Augmenting the efficacy of anti-cocaine catalytic antibodies through chimeric hapten design and combinatorial vaccination

Article abstract of DOI:10.1016/j.bmcl.2017.07.014

Given the need for further improvements in anti-cocaine vaccination strategies, a chimeric hapten (GNET) was developed that combines chemically-stable structural features from steady-state haptens with the hydrolytic functionality present in transition-state mimetic haptens. Additionally, as a further investigation into the generation of an improved bifunctional antibody pool, sequential vaccination with steady-state and transition-state mimetic haptens was undertaken. While GNET induced the formation of catalytically-active antibodies, it did not improve overall behavioral efficacy. In contrast, the resulting pool of antibodies from GNE/GNT co-administration demonstrated intermediate efficacy as compared to antibodies developed from either hapten alone. Overall, improved antibody catalytic efficiency appears necessary to achieve the synergistic benefits of combining cocaine hydrolysis with peripheral sequestration.

Full text of DOI:10.1016/j.bmcl.2017.07.014

Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Augmenting the efficacy of anti-cocaine catalytic antibodies through
chimeric hapten design and combinatorial vaccination
⇑
Cody J. Wenthur, Xiaoqing Cai, Beverly A. Ellis, Kim D. Janda
Departments of Chemistry and Immunology, The Scripps Research Institute, La Jolla, CA 92037, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Given the need for further improvements in anti-cocaine vaccination strategies, a chimeric hapten
(GNET) was developed that combines chemically-stable structural features from steady-state haptens
with the hydrolytic functionality present in transition-state mimetic haptens. Additionally, as a further
investigation into the generation of an improved bifunctional antibody pool, sequential vaccination with
steady-state and transition-state mimetic haptens was undertaken. While GNET induced the formation of
catalytically-active antibodies, it did not improve overall behavioral efficacy. In contrast, the resulting
pool of antibodies from GNE/GNT co-administration demonstrated intermediate efficacy as compared
to antibodies developed from either hapten alone. Overall, improved antibody catalytic efficiency appears
necessary to achieve the synergistic benefits of combining cocaine hydrolysis with peripheral
sequestration.
Received 21 June 2017
Accepted 4 July 2017
Available online xxxx
Keywords:
Vaccine
Cocaine
Catalytic antibody
Chimeric hapten
Addiction
Ó 2017 Elsevier Ltd. All rights reserved.
Cocaine abuse and addiction are persistent public health prob-
lems in the US.1 With millions of users reported annually, cocaine
was the fifth-most consumed illicit drug type in 2014.2 However,
despite this prevalence, there have not yet been any medications
approved by the FDA for the treatment of cocaine abuse or addic-
tion.3,4 Among the strategies now being explored for treatment of
these conditions are antibody-based approaches, sometimes ter-
med ‘cocaine vaccines’.5–7 These agents are designed to induce a
drug-specific immune response in order to sequester cocaine in
the periphery and block its psychoactive effects.8,9 Despite pre-
clinical successes of this approach and promising efficacy signals
in small-scale human studies, the overall efficacy of vaccination
against cocaine in late-stage human trials has been limited thus
far.10–13 Considering this challenge, substantial efforts have been
made to improve the technology, including investigations into vac-
cine composition, adjuvant identity, delivery methods, and hapten
design.14–21
Within this search for improved hapten designs, our laboratory
investigated the use of ‘catalytic haptens’ to chemically inactivate
cocaine upon binding.22 In this study, while the use of GNT (1), a
transition-state mimetic hapten, was able to generate antibodies
that could both sequester and degrade cocaine, it also appeared
that some of these antibodies underwent covalent modification
by cocaine over time, thus reducing the vaccine’s ultimate effec-
tiveness. The acylation appears to be due to activation of cocaine’s
methyl ester, followed by nucleophilic attack from amino acids in
the active site of the transition-state mimetic antibody. Therefore,
in a subsequent investigation, we assessed the impact of hapten
stability on the immunogenicity of the resulting vaccines. Intrigu-
ingly, replacement of this offending ester linkage with an amide
was able to drastically increase the half-life of an analogous steady
state hapten, GNE (2), which ultimately increased its immuno-
genicity.23 Considering these findings, we postulated that the effi-
cacy of a transition-state mimetic vaccine could be improved by
promoting the selection of catalytic antibodies whose active site
was directed away from recognizing and reacting with the methyl
ester in cocaine.
Furthermore, recent findings have demonstrated the presence
of synergistic gains in efficacy when cocaine hydrolyzing enzymes
are directly administered together with antibodies.24,25 Given that
the early clinical indications for applications of enzyme-mediated
cocaine-hydrolysis are favorable, it seems highly prudent to direct
efforts into further development and validation of additional ther-
apeutic pairings that could combine the benefits of rapid cocaine
catalysis and robust peripheral sequestratio.26,27
Therefore, in pursuit of improving the ‘catalytic’ vaccine strat-
egy, we developed a new chimeric hapten that incorporated both
the phosphate ester necessary to induce cocaine degradation and
the amide linkage that improves hapten stability and eliminates
covalent antibody modification by cocaine.28 This hapten, GNET
(3), was generated using a convergent synthesis wherein cocaine
⇑
Corresponding author.
E-mail address: kdjanda@scripps.edu (K.D. Janda).
http://dx.doi.org/10.1016/j.bmcl.2017.07.014
0960-894X/Ó 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Wenthur C.J., et al. Bioorg. Med. Chem. Lett. (2017), http://dx.doi.org/10.1016/j.bmcl.2017.07.014

2
C.J. Wenthur et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
was first boiled in 1.25 M HCl to quantitatively generate (ꢀ) ecgo-
nine (5), which was then coupled with the amine linker species (6)
using EDC/DMAP conditions to generate amide 7. Linker 6 was pre-
pared from Boc-6-aminohexanoic acid and intermediate 8 was pre-
pared from treatment of phenylphosphoric dichloride with benzyl
alcohol. Next, 7 was treated with LDA in the presence of 8 to pro-
duce intermediate 9. Finally, global deprotection of 9 using
hydrogenolysis provided GNET (3) in 18% overall yield (Scheme 1).
The haptens 1–3 were then independently conjugated to Keyhole
Limpet Hemocyanin (KLH) to generate the immunogens GNE-
KLH, GNT-KLH, and GNET-KLH. Bovine serum Albumin (BSA) con-
jugates were also generated for use in biochemical analysis using
the same method.
Each of these immunogens was then formulated with Alum and
Sigma Adjuvant System (SAS) and administered subcutaneously
(SC) to mice at 0, 3, and 6 weeks (Fig. 1A). Analysis of antibody
response at weeks 3 and 8 for each of these vaccines was then
assessed using an Enzyme-Linked Immunosorbent Assay (ELISA),
where titers were determined by measuring the binding of serum
from vaccinated animals to their corresponding hapten-BSA conju-
gate. While all three vaccines generated an antibody response over
this time period, the GNET-KLH group demonstrated the largest
response by far (Fig. 1B).
Next, we assessed the ability of purified antibodies from each of
these vaccinated groups to catalyze the degradation of cocaine
in vitro (Fig. 1C). As anticipated, the antibodies generated by GNE
showed no catalytic activity, while those generated by GNT were
able to covert cocaine to methyl ecgonine and benzoic acid
(kcat = 0.72 0.23 minꢀ1; Km = 235 104 mM). The antibodies from
the GNET-treated animals demonstrated similar catalytic activity
(kcat = 0.25 0.02 minꢀ1; Km = 38.3 7.83 mM) to that seen with
GNT, although in both cases this activity was poor in comparison
to naturally occurring enzymes.
When we measured the ability of vaccination to blunt hyper-
locomotor activity due to cocaine administration, it was seen that
GNE-KLH had the most robust response, with GNT-KLH having a
lesser effect. Surprisingly, even though vaccination with GNET-
KLH was generating active catalytic antibodies, it was not able to
alter the animals’ behavioral response to cocaine (Fig. 1D).
Given the poor efficacy of this chimeric hapten strategy, we
next attempted to determine whether the sequential administra-
tion of GNE and GNT could generate a superior outcome to that
seen with GNET. We hypothesized that sequential administration
could improve vaccination outcomes by incorporating a first round
of selection for antibodies that had either limited methyl ester
recognition or increased cocaine catalysis, and then incorporating
a
subsequent round of counter-selection for antibodies that
retained recognition of the parent drug structure. For this study,
the vaccination schedule was extended to four total injections in
order to provide a balanced dose of GNE and GNT in the combina-
tion groups, but the total dose of hapten received over the course of
the full study was not changed (Fig. 2A). As a means to maximize
the potential to identify differences between administration sched-
ules for this combination in this round, we used a highly-immuno-
genic vaccine formulation, where the GNE and GNT haptens were
conjugated to Tetanus Toxoid (TT) and administered with CpG
Oligodeoxynucleotide 1826 (CpG) and alum.
Serum for ELISA analysis was taken after the second and fourth
injections. Analysis of the serum collected from all the vaccination
groups using ELISA revealed that the antibodies initially induced
by GNE were not able to recognize GNT-BSA, and the antibodies
initially induced by GNT were not able recognize GNE-BSA. How-
ever, subsequent exposure to the opposing hapten did induce
expansion of the antibody repertoire, although GNT-TT mainly
Scheme 1. Synthetic route to access GNET, a chimeric hapten combining features of
GNE and GNT. Conditions: a) 1.25 M HCl, 115 °C. b) BzOH, EDC, DMAP, DCM, 23 °C.
c) TFA, DCM. d) EDC, DMAP, 4-methylmorpholine, DCM. e) BzOH, Pyridine, CHCl3. f)
LDA, THF. g) H2, Pd/C, MeOH.
Fig. 2. Ex vivo and in vivo measurements of antibody efficacy following combina-
torial vaccination with GNE and GNT. A) Vaccination schedule for each condition,
with dashed lines representing concurrent co-administration of GNE and GNT. B)
Midpoint IgG titers against each hapten, as measured by ELISA (n = 6). C and D)
Hyperlocomotor activity results from vaccinated animals treated with increasing
doses of cocaine (n = 6).
Fig. 1. Ex vivo and in vivo measurements of antibody efficacy following vaccination
with GNET. A) Vaccination schedule for each hapten. B) Midpoint IgG titers as
measured by ELISA (n = 6). C) Michaelis-Menton plot for antibody-mediated
breakdown of cocaine (n = 6). D) Hyperlocomotor activity results from vaccinated
animals treated with increasing doses of cocaine (n = 6).
Please cite this article in press as: Wenthur C.J., et al. Bioorg. Med. Chem. Lett. (2017), http://dx.doi.org/10.1016/j.bmcl.2017.07.014

C.J. Wenthur et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
3
Table 1
Characteristics of resulting antibodies following vaccination as measured by RIA.
Group
GNE-TT
GNT-TT
E ? T
T ? E
E + T
y
Cocaine Kd (nM)
Concentration (ng/mL)
237 97.2
134 116
–
285 33.9
154 98.5
84.8 28.9
38.6 18.9
72.3 27.5
66.4 36.1
y
–
y
Could not be determined due to complete catalytic breakdown of cocaine over the course of the assay.
generated an anti-GNT antibody pool, with little expansion of the
existing population of anti-GNE antibodies (Fig. 2B).
Supplementary Data
Regardless of these changes, however, the order of GNE and
GNT administration had no effect on the efficacy of the vaccines
in blunting cocaine-induced hyperlocomotion. Each of the combi-
nation schedules, including the concurrent administration of GNE
and GNT, had an efficacy that fell between that of GNT-TT alone
or GNE-TT alone (Fig. 2C–D). This indicates that the overall efficacy
of the combinations is primarily driven by the presence of inde-
pendent reservoirs of sequestering and catalytic antibodies, and
that there are no significant synergistic effects arising from altered
affinity maturation during sequential hapten administration.
Using RIA, it was found that each of the combination schedules
did result in the production of a set of stable cocaine-binding anti-
bodies with affinities similar to that seen for GNE-TT vaccination
alone (Fig. 2D). Interestingly, the apparent antibody affinities for
those schedules where GNT-TT was given earlier were lower than
those where GNT-TT was given later. However, this shift is likely
driven by lowered concentrations of cocaine in the RIA due to
ongoing catalysis by established anti-GNT antibodies. Since transi-
tion-state mimetic antibodies result in the breakdown of cocaine
over time, steady state binding studies can only provide a measure
for the affinity and concentration of the non-catalytic antibody
population (Table 1).
Overall, vaccination with GNE-TT was the most effective strat-
egy attempted in this study, as compared to use of the structurally
chimeric GNET hapten and the combinatorial dosing of GNT with
GNE. Since both GNET and combination GNT dosing were able to
induce measurable antibody responses, their ultimately limited
behavioral efficacy is illuminating. These results indicate that
improvement in the catalytic efficiency of transition-state mimetic
haptens is essential if they ever hope to synergistically support
steady-state hapten efficacy in a manner similar to that reported
for hydrolytic enzymes directed against cocaine.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2017.07.
014.
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The authors thank Joel Schlosburg for helpful input on behav-
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ing synthetic materials. Research reported in this publication was
supported by the National Institute on Drug Abuse under grant
R01DA008590-21.
Please cite this article in press as: Wenthur C.J., et al. Bioorg. Med. Chem. Lett. (2017), http://dx.doi.org/10.1016/j.bmcl.2017.07.014