Cocaine catalytic antibodies: The primary importance of linker effects

Article abstract of DOI:10.1016/S0960-894X(00)00659-4

Current treatments for cocaine addiction are not effective. The development of a catalytic monoclonal antibody (mAb) provides a strategy for not only binding, but also degrading cocaine, which offers a broad-based therapy. Hapten design is the central element for programming antibody catalysis. The characteristics of the linker used in classic transition-state analogue phosphonate haptens were shown to be important for obtaining mAbs that hydrolyze the benzoate ester of cocaine.

Full text of DOI:10.1016/S0960-894X(00)00659-4

Bioorganic & Medicinal Chemistry Letters 11 (2001) 87±90
Cocaine Catalytic Antibodies: The Primary Importance of
Linker E€ects
Masayuki Matsushita, Timothy Z. Ho€man, Jon A. Ashley, Bin Zhou,
Peter Wirsching* and Kim D. Janda*
The Scripps Research Institute and the Skaggs Institute for Chemical Biology, Department of Chemistry BCC-582,
10550 North Torrey Pines Road, La Jolla, CA 92037, USA
Received 18 October 2000; accepted 21 November 2000
AbstractÐCurrent treatments for cocaine addiction are not e€ective. The development of a catalytic monoclonal antibody (mAb)
provides a strategy for not only binding, but also degrading cocaine, which o€ers a broad-based therapy. Hapten design is the
central element for programming antibody catalysis. The characteristics of the linker used in classic transition-state analogue
phosphonate haptens were shown to be important for obtaining mAbs that hydrolyze the benzoate ester of cocaine. # 2001
Elsevier Science Ltd. All rights reserved.
Cocaine abuse continues to be prevalent. Recent surveys
for the United States indicate that more than 23 million
people have tried cocaine and approximately 2.5 million
are considered chronic abusers.1,2 Numerous medical
problems, including death, often accompany cocaine
use and the association of the drug with the spread of
AIDS is of concern.3,4 Despite intensive e€orts, the
development of e€ective therapies for cocaine craving
and addiction remain elusive. An improved pharmaco-
therapy would increase the e€ectiveness of rehabilitative
programs. One alternative might rely on immunological
reagents and the immune system. We, and others, have
shown that the antibody-mediated binding of cocaine
impeded passage of the drug into the central nervous
system that resulted in a suppression of its characteristic
actions.5 7 Administration of a monoclonal antibody
(mAb) endowed with not only binding, but also cataly-
tic activity to metabolize cocaine, would have enhanced
therapeutic e€ects if the kinetic properties of the mAb
were sucient.
methyl ester 2,10 it is an excellent target for an immuno-
pharmacological strategy (Fig. 1).
Landry and co-workers used a transition-state (TS)
analogue approach for hapten design and reported sev-
eral cocaine-hydrolyzing mAbs.11,12 In this model, the
benzoyl ester of the cocaine framework is replaced by a
phenylphosphonate that approximates the TS for ester
hydrolysis (Fig. 2). Subsequently, other workers also
used a phosphonate analogue to obtain hybridomas
which were subjected to high-throughput screening
using cocaine benzoyl thioester.13 However, it was more
than 10 years ago that our laboratory applied the TS
design to cocaine and prepared phosphonate haptens.
The ®rst two structures founded on this principle were 3
(code named GNP) and 4 (code named GNN) in which
the site of a linker attachment for coupling to carrier
proteins was di€erent.
Yet, despite screening nearly 1000 clones, no mAbs with
catalytic activity above the background rate were dis-
covered. In an e€ort to elicit a cocaine esterase that will
have utility for human use, we continue to examine TS-
analogue designs, as well as other approaches. Herein, we
report that a speci®c change in the linker composition of 3
and 4 is critical for obtaining cocaine catalytic mAbs,
which provides a foundation for further advances.
Catalytic antibodies have emerged as a powerful tool at
the interface of chemistry and biology.8,9 In this regard,
the hallmark reaction catalyzed by antibodies is ester
hydrolysis. Since cleavage of the benzoate ester of cocaine
1 produces the nonpsychoactive metabolite ecgonine
Having tested a number of other modi®ed phosphonate
TS structures related to 3 without success, we decided to
make a simple change in the linker. A b-alanyl unit was
*Corresponding author. Tel.: +1-858-784-2516; fax: +1-858-784-2595;
e-mail: kdjanda@scripps.edu
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00659-4

88
M. Matsushita et al. / Bioorg. Med. Chem. Lett. 11 (2001) 87±90
Remarkably, we detected catalysis in ꢀ25% of the total
mAbs tested (>3-fold over background in the initial
rate; 20 mM mAb, 500 mM cocaine), of which several
were considered to have good activity (Table 1). Sig-
ni®cantly, as facilitated by the low Km value, the most
ecient mAb, GNL3A6, was able to completely degrade
all o€ered cocaine (20 mM mAb, 900 mM cocaine).
Notably, the Km is the lowest reported to date for any
cocaine catalytic mAb at physiological pH. A low value
for this parameter is an essential contributor to a high
kcat=Km, the apparent second-order rate constant for the
reaction of antibody and cocaine, that dictates mAb
catalytic power.
Figure 1. The antibody-catalyzed hydrolysis of cocaine.
Our mAbs are similar in activity to all mAbs reported
by Landry, except one, in which kcat was ꢀ60-fold bet-
ter than GNL23A6 and kcat=Km ꢀ19-fold better than
GNL3A6.11 However, the conditions for the Landry
mAbs were optimized, which required an increase to pH
8. Cashman et al. reported the most ecient mAb
1
(kcat=Km ꢀ103 M
s
1),13 however this was at pH 8.4
and 37 ꢁC, so the value under our conditions would
likely be reduced ꢀ10-fold. What the results demon-
strate is that, despite e€orts by three laboratories invol-
ving numerous mAbs and methods, ecient clones are
rare and new approaches will be required.
From the standpoint of the cocaine hydrolysis problem,
but also catalytic antibody technology in general, the
e€ect incurred through a subtle change in the linker was
of great interest. Based on our experience with hapten
designs for a variety of hydrolytic reactions, the linker
lengths in 3 and 4 should be adequate to allow recogni-
tion of the cocaine framework, and certainly the phos-
phonate moiety. However, since the b-alanyl fragment
not only introduced a new amide functionality, but also
increased the linker length, it was necessary to separate
these characteristics and determine which contributed to
the ecacy of 9. The hapten 13 (GNK) was synthesized
in which the linker is an alkyl linker as in 3, but of the
same length as in 9 (Fig. 4).
Figure 2. The principle of TS stabilization and haptens based on this
concept.
appended at the linker terminus that a€orded the GNL
hapten 9 (Fig. 3).
We immunized mice with a GNL-KLH conjugate and
the mAbs were screened for the hydrolysis of cocaine by
following the release of benzoic acid using HPLC.
Figure 3. Reagents and conditions: (a) 1.25 M HCl, re¯ux; (b) 2-trimethylsilylethyl 6-bromohexanoate, NaOH, pyridine, 80 ꢁC; (c) (i) LDA, (ii) 11;
(d) (i) TFA, (ii) b-alanine benzyl ester, EDC, HOBt; (e) H2, Pd/C; (f) benzyl alcohol, NEt3; (g) PCl5, CHCl3.

M. Matsushita et al. / Bioorg. Med. Chem. Lett. 11 (2001) 87±90
89
Figure 4. Reagents and conditions: (a) benzyl 10-bromoundecanoate, Bu4NOH, Bu4NI, DMF; (b) (i) LDA, (ii) 11; (c) H2, Pd/C.
Only one mAb from a panel of 19 clones derived from
GNK-KLH was found with a signi®cant rate above
background (Table 1). Even though the activity was
low, the one clone and its catalysis was more than we
ever observed for GNP mAbs. Hence, the longer linker
length possibly promotes some elicitation of catalytic
activity. However, the internal amide bond seems prin-
cipally responsible for the linker-directed e€ects that led
to a `switching on' of an immune response that resulted
in catalytic mAbs. In order to provide a positive internal
control and further support for the hypothesis, a new b-
alanyl linker was introduced at the nitrogen atom as in 4
to give the GNJ hapten 17 (Fig. 5).
amide linkage might allow for more favorable hapten-
peptide fragment presentation by MHC II and/or
recognition by the T-cell receptor. Perhaps more tan-
gible, the hydrogen bonding of the linker amide bond
at the antibody binding site of B-cell surface immuno-
globulin could elicit amino acid residues for chemical
catalysis akin to the principle of `bait and switch'.14
Notably, the haptens of Landry et al. contain an amide-
based linker, in which an amino terminus is capped with
a succinyl unit, and their work has shown that mAb
catalytic activity exceeds that expected from a correlation
based only on TS stabilization.11 Elucidation of catalytic
mechanisms and further studies with new linkers will
help shed light on our hypothesis.
With GNJ-KLH three catalysts were found out of 24
tested (12.5%), fewer than with GNL-KLH. In addi-
tion, the best mAb, GNJ14G12, was less ecient than
the GNL mAbs (Table 1). But again, this single panel of
mAbs, derived from one fusion to produce a set of
hybridomas, contained several catalysts, where before
the GNN hapten yielded nothing from many fusions
and a large survey of candidates.
Both spontaneous15 18 and esterase-catalyzed16,19 21
hydrolysis of cocaine contribute to the short in vivo
half-life of ꢀ30 min in human blood, comparable to
that determined in our laboratory in rats.6 Yet, for an
enzyme or catalytic antibody therapy to be e€ective,
extensive clearance of cocaine must take place within
seconds. The administration of puri®ed human plasma
cholinesterase reduced cocaine toxicity in mice.22
Although the kinetic parameters for the enzyme are
currently superior to the best cocaine mAb,23 the e-
ciency is still not suitable to achieve cocaine clearance in
the human condition at clinically manageable concentra-
tions of enzyme. Landry et al. also reported some positive
results in animal models using their best catalytic mAb.24
However, high catalytic power is required to meet the
demands of hydrolyzing cocaine rapidly enough to alter
its pharmacokinetic pro®le and psychoactive e€ects in
the human condition.
Until further insight is gained from immunochemical
and immunological experiments, we can only speculate
as to the observed linker e€ect. One rationale is that an
Table 1. Data for some catalytic mAbs that hydrolyze cocainea
mAb
kcat
(min
Km
(mM)
kcat=Km
kcat=kuncat
1
1
1
)
(M
s )
GNL3A6
GNL4D3
GNL23A6
GNJ14G12
GNK28C9
0.030
0.010
0.038
0.023
55
70
830
5240
2080
9.2
2.3
0.77
0.083
0.050
3705
1210
4690
2840
745
An estimate can be made as to the requirements of an anti-
cocaine catalytic mAb during a period of rehabilitation
from cocaine abuse. We suggest that an administered
0.0060
aDetermined in 100 mM phosphate bu€er, pH 7.4, 21 ꢁC.
Figure 5. Reagents and conditions: (a) HCl, MeOH; (b) (i) LDA, (ii) 11; (c) (i) Troc-Cl, NEt3, (ii) Zn, formic acid; (d) t-butyl 6-bromohexanoate,
NEt3, CH3CN; (e) (i) TFA, (ii) b-alanine benzyl ester, EDC, HOBt, (iii) H2, Pd/C.

90
M. Matsushita et al. / Bioorg. Med. Chem. Lett. 11 (2001) 87±90
catalytic mAb, `humanized' or even `fully human' to
minimize an immune response,25,26 that is circulating at
a practical, long-term clinical level of ꢀ1 mg/mL (ꢀ15
mM in active sites for whole IgG) must have a minimum
kcat=Km ꢀ104 M 1 s 1. An mAb operating with this rate
constant would a€ord clearance of a typical single dose
of circulating cocaine (ꢀ10 mM) from the bloodstream
within a few seconds before transit into the brain. This
activity is in the range of the esterase family of enzymes
studied using various ester substrates, other than cocaine,
which again is indicative of the recalcitrant nature of
cocaine as a substrate and for hapten programming. Even
though our best mAb needs a 103-fold rate enhancement,
this is not unrealistic considering a similar factor was
attained in progressing from mAbs with no activity to
those resulting from only a change in linker chemistry.
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