A Novel Strategy for Development of Recombinant Antitoxin Therapeutics Tested in a Mouse Botulism Model
et al. (2012) A Novel Strategy for Development of Recombinant Antitoxin Therapeutics Tested
in a Mouse Botulism Model. PLoS ONE 7(1): e29941. doi:10.1371/journal.pone.0029941
A Novel Strategy for Development of Recombinant Antitoxin Therapeutics Tested in a Mouse Botulism Model
Jean Mukherjee 0
Jacqueline M. Tremblay 0
Clinton E. Leysath 0
Kwasi Ofori 0
Karen Baldwin 0
Xiaochuan Feng 0
Daniela Bedenice 0
Robert P. Webb 0
Patrick M. Wright 0
Leonard A. Smith 0
Saul Tzipori 0
Charles B. Shoemaker 0
Eric A. Johnson, University of Wisconsin, Food Research Institute, United States of America
0 1 Department of Biomedical Sciences, Tufts Cummings School of Veterinary Medicine, North Grafton, Massachusetts, United States of America, 2 National Institute of Allergy and Infectious Diseases, National Institutes of Health , Bethesda , Maryland, United States of America, 3 United States Army Medical Research Institute for Infectious Diseases , Frederick, Maryland , United States of America
Antitoxins are needed that can be produced economically with improved safety and shelf life compared to conventional antisera-based therapeutics. Here we report a practical strategy for development of simple antitoxin therapeutics with substantial advantages over currently available treatments. The therapeutic strategy employs a single recombinant 'targeting agent' that binds a toxin at two unique sites and a 'clearing Ab' that binds two epitopes present on each targeting agent. Co-administration of the targeting agent and the clearing Ab results in decoration of the toxin with up to four Abs to promote accelerated clearance. The therapeutic strategy was applied to two Botulinum neurotoxin (BoNT) serotypes and protected mice from lethality in two different intoxication models with an efficacy equivalent to conventional antitoxin serum. Targeting agents were a single recombinant protein consisting of a heterodimer of two camelid anti-BoNT heavy-chain-only Ab VH (VHH) binding domains and two E-tag epitopes. The clearing mAb was an anti-E-tag mAb. By comparing the in vivo efficacy of treatments that employed neutralizing vs. non-neutralizing agents or the presence vs. absence of clearing Ab permitted unprecedented insight into the roles of toxin neutralization and clearance in antitoxin efficacy. Surprisingly, when a post-intoxication treatment model was used, a toxin-neutralizing heterodimer agent fully protected mice from intoxication even in the absence of clearing Ab. Thus a single, easy-to-produce recombinant protein was as efficacious as polyclonal antiserum in a clinically-relevant mouse model of botulism. This strategy should have widespread application in antitoxin development and other therapies in which neutralization and/or accelerated clearance of a serum biomolecule can offer therapeutic benefit.
Funding: This project was funded in part with Federal funds from the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health
(NIH), and the Department of Health and Human Services, under Contract No. N01-AI-30050 and Award Number U54 AI057159. Some work was also supported by
the Intramural Research Program of the NIAID, Bethesda, MD, USA. The content is solely the responsibility of the authors and does not necessarily represent the
official views of NIAID or NIH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
. These authors contributed equally to this work.
The presence of toxins in circulation is the cause of a wide variety
of human and animal illnesses. Antitoxins are therapeutic agents
that reduce further development of symptoms in patients that have
been exposed to a toxin. Typically, antitoxins are the antisera
obtained from large animals that were immunized with inactivated
toxin [1,2]. More recently, some antitoxin therapies have been
developed using one or more antitoxin mAbs [3,4,5,6]. Antisera and
mAbs can be difficult to produce economically at scale, usually
require long development times and often have problematic quality
control, shelf-life and safety issues. New therapeutic strategies to
develop and prepare antitoxins are needed.
Antitoxins function through two key mechanisms; neutralization
of toxin function and clearance of toxin from the body. Toxin
neutralization can occur through processes such as inhibition of
enzymatic activity and prevention of binding to cellular receptors.
Antibody mediated clearance from serum is thought to occur
subsequent to the binding of multiple antibodies to the target
antigen [7,8,9,10]. Multimeric antibody decoration of the target is
considered necessary to permit binding to low affinity Fc receptors
[8,10]. An ideal antitoxin therapeutic will both promote toxin
neutralization to immediately block further toxin activity and
accelerate toxin clearance to eliminate future pathology if
neutralization becomes reversed.
Clostridium botulinum neurotoxin (BoNT) is a National Institute of
Allergy and Infectious Diseases (NIAID) Category A priority
pathogen which can cause botulism, a potentially lethal flaccid
paralysis. Currently, the only treatments for botulism are
antitoxins. Polyclonal antitoxin sera are available to treat infants
(BabyBIG ) or adults (HBAT ) that become exposed to
BoNT and these can prevent further development of paralysis.
Once serious paralysis has occurred, though, palliative care is the
only available option . Some laboratories are working to
develop monoclonal antibodies (mAbs) as possible antitoxin
alternatives to polyclonal antisera [3,14,15,16,17]. Nowakowski
et al  found that effective protection of mice against high dose
challenge of BoNT serotype A (BoNT/A) required
co-administration of three antitoxin mAbs, presumably to promote clearance.
We previously demonstrated that administration of a pool of three
or more small binding agents, each produced with a common
epitopic tag, dramatically reduced serum levels of a toxin when
coadministered with an anti-tag mAb . The tagged binding
agents directed the binding of anti-tag mAb to multiple sites on the
toxin, thus indirectly decorating the toxin with Ab Fc domains and
leading to its clearance through the liver.
The use of small binding agents to direct the decoration of toxin
with Ab permits new strategies for the development of agents with
improved commercial properties. One binding agent scaffold
with excellent properties is the camelid heavy-chain-only Ab VH
(VHH) domain. VHHs are small (,12 kD), easy to produce, and
generally more stable than conventional antibody fragments
[19,20]. They are often found to have unusual epitope specificities,
particularly an improved ability to bind active site pockets to
produce enzyme inhibition . Because of the many favourable
properties of VHHs, they have become widely used in research
and show clear commercial potential [22,23].
Here we show that a single recombinant heterodimeric binding
agent consisting of two high-affinity BoNT binding VHH agents
and two epitopic tags, co-administered with an anti-tag mAb,
protected mice from lethality with an efficacy equivalent to
conventional BoNT antitoxin serum in two different in vivo assays.
Studies comparing neutralizing or non-neutralizing binding
agents administered with or without clearing Ab provide a
unique method for evaluating the relative contributions of toxin
neutralization and toxin clearance to antitoxin efficacy. We show
that toxin neutralization and toxin clearance both contribute
significantly to antitoxin efficacy in mice. Using the heterodimer
antitoxin strategy, toxin neutralization or toxin clearance alone
proved to be sufficient to protect mice from BoNT intoxication in
a therapeutically relevant, post-intoxication assay.
Identification and characterization of anti-BoNT VHHs
Serum clearance of the protein, Botulinum neurotoxin serotype A
(BoNT/A), can be dramatically accelerated by administering a
pool of different epitopically-tagged single-chain Ig variable
fragment (scFv) domain binding agents together with an anti-tag
mAb . To determine whether similar results could be obtained
using a more commercially and clinically acceptable binding
agent, a panel of camelid heavy-chain-only Vh (VHH) binding
agents were obtained having high affinity for BoNT/A holotoxin.
In addition, VHHs were obtained that bind to BoNT serotype
B (BoNT/B) holotoxin to permit efficacy testing on a second
pathogenic serum protein. Competition ELISAs were used to
identify the VHHs with the highest apparent affinity for unique
epitopes on BoNT/A and BoNT/B leading to the selection of
seven BoNT/A VHHs (Figure S1A) and four BoNT/B VHHs
(Figure S1B). Each VHH was purified from E. coli as a thioredoxin
fusion protein containing a single carboxyl-terminal epitopic tag
(E-tag) (SDS-PAGE shown in Figure S2A).
The seven unique BoNT/A binding VHHs were further
characterized for their target affinity by surface plasmon resonance
(SPR) and their ability to prevent intoxication of primary neurons
in culture (Table 1; Figure 1). All VHHs displayed good affinity for
their toxin targets with Kd,3 nM. Three VHHs (ciA-B5, -C2 and
-H7) proved to be potent toxin neutralizing agents, preventing
intoxication of neurons with 10 picomoles (pm) BoNT/A at
concentrations near equimolar with toxin. Two VHHs (ciA-D12
and -F12) showed negligible toxin neutralizing activity on primary
neurons even at 10006 excess of toxin. Two VHHs (ciA-A5 and
G5), displayed intermediate neutralizing activity. Western and
ELISA data (not shown) demonstrated that three anti-BoNT/A
VHHs (ciA-H7, -D12 and -F12) recognize the light chain protease
domain on the holotoxin. VHH ciA-B5 and -C2 recognize the
heavy chain and the other VHHs recognize epitopes apparently
requiring both heavy and light chain domains.
Protection from BoNT/A lethality in mice using pools of
monomeric anti-BoNT/A VHHs
The epitopically tagged anti-BoNT/A VHHs were next tested
in mice for the ability to prevent toxin induced lethality in the
presence or absence of the clearing anti-tag mAb . Various
pools of two, three or four different anti-BoNT/A VHHs were
coadministered with BoNT/A holotoxin to mice and monitored for
symptoms of intoxication and time to death (Figure 2). In all
studies employing monomeric VHHs, the total dose of
antiBoNT/A VHH was 2 mg/mouse so only the complexity of the
VHH pool varied between groups. Mice receiving two
antiBoNT/A VHHs in which both VHHs are unable to neutralize
BoNT/A in cell assays (ciA-D12, -F12) did not survive toxin
challenge any longer than did control mice receiving no agents
(Figure 2A). Even when the anti-E-tag clearing antibody (aE) was
included (5 mg), death was only slightly delayed, indicating that
serum clearance mediated by the decoration of BoNT/A with two
antibodies provides little therapeutic benefit in the absence of toxin
neutralization. In contrast, administration of two BoNT/A
neutralizing VHHs (ciA-B5, -H7) delayed death from 100 LD50
of BoNT/A (,5 ng) for about a day in the absence of clearing
antibody. When clearing antibody was co-administered with the
same two neutralizing VHHs, mice fully survived the 100 LD50
BoNT/A dose and death was delayed about a day with 1000 LD50
BoNT/A. Thus a combination of toxin neutralization and
clearance provided greater therapeutic benefit than either
protective mechanism could alone. The influence of VHH affinity
cannot be excluded in this study (see below) although each VHH
had similar sub-nanomolar affinities (Table 1).
Administration of a pool of three different anti-BoNT/A VHHs
(ciA-B5, -H7, -C2), each capable of potent toxin neutralization,
delayed death less than a day in mice exposed to 1000 LD50
(Figure 2B). In the presence of clearing Ab, this pool of tagged
VHHs completely protected mice exposed to 1000 LD50 of
BoNT/A from any apparent symptoms of intoxication and
delayed death more than a day in mice exposed to 10,000 LD50
(,0.5 mg of BoNT/A). A different pool of three VHHs in which
only one VHH contained potent neutralizing activity was much
less effective even in the presence of clearing antibody. These
results extend evidence from previously reports [3,18] that toxin
clearance becomes much more effective when BoNT is decorated
by at least three Abs and further demonstrates that toxin
neutralization can make an important contribution to antitoxin
efficacy in this assay.
A pool of four anti-BoNT/A VHHs in which two VHHs were
capable of toxin neutralization only slightly delayed death in
mice exposed to 1000 LD50 BoNT/A (Figure 2C). The same pool
administered together with the clearing Ab fully protected from
1000 LD50 and delayed death for one or two days in mice exposed
AVHH epitopes are named arbitrarily based on their inability to compete with the binding of VHHs recognizing other epitopes.
BSubunit recognition was assessed by ELISA with purified BoNT light chain (Lc) or heavy chain (Hc). VHHs recognizing BoNT holotoxin without recognition of purified Lc
or Hc are indicated as none. RBD indicates recognition of the 50 kDa carboxyl end receptor binding domain of Hc.
CVHH neutralization was determined by the ability of the VHH to prevent intoxication of primary neurons by 10 pM BoNT/A (Figure 1). Strong indicates that the
presence of #0.1 nM VHH led to obvious toxin neutralization in primary neuron assays (see Figure 1). Weak indicates detectable toxin neutralization when the
medium contained #1 nM VHH. None indicates no toxin neutralization was detected when the medium contained #10 nM VHH.
DSurface plasmon resonance (SPR) studies were performed using chips coated with ciBoNTA for BoNT/A VHHs and ciBoNTB for BoNT/B VHHs as described in Methods
to 10,000 LD50. In another study, a pool of four anti-BoNT/A
VHHs was compared to a pool of six different VHHs. The pool of
six contained the same VHHs as the pool of four plus two
additional anti-BoNT/A VHHs and was administered in the
presence of clearing antibody (Figure S3). The pools of four or six
tagged VHHs, administered with anti-tag clearing Ab, both fully
protected mice from 1000 LD50 and delayed death from 10,000
LD50 with almost identical efficacy. Based on these results, and
those from several other similar studies (not shown), decoration of
BoNT with four Abs improves antitoxin efficacy compared with
three Abs while decoration with more antibodies provides little
Role of affinity in antitoxin efficacy
Both toxin neutralization and clearance mechanisms depend on
the binding of antitoxin agents to the toxin. The kinetics of toxin
binding (kon) and release (koff) by the antitoxin binding agents
would thus be expected to contribute to their efficacy. To
determine the role of toxin affinity to antitoxin efficacy required
the identification of multiple VHHs recognizing the same epitope
with different affinities. In the course of anti-BoNT/A VHH
screening, several VHHs (ciA-D1, H4 and H11) were identified
that recognized the same epitope as ciA-H7 based on competition
ELISA analysis (sequences shown in Figure S1C). SPR analysis
showed that the four VHHs recognized this epitope with affinities
ranging from ciA-H7 (KD 0.06 nM) to ciA-H11 (KD 4.3 nM)
(Figure 3A). These four VHHs were tested for their efficacy as
antitoxin VHHs, each in combination with the two VHHs
(ciAB5, C2) that recognize distinct, non-overlapping epitopes
(Figure 3B). The results comparing antitoxin efficacy with 100
and 1000 LD50 challenges showed that the antitoxin efficacy of the
VHHs was clearly related to their toxin affinities. The highest
affinity VHH ciA-H7 was superior to the other VHHs recognizing
the same epitope and suggests that sub-nanomolar affinities (KD)
for the tagged toxin binding agents is necessary to achieve
maximal antitoxin efficacy in the mouse lethality assay.
Antitoxin VHHs expressed as heterodimers
Camelid VHHs are stable domains that can be functionally
expressed as dimers. Combining two anti-BoNT/A VHHs into a
heterodimer would permit one molecule to bind to two different
sites on the toxin while likely improving toxin avidity . If the
heterodimer contains a single epitopic tag, the one molecule can
promote the decoration of BoNT/A with two anti-tag clearing Abs
(see diagram in Figure 4A). By addition of a second copy of the
epitopic tag to the heterodimer (see diagram in Figure S1D), it
should be possible to promote toxin decoration of the toxin with
four clearing Abs to yield near maximum clearing efficacy (see
diagram in Figure 4B). This hypothesis was tested by preparing
two anti-BoNT/A VHH heterodimers in which the two VHHs in
the heterodimer were either non-neutralizing (ciA-F12/D12) or
potent toxin neutralizing agents (ciA-H7/B5). The two different
heterodimers were expressed containing either one or two copies
of the epitopic tag (E-tag) (sequences shown in Figure S1E). SPR
analysis confirmed that the heterodimer KDs are in the range of
10100 picomolar; significantly higher than the affinities of the
component monomers (Table 1).
The antitoxin efficacies of the single-tagged heterodimers in
mice (Figure 4) were very similar to those achieved by two
corresponding monomers (Figure 2A). The single-tagged
heterodimer consisting of non-neutralizing VHHs, ciA-F12/D12(1E),
provided no protection from 1000 LD50 BoNT/A in the absence
of clearing Ab and only slightly delayed death in the presence of
clearing Ab. The toxin neutralizing single-tagged heterodimer,
ciA-H7/B5(1E), delayed death in mice exposed to 1000 LD50
BoNT/A for 12 days in the absence of clearing Ab and efficacy
was only slightly improved by the addition of clearing Ab. These
results are consistent with other data indicating that decoration of
toxin with two Abs is not very effective in promoting toxin
Much improved antitoxin efficacy occurred by the simple
addition of a second copy of the epitopic tag to the anti-BoNT/A
VHH heterodimers when each of these agents was co-administered
with clearing Ab. The hypothesis for using a double-tagged
heterodimer was that the heterodimer VHH would bind at two sites
on the toxin and each bound heterodimer would promote toxin
decoration with two clearing Abs, thus resulting in decoration of the
toxin with four Abs (see diagram in Figure 4B) which was previously
shown to be optimal for promoting clearance (see above). A
doubletagged heterodimer of non-neutralizing VHHs, ciA-F12/D12(2E),
provided virtually no antitoxin efficacy in the absence of clearing Ab
as expected for binding agents with little or no toxin neutralizing
activity. In the presence of clearing Ab, the same agent fully
protected mice from 1000 LD50 of BoNT/A and delayed death
about a day in mice receiving 10,000 LD50 (Figure 4B, Figure S4).
Thus the simple addition of a second epitopic tag to the heterodimer
dramatically improved the antitoxin efficacy.
In a separate study, the ciA-F12/D12 heterodimer was
expressed with one, two or three epitopic tags and tested for
antitoxin efficacy in the presence of clearing Ab (Figure S4). The
single-tagged heterodimer poorly protected mice from toxin
challenge while the double and triple-tagged heterodimers
were fully protective to a 100 LD50 challenge. There was little
improvement in efficacy using the triple-tagged heterodimer as
compared to the double-tagged heterodimer, consistent with the
prior observation (above) that near maximal clearance is achieved
by decorating the target with four antibodies. A titration of the
clearing Ab administered with the double-tagged ciA-F12/D12
heterodimer demonstrated that maximal antitoxin efficacy was
achieved when the number of Ab molecules administered was
approximately equivalent to the number of epitopic tags (Figure
An even more dramatic antitoxin effect was observed using the
double-tagged heterodimer, ciA-H7/B5(2E), in which both
antiBoNT/A VHHs possess potent neutralizing activity in cell culture
intoxication assays (Figure 1). In the absence of clearing Ab, this
agent produced the same antitoxin efficacy as the equivalent
single-tagged heterodimer (Figure 4A, B). When clearing Ab was
included, the neutralizing double-tagged heterodimer (40 pmoles)
became a highly potent antitoxin that fully protected mice from
lethality when co-administered with 10,000 LD50 BoNT/A
(,1 pmole of holotoxin). A dose-response study was performed
in which this agent was co-administered to mice with 1000 LD50
(,0.3 pm) (Figure S6) and demonstrated that 40 pmoles and
13 pmoles completely protected the mice. A dose of 4 pmoles of
this neutralizing double-tagged heterodimer had the same
protective efficacy for 1000 LD50 (Figure S6) as a dose of
40 pmoles did with 10,000 LD50 (Figure 4B). These results show
that about a 15 fold molar excess of the double-tagged H7/B5
heterodimer binding agent and clearing Ab was sufficient to
neutralize and/or clear the vast majority (.99.99%) of BoNT/A
when co-administered to a mouse.
Recombinant antitoxin efficacy in a clinically relevant
Assays in which varying doses of toxins are co-administered with
antitoxin agents permit sensitive quantification of antitoxin
efficacy but do not accurately reflect the typical clinical situation.
To test antitoxin agents in a more clinically relevant assay, mice
were administered with 10 LD50 of BoNT/A by intraperitoneal
administration and, at various times later, administered
intravenously with test agents. As a positive control, we used a potent
sheep anti-BoNT/A serum at a dose previously demonstrated to
protect 100% of mice from lethality when co-administered with
10,000 LD50 of BoNT/A (not shown). Two different anti-BoNT/
A VHH heterodimers were tested; the non-neutralizing ciA-F12/
D12(2E) heterodimer (Figure 5A) and the neutralizing ciA-H7/
B5(2E) heterodimer (Figure 5B). Each heterodimer contained two
copies of E-tag and was tested both with and without the
anti-Etag clearing Ab. The non-neutralizing heterodimer had little or no
antitoxin efficacy in the absence of clearing Ab, yet when in the
presence of this agent it displayed an efficacy nearly equivalent to
the positive control sheep antiserum. These results show that toxin
clearance alone is sufficient to protect mice from a low dose BoNT
challenge (10 LD50), even when the agents are administered
several hours post-intoxication.
Surprisingly, the neutralizing heterodimer was highly effective
as an antitoxin in this assay whether or not clearing Ab was
included (Figure 5B). The double-tagged toxin neutralizing
heterodimer possessed an antitoxin efficacy equivalent to
polyclonal antitoxin even when administered in the absence of anti-tag
clearing Ab. These data strongly suggest that BoNT neutralization
is sufficient for full antitoxin efficacy when tested in a clinically
relevant post-intoxication assay with low dose toxin challenge. The
results show that a single protein composed simply of two
toxinneutralizing VHHs has the potential to be as effective as antitoxin
sera in clinical situations.
Antitoxin efficacy of a double-tagged heterodimer
The use of double-tagged heterodimer antitoxins was extended
to a different toxin target by using VHHs recognizing unique
epitopes on BoNT/B holotoxin (Figure S1B). Two of the VHHs,
ciB-A11 and B5, were the most potent in vivo in monomer pool
studies (not shown) and were selected for expression as a
doubletagged heterodimer (ciB-A11/B5(2E)) (sequence in Figure S1E).
This agent fully protected mice against 1000 LD50 of BoNT/B in
the presence of clearing Ab (Figure 6A). In the clinically relevant
post-intoxication assay, ciB-A11/B5(2E) was only partially
effective in the absence of clearing Ab indicating that the heterodimer
is not potent at toxin neutralization. Furthermore, the affinity of
this heterodimer for BoNT/B (Kd,5 nM, Table 1) was weaker
than the affinity of either of the two component monomers
(Kd,1 nM each) suggesting that the recombinant heterodimer
was not fully functional. Despite this, when the heterodimer was
administered with clearing Ab, the treatment was at least as
effective as sheep anti-BoNT/B polyclonal antiserum in
preventing BoNT/B lethality (Figure 6B). These results demonstrate the
efficacy of the heterodimer VHH antitoxin strategy for a second
BoNT serotype and suggest the strategy will be effective for
treating exposure to the other BoNT serotypes as well as to other
pathogenic biomolecules that may occur in patient serum.
This manuscript reports a new approach to the development of
antitoxins that employs a single recombinant protein
(doubletagged VHH heterodimer) to promote toxin decoration with
multiple copies of a single monoclonal antibody (anti-tag mAb)
leading to its neutralization and clearance from the body. The
approach should have general applicability for clinical situations in
which neutralization and/or clearance of a circulating pathogenic
biomolecule will result in therapeutic benefit.
Earlier studies had shown that a pool of scFv domain binding
agents with specificity for BoNT/A, each containing a common
epitopic tag, could direct the decoration of the toxin with multiple
anti-tag Abs leading to its clearance via the liver with an efficacy in
mouse assays equivalent to conventional polyclonal antitoxin sera
. The scFvs served as the toxin targeting agents and the
antitag mAb served as the clearing agent. Here we show that camelid
Figure 3. Anti-BoNT/A VHH protection from lethality improves with higher affinity VHHs. (A) The KDs for four anti-BoNT/A VHHs that
each recognize the same epitope (epitope A1; Table 1) based on competition analysis. (B) Time to death is plotted as % survival following injection of
the indicated dose of BoNT/A in groups of five mice co-administered with pools of different anti-BoNT/A VHHs and clearing Ab. Each VHH pool
contained VHHs ciA-B5 and ciA-C2 and one of the four different VHHs recognizing BoNT/A epitope A1. The pool of ciA-VHHs or control (no agents)
that were administered to the mice is indicated by arrows.
VHH binding domains, with multiple commercial advantages
over scFvs [22,23], also serve effectively as the toxin targeting
One important advantage of VHHs is the ability to express
these agents as heterodimers in which each VHH remains fully
functional. This makes possible the fusion of two VHHs that bind
to different epitopes on the same toxin target. Incorporation of two
epitope tags on the heterodimer permits decoration of the toxin
with two clearing Abs at each epitope, a total of four mAbs on the
toxin to promote efficient toxin clearance. Since each
doubletagged heterodimeric binding agent binds to only two mAbs, the
agent itself should not be effectively cleared by low affinity Fc
receptors unless bound to toxin. The heterodimers also proved to
have much greater apparent affinity for toxin than the equivalent
pool of two monomers and this likely contributed to the substantial
gains in antitoxin efficacy achieved with the heterodimer
antitoxins in mouse models of BoNT intoxication.
The ability of antitoxin antibodies to protect animals from the
symptoms of toxin exposure can be influenced by several factors.
The dose of antitoxin agent and the timing of antitoxin
administration relative to exposure to toxin are obvious factors
that influence efficacy. In addition, the affinity of the antibodies for
the toxin will influence the ability of the antibody to bind (kon) and
remain bound (koff) to the toxin and exert its effect. The further
ability of the antibody to inhibit the enzymatic activity of the toxin
and/or prevent its entry into target cells (i.e. neutralization) also
must be expected to play a key role. Finally, the ability of the
antibodies to promote the clearance of the toxin from the serum
will permit the antitoxin to reduce the pool of toxin in circulation.
Little is known about the relative importance neutralization vs
clearance mechanisms to antitoxin efficacy.
In this study, insight into antitoxin mechanism was possible
since experiments were performed that separately tested the roles
of toxin neutralization and clearance in determining antitoxin
efficacy. The role of neutralization could be assessed by comparing
the efficacy of VHHs that are non-neutralizing with toxin
neutralizing agents. The role of clearance could be analyzed by
comparing antitoxin efficacy of identical non-neutralizing VHH
agents in the presence or absence of the anti-tag clearing antibody.
The results clearly demonstrate that agents possessing potent
BoNT neutralizing activity can be very effective in mouse
intoxication models in the absence of clearance. Similarly, agents
that have little or no antitoxin efficacy in the absence of clearing
Ab can become highly effective simply by co-administration of
clearing Ab to promote toxin clearance. Furthermore, our results
indicate that combining neutralization and clearance is essential
for maximal antitoxin efficacy when the toxin doses are very high.
Interestingly, when low BoNT doses were employed, such as the
10 LD50 dose used in the clinically-relevant post-intoxication
model, toxin clearance or neutralization alone were each sufficient
and about equally effective. Presumably this occurs because
clearance or neutralization alone are each capable of sufficiently
Figure 6. BoNT/B intoxication is prevented by heterodimer antitoxin agents in two models of BoNT/B intoxication in mice.
Protection of mice from BoNT/B lethality by administration of a double-tagged heterodimer of anti-BoNT/B VHHs ciB-A11 and ciB-B5 (A11/B5(2E))+/
2clearing Ab. Time to death is plotted as % survival as a function of time. An asterisk indicates that mice did not display any symptoms of
intoxication. The results shown are combined from two replicate studies. (A) Groups of mice were co-administered the indicated LD50 dose of BoNT/B
together with no agents or the A11/B5(2E) heterodimer VHH with or without anti-E-tag clearing Ab (aE). (B) Groups of mice were administered a 10
LD50 dose of BoNT/B and three hours later administered no agents, sheep anti-BoNT/B antiserum or the A11/B5(2E) heterodimer VHH with or without
anti-E-tag clearing Ab (aE).
reducing the level of active BoNT when the antitoxin is
administered during the window of opportunity  that exists
between exposure and the onset of irreversible symptoms. At
much higher BoNT doses, both toxin clearance and neutralization
appear necessary to permit survival.
The toxins employed in this study were Botulinum neurotoxins
(BoNTs) and additional studies will be necessary to assess the
extent to which the heterodimer binding agent antitoxin strategy
described here will prove efficacious for other toxins. BoNTs are
extremely potent with exquisite specificity for neurons and
normally remain in circulation until they enter a neuron or are
naturally cleared. Because of the high potency of BoNT,
measurable toxicity occurs with extremely small amounts of
circulating toxin. With less potent toxins, intoxication requires
higher doses of toxin and thus higher concentrations of Ab are
required for antitoxin efficacy.
For toxins with less target cell specificity, such as those from
Clostridium difficile and Escherichia coli or ricin, the toxins are likely to
spend shorter time in circulation and, in these cases, toxin
neutralization may be more important to efficacy than toxin
clearance. Where the toxins are active on cells with low affinity Fc
receptors involved in toxin clearance, antitoxins that promote
clearance may not be of benefit. For these reasons, it is difficult to
predict whether the most effective heterodimer antitoxin strategy
should promote toxin clearance or focus entirely on toxin
One concern with the use of heterodimer binding agents is the
possibility that the binding agents will be immunogenic and elicit
an immune response that reduces or eliminates the therapeutic
efficacy. VHHs are not considered to be strongly immunogenic
and the immunogenicity can by reduced further by introducing
targeted mutations . Alternative non-Ab binding agents such
as DARPins, Anticalins or AdNectins  should be able to
replace VHHs if sufficient target affinity can be achieved and these
are specifically designed to be poor immunogens. Some
immunogenicity may be tolerable and may even improve efficacy by
promoting target clearance.
Studies reported here have demonstrated that a single
heterodimer protein composed of two distinct toxin neutralizing
VHHs has efficacy equivalent to polyclonal antitoxin serum in a
clinically-relevant post-intoxication BoNT lethality assay. The
ability to prevent intoxication with a single polypeptide
substantially simplifies the commercial production of the antitoxin and
makes possible genetic delivery approaches such as with DNA or
viral vectors. Improved therapeutic efficacy is possible by
promoting the clearance of the pathogenic biomolecule target
and this can be achieved by producing the heterodimer with two
copies of an epitopic tag and co-administering the agent with an
anti-tag clearing mAb. This results in the decoration of the target
with up to four mAbs which leads to clearance presumably by a
low affinity FcR-dependent pathway. The clearing mAb itself
could be made unnecessary by producing the heterodimer fused to
a peptide or VHH that binds to low-affinity FcR. Using these
varied strategies, it should be possible to develop new and versatile
therapeutic approaches that permit the neutralization and/or
clearance of one or more targeted pathogenic biomolecules from
the circulation of patients.
Materials and Methods
All studies were carried out in strict accordance with the
recommendations delineated in the Guide for the Care and Use of
Laboratory Animals of the National Institutes of Health. The
procedures used were approved by the Tufts University
Institutional Animal Care and Use Committee (IACUC) and were
performed under Protocols #G2010-60 and G874-07.
Toxins and reagents
Botulinum neurotoxin serotype A1 (BoNT/A) and serotype B
(BoNT/B) were obtained from Metabiologics Inc. Each batch of
toxin was assayed to establish LD50 dose. BoNT complex was used
for animal studies and BoNT holotoxin was used for the cell-based
studies. Purified recombinant BoNT serotype A1 and B holotoxins
containing mutations rendering them catalytically inactive
(ciBoNTA, ciBoNTB) were produced as previously described .
Sheep anti-BoNT/A1 antiserum was produced by immunization
of sheep with BoNT/A1 toxoid followed by BoNT/A1 holotoxin.
Less than 1 ml of this sheep antitoxin serum protects mice from
lethality when co-administered with 10,000 LD50 of BoNT/A1.
Reagents for Western blotting were purchased from KPL.
Antibodies used were rabbit anti-SNAP25 antibody (Sigma); goat
anti-rabbit HRP antiserum (Sigma); anti-E-tag mAb (Phadia);
HRP anti-E-tag mAb (GE Healthcare). All studies with holotoxin
were performed within a Select Agent laboratory registered with
Alpaca immunization and VHH-display library
Two alpacas were immunized with ciBoNTA and two with
ciBoNTB. The immunization regimen employed 100 mg of
protein in the primary immunization and 50 mg in three
subsequent boosting immunizations at about 3 weekly intervals
in alum/CpG adjuvant. Five days following the final boost, blood
was obtained for lymphocyte preparation and VHH-display phage
libraries were prepared from the immunized alpacas as previously
described [29,30]. More than 106 independent clones were
prepared from B cells of alpacas successfully immunized with
each of the BoNT immunogens.
Anti-BoNT VHH identification and preparation
The VHH-display phage libraries were panned for binding to
ciBoNTA or ciBoNTB targets that were coated onto a well of a 12
well plate. Coating was performed by overnight incubation with
one ml of a 5 mg/ml target solution in PBS at 4uC followed by
washing with PBS and 2 hrs incubation at 37uC with blocking
agent (4% non-fat dried milk powder in PBS). Panning, phage
recovery and clone fingerprinting were performed as previously
described [29,30]. A total of 192 and 142 VHH clones were
identified as strong positives for binding to BoNT/A and BoNT/B
respectively based on phage ELISA signals. Of the strong positives,
62 unique DNA fingerprints were identified among the VHHs
selected for binding to BoNT/A and 32 for VHHs selected for
binding to BoNT/B. DNA sequences of the VHH coding regions
was obtained for each phage clone and compared for homologies.
Based on this analysis, 12 of the anti-BoNT/A VHHs and 11
antiBoNT/B were identified as unlikely to have common B cell clonal
origins and selected for protein expression.
Expression and purification of VHHs in E. coli as recombinant
thioredoxin (Trx) fusion proteins containing hexahistidine was
performed as previously described . For heterodimers, DNA
encoding two different VHHs were joined in frame downstream of
Trx and separated by DNA encoding a 15 amino acid flexible
spacer ((GGGGS)3). All VHHs were expressed with a carboxyl
terminal E-tag epitope. Some expression constructions were
engineered to contain a second copy of the E-tag by introducing
the coding DNA in frame between the Trx and VHH domains.
An example of a Trx fusion to a VHH heterodimer with two
Etags is shown in Figure S1C. A third E-tag was introduced in
frame within the DNA encoding the flexible spacer of the
heterodimer containing ciA-D12 and ciA-F12 to create a triple
tagged heterodimer (D12/F12(3E)).
VHH target binding competition analysis
Phage displaying individual VHHs were prepared and titered
by phage dilution ELISA  for recognition of ciBoNTA or
ciBoNTB using HRP/anti-M13 Ab for detection. A dilution was
selected for each phage preparation that produced a signal near
the top of the linear range of the ELISA signal. The selected phage
dilution (100 ml) for each VHH-displayed phage preparation were
added to a 96 well plate that has been coated with ciBoNTA or
ciBoNTB and then pre-incubated for 30 minutes with 100 ml of a
10 mg/ml solution containing a purified Trx/VHH fusion protein
test agent or control in PBS. After an hour, the wells were washed
and phage binding was detected as above. Test VHHs that
reduced target binding of phage-displayed VHHs by less than two
fold vs controls were considered to recognize distinct epitopes.
Positive controls were performed in which the Trx/VHH
competitor contained the same VHH as displayed on phage and
typically reduced the ELISA signal .95%.
Characterization of VHH binding properties
VHHs were tested for binding to native or atoxic mutant BoNT
holotoxins by standard ELISA using plates coated with 100 ml of
1 mg/ml protein. VHHs were also tested for recognition of BoNT
subunits by a dilution series ELISA (10 mg/ml, 1:5 dilutions) using
plates coated by overnight incubation at 4uC with 5 mg/ml
purified recombinant BoNT light chain  or 1 mg/ml BoNT
heavy chain. VHH binding was detected with HRP-anti-E-tag
mAb (GE Healthcare). VHHs were also characterized for
recognition of subunits by Western blotting on BoNT holotoxin
following standard SDS-PAGE (420% gel) with samples boiled in
SDS sample buffer under reducing conditions (5% bME). VHHs
were incubated with filters at 10 mg/ml and bound VHH detected
with HRP-anti-E-tag mAb (GE Healthcare) by standard
Kinetic analysis by surface plasmon resonance
Studies to assess the kinetic parameters of the VHHs were
performed using a ProteOn XPR36 Protein Interaction Array
System (Bio-Rad, Hercules, CA) after immobilization of ciBoNTA
or ciBoNTB by amine coupling chemistry using the manufacturer
recommended protocol. Briefly, after activation of a GLH (high
protein immobilization capacity) chip surface with a mixture of
0.4 M ethyl(dimethylaminopropyl) carbodiimide (EDC) and
0.1 M N-hydroxysulfosuccinimide (sulfo-NHS) injected for 300 s
at 30 mL/min, ciBoNTA or ciBoNTB was immobilized by passing
a 60 mg/mL solution of the protein at pH 5 over the surface for
180 s at 25 mL/min. The surface was deactivated with a 30 mL/
min injection of 1 M ethanolamine for 300 s. A concentration
series for each VHH (between 2.5 nM and 1000 nM, optimized
for each antibody fragment) was passed over the surface at
100 mL/min for 60 s, then dissociation was recorded for 600 s or
1200 s. The surface was then regenerated with a 36 s injection of
10 mM glycine, pH 2.0 at 50 mL/min. Running buffer for these
studies was 10 mM Hepes, pH 7.4, 150 mM NaCl, 0.005%
Tween-20. Data was evaluated with ProteOn Manager software
(version 2.1.2) using the Langmuir interaction model.
BoNT neutralization assay using primary neurons
Neuronal granule cells from the pooled cerebella of either 78
day old Sprague-Dawley rats or 57 day old CD-1 mice were
harvested as described by Skaper et al  and cultured in 24 well
plates as described by Eubanks et al . After at least a week of
culture the well volumes were adjusted to 0.5 ml containing
various VHH dilutions or buffer controls followed immediately by
addition of BoNT/A in 0.5 ml to a final 10 pM. After overnight at
37uC, cells were harvested and the extent of SNAP25 cleavage
assessed by Western blot as previously described .
Standard mouse toxin lethality assay
Female CD1 mice 1517 g each (Charles River Labs) were
received 5 days prior to use. One day prior to initiation of study,
mice were weighed and placed into groups in an effort to minimize
inter-group weight variation. Appropriate dilutions of the test
agents were prepared in PBS. BoNT holotoxins were separately
prepared in PBS+0.2% gelatin (Sigma) at the desired doses. 600 ml
of test agent and 600 ml of the toxin were combined and incubated
at room temperature for 30 minutes. 200 ml of the mixture was
administered by intravenous injection at time 0 to mice in groups
of five. Mice were monitored at least four times per day and scored
for overall disposition, severity of abdominal breathing, presence
of open-mouth breathing, activity level, presence of lethargy, and
mortality. Moribund mice were euthanized. Time to onset of
symptoms and time to death were established for each mouse .
Mouse toxin lethality assay with agents administered
This assay is a modification of an assay developed by Cheng
et al . Groups of mice were prepared as above. Mice were
administered 10 LD50 of BoNT/A by intraperitoneal injection. At
indicated times post-intoxication, mice were administered 200 ul
of test agent in PBS by intravenous injection. Mice were
monitored for symptoms of intoxication as above.
Figure S1 Protein sequences of anti-BoNT/A and
antiBoNT/B VHH monomers and heterodimers. (A) Protein
sequences of VHHs recognizing unique epitopes on BoNT/A (ciA)
are shown aligned for homology. Regions represented by dashes
are gaps. (B) Protein sequences of VHHs recognizing unique
epitopes on BoNT/B (ciB) are shown aligned for homology.
Regions represented by dashes are gaps. (C) Protein sequences of
three VHHs recognizing the same BoNT/A epitope as ciA-H7 are
shown aligned for homology. VHHs in A and B also contain Q(L/
V)QLVE at the amino end that is encoded by the PCR primer
used to generate the VHH-display library . The eight amino
acids shown at the carboxyl end are encoded by either the short
hinge or long hinge PCR primer that were used to generate the
library . (D) Schematic diagram of the domain structure of a
double-tagged VHH heterodimer protein. Proteins were expressed
in pET32b with an amino-terminal E. coli thioredoxin. Domain
abbreviations used were: Trx, E. coli thioredoxin; 6H,
hexahistidine domain including enterokinase cleavage site (DDDDK);
E, E-tag peptide; VHH-1, first VHH; fs, flexible spacer domain
((GGGGS)5); VHH-2 second VHH. Relative domain sizes in the
diagram are approximate. (E) Protein sequences of the entire
translation product of three recombinant VHH heterodimers
containing two copies of E-tag. The E-tag sequences
(GAPVPYPDPLEPR) are underlined. The amino acid sequences
preceding the first E-tag in each protein contains the thioredoxin
fusion partner and hexahistidine encoded by the pET32b
expression vector. The VHH sequences are flanked by the two
E-tag peptides and separated by the unstructured spacer
Figure S2 SDS-PAGE analysis of purified VHH
monomers and heterodimers. Following gel electrophoresis of 1 mg
of the indicated purified proteins, gels were stained for protein.
(A) VHH monomers recognizing BoNT/A (ciA-). (B) VHH
heterodimers recognizing BoNT/A (ciA-) or BoNT/B (ciB-) in
which the two indicated VHHs are expressed with the first VHH
at the amino end and the second VHH at the carboxyl end. An E
indicates the presence and position of peptide E-tags relative to the
VHHs. The migration positions of molecular weight markers are
shown with arrows.
Figure S3 Time to death plots following co-injection
BoNT/A and pools of four or six anti-BoNT/A
VHHs+clearing Ab (aE). The contents of the pool of
ciAVHHs or control (no agents) that was administered to the mice is
indicated by arrows. An asterisk indicates that mice did not display
any symptoms of intoxication.
Figure S4 Antitoxin efficacy of non-neutralizing
antiBoNT/A VHH heterodimer ciA-F12/D12 containing one,
two or three E-tag epitopes when co-administered with
anti-E-tag clearing Ab and BoNT/A. The % survival is
plotted as a function of time for groups of five mice. Groups of
mice were administered 20 pmoles of the heterodimer of VHHs
ciA-F12 and ciA-D12 (F12/D12) containing one (1E), two (2E) or
three (3E) copies of the E-tag epitope as indicated by arrows.
Another group of mice received a pool of the two monomer VHHs
(20 pm each), ciA-F12 and ciA-D12. The toxin dose is indicated in
LD50. All mice received 60 pm of anti-E-tag clearing Ab (aE).
Figure S5 Antitoxin efficacy of non-neutralizing
antiBoNT/A VHH heterodimer ciA-F12/D12 containing two
copies of E-tag and co-administered with BoNT/A and
varying doses of anti-E-tag clearing Ab. The % survival is
plotted as a function of time for groups of five mice. Groups of
mice were co-administered BoNT/A, 20 pmoles of the
nonneutralizing VHH heterodimer ciA-F12/D12 containing two
copies of E-tag (F12/D12(2E)) or no agents and anti-E-tag mAb
as the indicated dose. The toxin dose is indicated in LD50.
Figure S6 Titration of the BoNT/A antitoxin efficacy of
the neutralizing anti-BoNT/A VHH heterodimer
coadministered with clearing Ab. The % survival is plotted
as a function of time for groups of five mice. Groups of mice were
administered 1000 LD50 of BoNT/A (,0.3 pmoles) and either no
We thank Dr. Patrick Skelly for helpful suggestions during the course of
this project and Dr. Gerald Beltz for critical reading of the manuscript.
Conceived and designed the experiments: JM CEL CBS. Performed the
experiments: JM JMT CEL KO KB XF DB. Analyzed the data: JM JMT
CEL XF ST CBS. Contributed reagents/materials/analysis tools: RPW
PMW LAS. Wrote the paper: CBS.
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