum toxin type A (BoNT/A) is the most potent toxin known to man. Due to their potency and ease of production, these toxins were classified by the Centers for Disease Control and Prevention (CDC) as Category A biothreat agents. For several biothreat agents, like BoNT/A, passive immunotherapy remains the only possible effective treatment allowing in vivo neutralization, despite possible major side effects. Recently, several mouse monoclonal antibodies directed against a recombinant fragment of BoNT/A were produced in our laboratory and most efficiently neutralised the neurotoxin. In the present work, the most powerful one, TA12, was selected for chimerisation. The variable regions of this antibody were thus cloned and fused with the constant counterparts of human IgG1 (kappa light and gamma 1 heavy chains). Chimeric antibody production was evaluated in mammalian myeloma cells (SP2/0-Ag14) and insect cells (Sf9). After purifying the recombinant antibody by affinity chromatography, the biochemical properties of chimeric and mouse antibody were compared. Both have the same very low affinity constant (close to 10 pM) and the chimeric antibody exhibited a similar capacity to its parent counterpart in neutralising the toxin in vivo. Its strong affinity and high neutralising potency make this chimeric antibody interesting for immunotherapy treatment in humans in cases of poisoning, particularly as there is a probable limitation of the immunological side effects observed with classical polyclonal antisera from heterologous species.
Botulinum neurotoxins (BoNTs) are the most toxic substances
known . The seven serotypes, BoNT/A to BoNT/G, are
produced by different strains of Clostridium botulinum. Subtypes
have been identified within some serotypes based on amino acid
variation from approximately 2 to 32% , like BoNT/A1 to
BoNT/A4 for serotype A. This anaerobic spore-forming
bacterium is ubiquitous in the environment and can germinate under
suitable conditions to yield the vegetative bacterium which
synthesise the toxin . Human botulism is mainly caused by
ingestion of contaminated food with botulinum toxin (foodborne
botulism), by contamination of a wound with C. botulinum spores
(wound botulism) or by intestinal colonization and toxin
production in infants ,1 year (infant botulism) . Clostridia release
their neurotoxins as protein aggregates in culture or food. These
aggregates, or progenitor toxins, are formed by a complex of an
inactive polypeptide toxic chain (150 kDa) and other
neurotoxinassociated proteins (haemagglutinin and/or other proteins
depending on serotypes) [5,6] which stabilise neurotoxins . After
proteolytic cleavage, the active form consists of a 100 kDa heavy
chain (HC) linked by a disulfide bridge to a 50 kDa light chain
(LC). The HC allows the toxin to bind irreversibly to nerve cells at
the neuromuscular junction and mediates translocation across the
membrane. The LC bears the catalytic activity and, as a Zn2+
endopeptidase, cleaves protein member(s) of the SNARE complex
involved in the release of acetylcholine . The neuromuscular
blockade results in flaccid paralysis , generates similar
symptoms regardless of BoNT type and may cause death due to
respiratory failure or cardiac arrest. Recovery depends on the
capacity of new motor axons to reinnervate paralysed muscle
fibres. This takes weeks or months according to the quantity and
type of toxin . During this period, intensive care is crucial,
especially artificial ventilation. Human cases are caused by toxin
types A, B and E. Serotype B is the most widely encountered,
while serotype A gives the gravest symptoms because of its higher
toxicity and longer persistence in the body [11,12]. The lethal dose
of crystalline toxin A is estimated at 1 mg/kg when introduced
orally and the dissemination of a single gram could kill more than
1 million people .
Because of its extreme toxicity, potency, lethality, ease of
production and the lack of an effective treatment, BoNTs have
thus been classified by the Centers for Diseases Control and
Prevention (CDC) among the 6 major agents (category A) that
could be used in bioterrorism . The potential threat of
biological warfare and bioterrorism has stimulated renewed efforts
to generate vaccines and therapies against agents such as BoNTs.
Preventing the effects of such threats requires the development of
specific pharmaceutical compounds to protect the general
population and the military .
Among the different strategies, the use of a protective antibody
as a countermeasure appears the most suitable therapy since
antibodies are less toxic and more specific than other chemical
drugs . Moreover, passive immunotherapy provides
immediate protective immunity in the case of emergency after an attack,
as compared with vaccination . Two immunotherapies against
botulism have reduced botulism mortality rates from
approximately 60% to less than 10% . The most frequent antitoxin
preparations are equine products such as the bi- or trivalent
antitoxin (type AB or ABE) introduced by the FDA in the 1970s
. The US Army Medical Research Institute of Infectious
Diseases also developed a heptavalent preparation from horse IgG
antibodies against serotypes A, B, C, D, E, F and G, with and
without their Fc fragment . The other type of antitoxin is the
human Botulism Immune Globulin (BabyBIG) approved by the
FDA in 2003 as BIG-IV to treat infant botulism caused by type A
or B toxins. It was produced from immune plasma of donors who
had been immunised with pentavalent (AE) botulinum toxoid
. Although treatments cannot reverse existing paralysis once
the toxin has entered the synaptic button, antitoxins can minimise
nerve damage, preventing progression of paralysis, and decrease
the duration of supportive care [18,19]. Use of BIG-IV has thus
largely reduced hospitalisation costs (by $88 600 per patient).
Furthermore, equine antitoxin may cause adverse effects ranging
from moderate hypersensitive immune reactions to anaphylactic
shock . Protection by therapeutic agents can also differ
according to subtype within the BoNT/A serotype. Indeed,
reduction in binding affinity and neutralisation between BoNT/
A1 and BoNT/A2 has already been noted .
Recent publications report the production of mouse monoclonal
antibodies (mAbs) with neutralising activity. Most are directed
against the HC domain and a recent study described mAbs binding
the LC part of BoNT/A [22,23]. In this context, we have recently
produced several mouse mAbs , using a recombinant protein
corresponding to the C-terminal binding domain of Botulinum
neurotoxin A1 (Fc-BoNT/A1, 50 KDa) which has protective
antigenic properties . Among the different mAbs neutralising
BoNT/A1 in vivo , the most efficient, murine TA12 (mTA12),
was selected to construct a chimeric antibody combining the TA12
variable regions with the constant regions of the human IgG1. The
corresponding chimeric TA12 antibody (cTA12) was produced
and characterised for further comparison with the initial mouse
mAb. Different parameters, i.e. binding affinity, pharmacokinetic
parameters and in vivo neutralising titre, were determined for both
mouse and chimeric antibodies. The results show that the
recombinant mAb retained a capacity for highly efficient
neutralisation together with a very low dissociation constant (close
to 10 pM). Thus, the cTA12 antibody may possibly be considered
as a promising candidate of therapeutic tool and could be
combined with other mAbs for the treatment of human botulism.
Construction of the chimeric monoclonal TA12 antibody
14 monoclonal antibodies were obtained after immunising mice
with the recombinant C-terminal domain of BoNT/A1 heavy
chain, Fc-BoNT/A1 . 12 out of the 14 antibodies neutralized
toxin activity in vivo . TA12 mAb, the most efficient antibody,
was selected for chimerisation. VH and VL cDNAs were cloned
from hybridoma cells secreting TA12 and sequences were verified
using scFv production. Functional scFv were selected thanks to
their Fc-BoNT/A1 binding, and chimeric light and heavy chain
genes were constructed in a specific vector for expression in the
baculovirus system or in mammalian cells (see methods).
Comparison of the chimeric antibody produced in Sf9
cells and in SP2/0-Ag14 cells
Production in insect cells. Sf9 cells were transfected with
recombinant bacmids (including genes encoding heavy and light
cTA12 chains) to produce recombinant baculovirus. Conditions of
antibody production were optimised after a second round of
infection and were obtained with cells seeded at a density of
1.86105 cells/cm2 (data not shown). After infection of cells at
different MOI (0.04, 0.12, 0.4, 1.2), supernatants were harvested
every 24 h and the concentration of secreted cTA12 was measured
by EIA using Fc-BoNT/A1 as antigen (Figure 1). The production
of cTA12 reached a plateau and thus appeared optimum 34 days
post-infection. The greater the MOI, the lower the plateau,
probably due to the rapid cell lysis. Using purified cTA12 as
standard, the concentration of cTA12 in the supernatant was
calculated as 5 mg/l (corresponding to 3.75 pg/cell 4 days after
infection). The Sf9 supernatant was analysed by western blotting
(Figure 2). The light and heavy chains secreted in the supernatant
exhibited approximately the expected 1:1 stoichiometry (Figure 2B,
lane 1). These observations are confirmed by the results obtained
after affinity chromatography purification, showing in reducing
conditions the heavy and light cTA12 chains (lane 2, Figure 2B)
that are correctly assembled into an Ig molecule, since a single
band with the expected molecular weight is detected in
nonreducing conditions (lane 2, Figure 2A).
Production in mammalian myeloma cells. Stable
expressing Sp2/O-Ag14 cells were established after
cotransfection with the two pcDNA3 plasmids encoding cTA12
light and heavy chains using the limiting dilution cloning method.
Screening of the supernatants by EIA allowed the selection of 12
different clones secreting cTA12. A kinetic study of the
productivity of these clones was done to evaluate the secreted
recombinant cTA12 concentration. As seen in Figure 3, the
production curves showed a general pattern similar to that of
previous observations with the Sf9 cells and presenting a maximum
at 4 days of culture. The production yields strongly differ
according to the clone, probably because of the number of
recombinant cDNA copies integrated in the cell genome, the
localisation of integration and the ratio between the two cDNA
encoding chains. Clones 16G6 and 16G7 proved to be the two
best producers, with a production yield (using non-optimised
conditions) of 0.8 mg/ml. For 16G7 clones, productivity reached
1 pg/cell at day 4. When optimising the culture conditions, the
maximal concentration of functional cTA12 in the 16G7
supernatant reached 1.5 mg/l (data not shown). To increase
production of chimeric antibody, 16G7 cells secreting cTA12 were
injected into irradiated Balb/c mice for production of ascitic fluids.
Total chimeric antibody was evaluated at 1.2 g/l concentration in
these ascitic fluids and was purified by affinity chromatography.
cTA12 is secreted as a whole antibody (Figure 2A, lane 1,) based
on western blot experiments. In the ascitic fluid, the light and
heavy chains are thus secreted with a correct 1:1 stoichiometry and
are all assembled into Ig molecules since no fragment other than
the two chains was detected using either non-reducing or reducing
conditions (Figure 2A, lane 1 and figure 2B, lanes 3 and 4).
Characterisation of TA12 antibody binding kinetics
Kinetic parameters of the TA12 mAb were measured by
Surface Plasmonic Resonance biosensor technology using
FcBoNT/A1 as antigen (Table 1). The calculated KD of 15.4 pM for
cTA12 produced by Sf9 cells appears very similar to the value of
14.6 pM obtained for cTA12 produced by murine myeloma cells
(ascitic fluids). These sub-nanomolar results are comparable to
those obtained for mTA12, which has a KD of 19.1 pM. These
observations demonstrate that the chimerisation of TA12 mAb did
not modify antibody affinity or kinetics. These strong affinities
result partly from a fast association rate constant (kon of 3.76106
and <26106 M21s21 for mTA12 and cTA12, respectively, see
Table 1 and Figure 4) combined with a very low dissociation rate
constant (koff), particularly for the cTA12 antibody showing a koff
value at the limit of detection for the Biacore calculation (Table 1
and Figure 4). This very slow koff allows us to estimate a minimum
half-life of 5.12 h at RT for the cTA12/Fc-BoNTA1 complex.
Functionality of the recombinant cTA12
Migration profiles of cTA12 light and heavy chains produced by
either insect cells or by myeloma murine cells do not present any
differences (Figure 2). However, the chimeric recombinant
antibody may be less stable than the murine wild-type mAb and
the purification process may also lead to some loss of functional
antibody. To evaluate better the possible discrepancy between
chimeric antibody concentration measured by UV absorbance
and the true corresponding functional concentration, a
comparative experiment was designed based on a sandwich enzyme
immunoassay taking the murine wild-type antibody as reference
Assuming that potential denaturation during adsorption to the
solid phase is similar for the two types of purified antibodies, and
KD (M)610211 kon (M21.s21)6106
KD was calculated from kon and koff with n = 4 for all experiments.
since the affinity of the two mAbs is identical (as deduced from
Biacore experiments), the purified chimeric mAbs (from
mammalian and insect cells) were compared with purified mTA12 for their
capacity to recognise native BoNT/A1 in quasi equilibrium
conditions (considering their kinetic parameters). As shown in
Figure 5, for the same UV absorbance, there was a loss of signal of
more than 50% for cTA12 purified from insect cell supernatants as
compared with mTA12, showing that a major part of the
recombinant mAb produced by the insect cells might not be
functional. The loss of signal is less important for purified cTA12
from ascitic fluids (close to 30%) Taking into account these results,
further experiments were performed with the cTA12 purified from
ascitic fluids rather than from Sf9 culture supernatants.
Pharmacokinetic studies of murine and chimeric TA12
antibodies in mouse
During previous pharmacokinetic studies, mTA12 antibody
proved to exhibit a slower clearance than its F(ab9)2 counterpart
(4 h vs 22 days), resulting in a 100-times longer in vivo half-life .
It thus seemed important to compare the half-life of the murine
and chimeric TA12 antibodies. After injecting antibodies
intraperitoneally into mice, plasma concentrations of cTA12 and
mTA12 were determined at different times by competitive
immunoassay. As shown in Figure 6, an initial rapid increase
was observed within the first hours after injection for both
antibodies, corresponding to antibody transfer from the
peritoneum to the blood compartment. A maximum was reached
approximately 24 h post-injection, followed by a decrease during
the subsequent five weeks. Whereas the plasma concentration
appears a bit lower for cTA12 than the mouse mAb during the
first day after injection, the maximum concentrations were quite
close. However, the plasma clearance of cTA12 was clearly faster
than that of the parent mAb, and less than 1% of the maximum
concentration remained after 700 h, as compared with about 95%
for mTA12. These discrepancies were predictable since the
chimeric antibody included a majority of human sequences which
Figure 4. Kinetic analyses of cTA12 and mTA12 binding to Fc-BoNT/A1 by SPR. mTA12 was directly immobilized on a sensor chip at 200 RU
(A), and cTA12 was captured at 150 RU using a sensor chip prepared with immobilized anti-human antibody (B). Fc-BoNT/A1 (range 0.8 to 20 nM) was
injected at 50 ml/min for 3 min (association phase). Dissociation was monitored over a period of 30 min before the chip was regenerated with 10 mM
glycine pH 2. BiaEvaluation Software 3.2 was used to calculate kinetic constants with the Langmuir 1:1 model fit.
further induced faster elimination in the mouse. Nevertheless,
values can be included in a pharmacokinetic model (without
compartment) to evaluate the half-lives of both antibodies. The
cTA12 half-life was close to 7 days, which is 2.5 times shorter
than that of mTA12, but much longer than that of the
(Fab92) fragments. When used in a more favourable environment
such as immunotherapy in humans, the cTA12 half-life should
greatly increase and become comparable to that of mTA12 in
The neutralising activity of TA12 corresponds to a protection of 1,000 estimated
MLD50 of BoNT/A1 with 50 mg of antibody or to 286 IU/mg according to the
method of Hatheway and Dang as described by Mazuet et al. .
In vitro and in vivo neutralisation of the neurotoxin with
murine and chimeric antibodies
When studying the ability of mouse and chimeric antibodies to
neutralise the neurotoxin, the cTA12 antibody retained the very
efficient neutralising power of mTA12 with a neutralisation titre of
nearly 1314 IU/mg, as compared with 6.2 to 20.8 IU/mg for the
mouse antibody (Table 2).
In addition, the protective activity of cTA12 was tested in vivo by
the co-injection protocol consisting in separate injections of the
toxin and antibody without any in vitro preincubation step. As
shown in Table 3, an intraperitoneal injection of 2.5 mg or 250 ng
of cTA12 was able to completely protect all the mice challenged
with a lethal injection of 5 estimated MLD50/mouse of BoNT/A1.
A dose of 25 ng of cTA12 per mouse only partially protected the
mice challenged with 5 estimated MLD50. The in vivo protective
activity of cTA12 is identical to the one obtained with mTA12
antibody  and comparable to the in vitro protective activity
against BoNT/A1 (Table 4).
Since variability of the BoNT gene and protein sequence within
botulinum neurotoxin subtypes A has been reported and these
sequence variations impact on antibody binding and neutralisation
, the capacity of mouse and chimeric antibodies to neutralise
BoNT/A2 and /A3 was evaluated using a mouse neutralisation
assay with 5 estimated MLD50 per mouse of each toxin subtype.
The two forms of antibody showed a marked reduction in
neutralising capacity of 10- and 100-fold for BoNT/A3 and /A2,
respectively (table 4). These results appear in accordance with
published data  reporting that mAbs directed against BoNT/
A1 showed a 500- to 1,000-fold reduction in binding affinity and a
minimal neutralisation capacity for A2 toxin.
There is an urgent need to produce neutralising antibodies
devoid of side effects and directed against botulinum neurotoxins,
especially the most toxic for humans, the serotype A. In this
context, the mAbs produced in our lab against BoNT/A1 for
The neutralizing activity was determined using the mouse protection assay with
5 estimated MLD50/mouse of BoN/A1. BoNT/A1 (0.5 ml) and serial dilutions of
cTA12 (0.5 ml) were injected separately into each mouse of a group of 810.
Results are expressed as surviving mice versus the total number of mice.
mAb quantity (ng/mouse)
MAb protection activity was determined using the mouse protection assay with
5 estimated MLD50/ml of BoNT/A1, BoNT/A2 or BoNT/A3 and serial dilutions of
mAbs. 5 estimated MLD50/mouse were incubated for 30 min at room
temperature with 2,500 to 0.25 ng of Mab and the mixture (0.5 ml) was injected
intraperitoneally in each mouse of a group of 8 to 10 mice. Results are
expressed as the number of surviving mice versus the total number of mice. ND,
diagnostic purposes were evaluated for their potential neutralising
ability . In this recent study, TA12 mAb (mTA12) was
characterised as the most powerful due to its great efficiency in
neutralising the toxin in vivo even alone. Moreover, its high affinity
and good half-life highlighted its potential for human
To limit or avoid possible side effects linked to the murine origin
of the Ig , a cloning strategy replacing most of the mouse
sequences by the human counterparts, i.e. production of a
chimeric antibody, was initiated. The chimeric antibody (cTA12)
was produced and characterised either in insect cells (Sf9) infected
with a recombinant baculovirus or in murine myeloma cells (SP2/
0-Ag14) transfected with recombinant vectors. In the optimal
culture conditions, cTA12 reached a concentration of 1.5 mg/ml
in the supernatant of SP2/0-Ag14 cells. This value is of the same
order as in a previous publication reporting the production in
CHO cells of a chimeric antibody directed against BoNT/A,
ranging from 0.5 to 3 mg/ml in the supernatant . For
production of chimeric antibody using the baculovirus system,
cTA12 reached a concentration of 5 mg/ml, corresponding to the
lower part of the range (between 6 and 18 mg/ml) previously
published . Without optimising conditions, antibody synthesis
per cell was 5-fold greater in Sf9 cells than in Sp2/O-Ag14 cells
(3.75 pg/cell vs 0.8 pg/cell at day 4). This difference in
productivity of the two systems of production is therefore
consistent with the literature values.
As Sp2/O-Ag14 myeloma cells derive from B lymphocytes, cells
dedicated to antibody synthesis and secretion, one might expect a
greater efficiency of these cells, allowing production of high
quantities of antibody. However, due to immortalisation, synthesis
might have been attenuated. Moreover, during transfection some
bias could have been introduced in the selection of positive clones
that was based on two criteria: the production of functional
antibody and obviously cell growth. Clones growing very slowly
were discarded for practical reasons, even if they may be among
the best secreting ones. Even if the antibody yield does not appear
in favour of myeloma cells, two other arguments partially
counteract the use of insect cells. First, Sf9 cells do not perform
the same post-translational modifications as the mammalian cells,
especially for glycosylation. Since modified glycosylated moieties
are a well-known source of immune response in mammalian hosts,
these may at least shorten the half-life of the antibody in humans,
thus probably decreasing the neutralising efficiency, or even worse,
inducing immunological side effects, which we try to limit by
creating a chimeric antibody. On the other hand, the lytic
baculovirus cycle of Sf9 cells not only releases intracellular
proteases that may alter the stability of secreted functional
antibodies, but also unfolded or unfinished intracellular chains of
antibodies that could interfere with functional antibody
purification and qualification. To evaluate the presence of unfinished or
degraded fragments of light and heavy chains in cTA12 purified
from Sf9 supernatants or ascitic fluids, western blot experiments
were performed in non-reducing or reducing conditions. No
degraded antibody was detected, leading to the conclusion that for
both production systems these unwanted forms may account for
less than 10% of recovered antibody. However, this absence of
characterisation of incomplete antibody does not guarantee that
all synthesised antibody was fully functional, particularly after
purification, which could denature recombinant chimeric antibody
more than the initial mouse antibody. Functional chimeric
antibodies recovered from both production systems were
characterised using the same sandwich immunoassay and purified
mTA12 as standard (assuming it was 100% functional). Part of the
purified cTA12 from baculovirus (up to 70%) appeared not to be
functional as compared with mTA12, while most of the chimeric
mAb recovered from mammalian system (.70%) appeared
functional. The secretion process (particularly post-translational
modifications) in insect cells might lower the stability and thus lead
to difficulties in maintaining the correct conformation at low pH
Interestingly, recombinant chimeric antibody retained all the
properties of its murine counterpart. First, cTA12 exhibited a
remarkably high kon and a koff even lower than that observed for
the mouse mAb. The resulting KD was nearly impossible to
deduce, since the dissociation rate constant was almost too slow (in
the order of 1025s21) to calculate. However, TA12 is clearly a
high-affinity antibody with a dissociation constant of at least of
10610212 M21. This combination of high kon and low koff
presents a real advantage in neutralising the target, notably when
circulating at low concentrations in the whole body. This high
affinity also limits the number of antitoxin injections, thus
minimising the potential immune side effects. This advantage is
emphasised by the relatively long half-life of the antibody as
observed during pharmacokinetic studies. The half-life of the
whole mouse antibody is similar to that obtained in a previous
study (18.5 days versus 22 days). As expected, the half-life of
chimeric TA12 is shorter than that of the mouse mAb in the
mouse model (7 days), but much longer than that of the mouse
F(ab)92 (4 h). The murine immune system is probably not as
favourable as the human one for the chimeric antibody and better
stability of cTA12 should be observed after administration to
humans. It should be kept in mind that a previous report described
a half-life of 6.5 days for the equine antitoxin in a patient . We
might expect a longer half life for the present cTA12.
The high affinity of mAb TA12 could explain the strong
neutralising effect observed in vivo. Correlation between antibody
affinity and serum neutralisation has been established for tetanus
toxin  and verified with anthrax toxin . The better the
affinity, the better the neutralisation, whatever the mechanism(s)
involved in protection against intoxication using antibodies. Among
the 12 neutralising anti-BoNT/A1 mAbs, mTA12 exhibits the
strongest neutralisation capacity, evaluated between 6.2 and
20.8 IU/mg, ranking this reagent as a good candidate for
immunotherapeutic purposes. The chimeric antibody described
here had a neutralising efficiency close to 13 IU/mg. Even if
neutralization of BonT/A2 and A3 is less effective, TA12 is still able
to neutralise this subtype in vivo. This might be improved if this
antibody is combined with antibodies neutralising A2 and A3 toxins.
Very promising human or chimeric antibodies have also been
described, such as an oligoclonal recombinant antibody, involving
3 mAbs cloned by phage display . This combination inhibits
BoNT/A1 with a great potency (45 IU/mg), when each separated
antibody was unable to neutralise BoNT/A1. More recently, two
other mAbs directed against the HC and the LC parts of BoNT/A
proved to protect mice at very low concentrations when they were
combined . Two distinct human neutralising antibodies,
proved to be efficient alone in the mouse protection assay [22,34].
However, it seems difficult to compare antibodies with each
other, due to the lack of a gold standard for in vivo neutralisation
tests, in spite of the reference test (L+10) recommended by the
European Pharmacopeia . As an illustration, our mTA12
antibody, in the standard mouse neutralisation bioassay, has a
neutralising titre of 450 IU/mg of antibody , whereas in the
L+10 neutralising bioassay the titre falls to 6 to 20 IU/mg.
To the best of our knowledge, TA12 has great potential for
therapeutic use, for four main reasons: i) this antibody possesses an
extremely low KD, which is favourable for immunotherapeutic
purposes, ii) its neutralising titre is very high, iii) it can be used
alone (and obviously combined with other neutralising antibodies),
iv) its long half-life in mice (which should be even better in
humans) should avoid repeated injections.
To conclude, the present recombinant chimeric antibody seems to
be a good candidate for passive human immunotherapy due to its
high affinity in the picomolar range and its in vivo neutralising
efficiency alone, as characterised by the fast on-rate, very low off-rate
and long half-life. This TA12 mAb could be mixed with other
chimeric antibodies under development at the CEA (notably
antiBoNT/B and anti-BoNT/E antibodies), to prepare an oligoclonal
preparation especially for use against human botulinum intoxication.
Materials and Methods
All experiments were performed in accordance with French and
European Community guidelines for laboratory animal handling.
The protocols of experiments were approved by the Pasteur
Institute (Agreement of laboratory animal use nu 75279).
Enzyme Immunoassays (EIAs)
Sandwich immunoassays. Two types of sandwich
immunoassays were used throughout this study:
Quantitative immunoassay: The concentration of the different
forms of TA12 antibody was determined via their capacity to bind
the recombinant Fc-BoNT/A1 protein. This EIA was performed
in 96-well microtitre plates coated with different antibodies
depending on the form of TA12 evaluated. Polyclonal goat
antimouse IgG or rabbit anti-human IgG antibodies were used
(0.6 mg/well) to measure the initial mTA12 or the chimerised one.
Supernatants or purified antibodies were incubated overnight at
4uC in EIA buffer (0.1 M phosphate buffer pH 7.4, 0.15 M NaCl,
0.1% BSA and 0.01% sodium azide). After 3 washing cycles (in
10 mM phosphate buffer pH 7.4, 0.1% Tween20), 100 ml/well of
the biotinylated Fc-BoNT/A1 (25 ng/ml) was added and the
reaction was allowed to proceed for 2 hours (h) at room
temperature (RT). After 3 washing cycles, 100 ml/well of streptavidin
labelled with 2 EU/ml (for Ellman Unit (EU) definition, see )
acetylcholinesterase (AChE) was added for 1-h reaction at RT.
After 3 washing cycles, 200 ml of AChE substrate (Ellmans
reagent)  was added to each well and absorbance was
measured at 414 nm after 30-min reaction at RT.
Functional immunoassay: This second sandwich method was
used to evaluate the ability of TA12 to bind to the entire native
toxin in vitro. 96-well microtitre plates were coated overnight at RT
with diluted cTA12 or parent mTA12 (in 0.05 M phosphate
buffer). After 2 h of saturation in EIA buffer, 100 ml/well of native
BoNTA1-containing culture supernatant (at 300 LD100, kindly
provided by the Pasteur Institute) was incubated overnight. After 3
washing cycles, 100 ml/well of 5 EU/ml AChE-labelled TA13 (a
tracer antibody directed against BoNT/A1 ) was added for a
2-h reaction. After 3 washing cycles, 200 ml of Ellmans reagent
was added to each well and absorbance at 414 nm was measured
after 1.5 h of incubation at RT.
Competitive immunoassay. During pharmacokinetic studies,
plasma concentrations of mouse and chimeric TA12 antibodies were
determined with a competition assay using 96-well microtitre plates
coated with TA5, an anti-BoNT/A1 mAb . 100 ml of sample
dilutions (plasma dilutions in EIA buffer, ranging from 1:100 to 1:1000
for mTA12 and from 1:5 to 1:20 for cTA12, or purified antibody for
calibration) was added to the wells with 50 ml of 2 EU/ml competitor
(mTA12 tracer antibody) and 50 ml of 0.1 nM Fc-BoNT/A1. After an
overnight incubation at 4uC, plates were washed before adding 200 ml
of Ellmans reagent to each well. Absorbance at 414 nm was
measured after 1-h incubation.
The murine myeloma SP2/0-Ag14 cell line  (a generous gift
of Dr. Mazie from the Pasteur Institut ) and hybridoma cells
secreting mouse TA12 monoclonal antibody were maintained at
37uC with 7% CO2, in RPMI medium supplemented with 10%
foetal calf serum, 1% glutamine, 1% sodium pyruvate, 1%
penicillin/streptomycin. Sf9 insect cells, derived from Spodoptera
frugiperda (Invitrogen), were grown as monolayer culture in
synthetic InsectExpress medium (PAA). Cells were cultured at
28uC in a humidified incubator. These cells were used for the
infection with the recombinant baculovirus.
Cloning of variable heavy (VH) and light (VL) regions and
construction of recombinant antibody
Cloning of VH and VL. Total RNA was extracted from
mTA12 hydridoma cells (56106 cells) using the GenEluteTM
Mammalian total RNA Miniprep kit (Sigma-Aldrich). After
preparation of the RNA using the GeneRacer Kit (Invitrogen),
reverse-transcription was achieved using heavy chain upstream
(59-CCAGGAGAGTGGGAGAGGCTCTTCTCAGTATGGTGG-39) or light chain upstream primer Kappa
was then amplified by Race-PCR using the downstream
GeneRacerTM primer and the upstream heavy chain primer
IgG1 Nested (59-GGCTCA
GGGAAATAGCCCTTGACCAGGCATCC-39) or light chain primer Kappa Nested
(59-GTGAGTGGCCTCACAGGTATAGC-39). The VH and VL genes
were cloned in pCR2.1 vector with the TOPO TA cloning kit
(Invitrogen) and sequenced.
ScFv production. After characterising each sequence,
specific primers were designed to amplify the entire variable
domains VL and VH (V-J and V-D-J regions) for light and heavy
chains, respectively. VL and VH products were assembled by
overlap extension PCR (SOE-PCR)  with a 20-amino-acid
linker [Gly4Ser]4 . Each product was cloned into the SPI 3.0
vector, a prokaryotic expression plasmid derived from the pET26b
vector (Novagen, Madison, WI) including extra N-terminal
HATag and C-terminal His-Tag for detection and purification
purposes . TA12 scFv were further produced using the
prokaryotic expression vector SPI 3.0 and the E. coli BL21 (DE3)
expression strain (Novagen, Madison, WI). Supernatant
containing the soluble periplasmic extract was collected and stored at
220uC until use. Periplasm extracts, containing HA-scFv-His
products, were screened using two different immunoassays. The
first one detects full-length scFv using double Tag detection: an
anti-HA-Tag as capture antibody (mAb 12CA5) and an
AChElabelled anti-His-Tag antibody was used as tracer. The second one
detects functional scFv using the same coated solid phase, together
with biotinylated-FcBoNT/A1 as tracer revealed using
AChElabelled streptavidin. Briefly, 50 ml of periplasmic extract were
incubated with 50 ml of tracer antibody in microtitre plates coated
with the capture antibody. Comparison of both screening tests
allows selection of full-length scFv specific to Fc-BoNT/A, thus
presenting correct folding.
Cloning and production of the chimeric antibody in
insect cells using the baculovirus system
DNA encoding variable gene fragments (VH and VL) was
reamplified by PCR (RedAccuTaq DNA polymerase, Sigma) using
oligonucleotides containing restriction sites. PCR products were
digested (by XhoI/NheI for VH and SacI/HindIII for VL) and
inserted in frame with the human heavy chain constant region
(IgG1) for VH and the kappa light chain constant region for VL, in
pAc-K-CH3 vector (Progen Biotechnik) . Constructions were
verified by sequencing. To express the chimeric antibody in the
baculovirus system (Bac-to-Bac baculovirus expression system,
Invitrogen), chimeric heavy and light chains were subcloned in
pFastBacDUALTM expression vector (Invitrogen), using BamHI
and NcoI/PvuII restriction sites for heavy and light chain,
respectively. Recombinant bacmids, corresponding to the
genetically modified baculovirus (Autographa californica) genome containing
cTA12 genes, were generated according to the manufacturers
instructions. Briefly, DH10BAC bacterial cells were transformed by
the recombinant vector for transposition of the heavy and light
chain DNA into the bacmid. Positive recombinant bacmids were
used to transfect Spodoptera frugiperda (Sf9) insect cells for viral particle
formation. All procedures for the production of viral particles were
performed according to the manufacturers instructions. Briefly,
0.5 mg of the purified bacmid was complexed with Escort Reagent
(Sigma) to transfect 16106 Sf9 cells seeded in 6-well plates. 72 h
later, the first stock of recombinant baculovirus (P1) containing
supernatant was harvested. The titre of the virus was determined by
the modified endpoint dilution assay . The virus stock solution
was filtered through 0.22 mm filters and stored at 4uC, protected
from light. To amplify baculovirus stock, this P1 low titre viral stock
was used at a multiplicity of infection (MOI) of 0.1 to infect cells
seeded at 26106 cells/well in 6-well plates or 1.356107 cells in T-75
flasks. P2 viral stock was harvested 72 h after cell infection.
For antibody production (P3), Sf9 cells were infected with the
high titre (P2) viral stock and 1.356107 cells in T-75 flasks.
Supernatant (containing 1 mM protease inhibitor AEBSF (Fluka))
was harvested from 0 to 7 days post-infection, clarified by
centrifugation (5 min at 500g), filtered through 0.22 mm filters and
stored at 4uC until purification. To determine the optimal
production, 0.2 ml of supernatant was removed every day and
the functional antibody concentration was checked by EIA.
Cloning in mammalian expression vector and production
of the chimeric antibody in mammalian cells
Chimeric heavy and light chains from pAc-K-CH3 vector were
subcloned separately in pcDNA3 expression vector (Invitrogen)
using digestion by BamHI and EcoRV, respectively (further
named pcDNA3-H and pcDNA3-L, respectively). SP2/0-Ag14
cells were co-transfected with pcDNA3-H and pcDNA3-L
recombinant vectors using the standard method with
LipofectamineTM reagent (Gibco). To generate stable cell lines secreting the
recombinant cTA12 antibody, transfected cells were selected with
geneticin (1 mg/ml, Invitrogen) 3 days post-transfection. To assess
the production of functional recombinant TA12 antibody,
supernatants of geneticin-resistant cells were tested by EIA.
EIApositive cells were cloned by limiting dilution method and
supernatants were harvested for the characterisation of antibodies.
Purification of the recombinant antibody
Recombinant and wild-type antibodies were purified using
protein A affinity chromatography . Samples containing the
antibody were mixed (1:1 v/v) with binding buffer (20 mM
phosphate buffer pH 7.4, 0.15 M NaCl ) and incubated overnight
at 4uC with protein A gel (Millipore). The bound antibody was
eluted in 0.1 M sodium citrate buffer (pH 3) and each fraction
(750 ml) was neutralised with 250 ml of potassium phosphate buffer
(1 M pH 8.8). Fractions containing antibody were pooled and
dialysed against 50 mM potassium phosphate buffer pH 7.4,
0.15 M NaCl. The total IgG concentration was determined by
measuring UV absorbance at 280 nm (taking as reference 1 mg/
ml = 1.4 AU) while the functional IgG was characterised using
Cell culture supernatants, ascitic fluids or purified antibodies
were denatured in Laemmli buffer (0.25 M Tris-HCl pH 6.8, 4%
SDS, 40% glycerol, 0.1% bromophenol blue, and 10%
bmercaptoethanol for reducing conditions)  for 5 min at
95uC. After SDS-PAGE (13% gel resolving), proteins were blotted
onto PVDF membrane. After saturation in 5% dried milk,
membranes were incubated with a rabbit anti-human (RAH)
polyclonal antibody (0.5 mg/ml, Jackson Immuno Research) for
1 h at RT. After several washings in PBS-0.1% Tween20,
membranes were reacted with a secondary HRP-conjugated
anti-rabbit IgG for 20 min and the protein bands were detected
via chemiluminescence (ECL, Amersham Biosciences) using a
Versadoc apparatus (BioRad).
The affinities of mouse and chimeric TA12 antibodies were
determined by surface plasmonic resonance (SPR) using a BIAcore
2000 instrument (Biacore, Sweden). All analyses were performed
at 25uC on CM5 sensor chip in the running buffer HBS-EP
(10 mM Hepes, 0.15 M NaCl, 3 mM EDTA and 0.005% P20
surfactant, pH 7.4). Experiments were performed assuming the
existence of a high-affinity antigen/antibody complex .
mTA12 antibody was immobilised on the chip at 200 RU using
the amine coupling method. cTA12 antibody was indirectly
immobilised on a RAH antibody-conjugated chip by injection of a
25 nM solution in running buffer at 5 ml/min for 3 min (to
immobilise 150 RU). For both antibodies, kinetic analyses were
performed by injecting different concentrations (ranging from 0.8
to 20 nM) of the ligand, Fc-BoNT/A1, for 3 min at 50 ml/min.
After 30 min of dissociation, the chips were regenerated with two
30-sec pulses of 10 mM glycine pH 2. The equilibrium
dissociation constant (KD) was calculated using the ratio between the
dissociation rate constant (koff) and the association rate constant
(kon), obtained with global Langmuir 1:1 fit (BIA evaluation
Male Swiss mice of 68 weeks (body weight from 28 to 32 g)
were used for the pharmacokinetic study. 50 mg (100 ml at 500 mg/
ml) of 0.22 mm-filtered antibodies was administered
intraperitoneally to mice. Blood was collected at 2, 7, 24, 48, 98, 313, 553
and 984 h after injection and centrifuged for 30 min at 3500g at
4uC. Plasma was recovered and stored at 220uC until use.
Pharmacokinetic parameters were determined based on the mean
antibody concentration values from 34 animals per time point.
For calculation of in vivo blood clearance, data values were fitted
using WinNonLin professional software (PharsightH).
Mouse protection assay
In vitro neutralising activity. The ability of the mouse and
chimeric antibodies to neutralise the neurotoxin was measured in
vivo with the mouse lethality assay according to the European
Pharmacopeia . For the mouse protection assay, male EOPS
mice (Charles River) weighing 1618 g were used. Botulinum
toxin was prepared from culture of C. botulinum type A1 strain Hall
grown in TGYH medium for four days under anaerobic
conditions at 37uC. The culture was centrifuged at 10,000g for
15 min at 4uC to separate bacteria and bacterial debris from the
culture supernatant, which was subsequently acidified to pH 3.5,
stabilised with 5% sterile glycerol, and stored at 4uC. Neutralising
antibodies were assayed by the method recommended in the
European Pharmacopeia known as the L+ test. The test dose
determines the relationship between a toxin concentration and a
reference antitoxin serum. By definition, one L+ toxin dose is the
smallest quantity of toxin, when mixed with one international unit
of reference antitoxin, which kills 100% of injected mice by the
intraperitoneal route within 96 h. International standard for
botulinum type A (NIBSC 59/021) was obtained from NIBSC.
The test dose, which is the smallest amount of toxin in a volume of
0.5 ml when incubated with standard serum (representing 0.5 IU/
ml with the preparation used in this study) causing the death of a
group of four mice (0.5 ml injected intraperitoneally into each
mouse), was 5 L+10 (30,000 LD100/ml). Variable amounts of
mouse and chimeric antibodies diluted in phosphate buffer
(pH 6.3) containing 0.2% gelatine (PB-G) were mixed with the
test dose (2 ml) and the final volume was adjusted to 5 ml buffer.
The mixtures were homogenised and incubated for 1 h at RT.
500 ml of each mixture was injected by intraperitoneal route into
each mouse of a group of 4. The mice were observed every day for
96 h. The mixture, containing the largest volume of antitoxin and
which fails to protect the mice from death, contained 0.5 IU.
Neutralising antibody titres are given as international units per
milligram of antibody (IU/mg), one IU of antitoxin being defined
as neutralising 104 mouse IP LD50 .
In vivo neutralizing activity using the co-injection
protocol. The neutralizing capacity of the antibody was
evaluated in the in vivo mouse lethality test. 5 estimated MLD50
of BoNT/A1 in PB-G (0.5 ml) were injected i.p. into Swiss mice
weighing 2022 g (10 mice per group). Concurrently, 2.5 mg of
cTA12 (0.5 ml in PB-G) was injected i.p. into a close but different
site. Mice were observed and any death was recorded every day
during 4 days.
Conceived and designed the experiments: SS. Performed the experiments:
JP CM DB PL. Analyzed the data: HV MRP CC SS. Contributed
reagents/materials/analysis tools: CM MRP. Wrote the paper: JP CC SS.
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