WO2002046234A1

WO2002046234A1 - Cocaine haptens

Info

Publication number: WO2002046234A1
Application number: PCT/US2001/047471
Authority: WO
Inventors: Kim D. Janda; Peter Wirsching
Original assignee: The Scripps Research Institute
Priority date: 2000-12-07
Filing date: 2001-12-07
Publication date: 2002-06-13
Also published as: WO2002046234A8; AU2002226055A1

Abstract

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

Description

COCAINE HAPTENS

Description

Background:

Despite intensive efforts, the development of effective therapies for cocaine craving and addiction remain elusive. An improved pharmacotherapy would increase the effectiveness of rehabilitative programs. One approach to the development of therapies for cocain addiction has relied on immunological reagents and the immune system. It has been 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 (Carrera, M. R. A.; et al. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 6202; Carrera, M.; et al. Nature 1995, 378, 727; Fox, B. S.; et al. Nat. Med. 1996, 2, 1129). Administration of a monoclonal antibody (mAb) endowed with not only binding, but also catalytic activity to metabolize cocaine, would have enhanced therapeutic effects if the kinetic properties of the mAb were sufficient.

Catalytic antibodies have emerged as a powerful tool at the interface of chemistry and biology (Wentworth, Jr., P.; Janda, K. D. Curr. Opin. Chem. Biol. 1998, 2, 138; Schultz, P. G.; Lerner, R. A. Science 1995, 269, 1835). 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 methyl ester 2(Misra, A. L; et al. J. Pharm. Pharmacol. 1975, 27, 784), it is an excellent target for an immunopharmacological strategy (Figure 1 ).

Landry and co-workers used a transition-state (TS) analog approach for hapten design and reported several cocaine-hydrolyzing mAbs (Yang, G. X.-Q.; et al. J. Am. Chem. Soc. 1996, 118, 5881 ; Landry, D. W.; et al. Science 1993, 259, 1899). In this model, the benzoyl ester of the cocaine framework is replaced by a phenylphosphonate that approximates the TS for ester hydrolysis (Figure 2). Subsequently, other workers also used a phosphonate analog to obtain hybridomas which were subjected to high-throughput screening using cocaine benzoyl thioester (Cashman, J. R.; et al. J. Pharmacol. Exp. Ther. 2000, 293, 952).

More than 10 years ago, the TS design was applied by the present investigators to the preparation of phosphonate based cocaine haptens. The first 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 different. Yet, despite screening nearly 1000 clones, no mAbs with catalytic activity above the background rate were discovered. In an effort to elicit a cocaine esterase that will have utility for human use, the present investigators continue to examine TS-analog designs, as well as other approaches. Herein, it is reported that a specific change in the linker composition of 3 and 4 is critical for obtaining cocaine catalytic mAbs, which provides a foundation for further advances.

Summary of Invention:

One aspect of the invention is directed to a cocaine hapten represented by the following structure:

In the above structure, m and n are integers such that 0<m≤5 and 1 <n≤3. A preferred cocaine hapten is represented by the following structure:

Another aspect of the invention is directed to a process for eliciting a catalytic antibody from an immune responsive animal, the catalytic antibody having a catalytic activity for converting cocaine to ecgonine methyl ester by hydrolysis. The process comprises the step of immunizing the immune responsive animal with a sufficient quantity of an immunogen for eliciting an immune response, the immunogen having a hapten represented by the following radical:

In the above structure, m and n are integers such that 0≤m≤5 and 1 <n<3. In a preferred mode, the hapten is represented by the following radical:

Another aspect of the invention is directed to a catalytic antibody having a catalytic activity for converting cocaine to ecgonine methyl ester by hydrolysis. The catalytic antibody is of a type that is isolated from an immune responsive animal that has been immunized with a quantity of an immunogen sufficient to elicit an immune response. More particularly, the immunogen has a hapten represented by the following radical:

In the above structure, m and n are integers such that 0<m≤5 and 1≤n≤3. A preferred hapten is represented by the following radical:

Another aspect of the invention is directed to a process for obtaining a catalytic monoclonal antibody having a catalytic activity for converting cocaine to ecgonine methyl ester by hydrolysis. The process comprises the following steps: Firstly, an immune responsive animal is immunized with an immunogen having a hapten represented by the following radical:

In the above structure, m and n are integers such that 0≤m<5 and 1 ≤n≤3. In a preferred mode, the hapten is represented by the following radical:

Secondly, antibody producing cells that express catalytic antibody having a catalytic activity for converting cocaine to ecgonine methyl ester by hydrolysis are isolated from the immune responsive animal. Thirdly, the antibody producing cells are cloned and the catalytic monoclonal antibody is isolated therefrom.

Another aspect of the invention is directed to a catalytic monoclonal antibody having a catalytic activity for converting cocaine to ecgonine methyl ester by hydrolysis. The catalytic monoclonal antibody is of a type that is isolated from an antibody producing cell obtained by the following process. Firstly, an immune responsive animal is immunized with an immunogen having a hapten represented by the following radical:

In the above structure, m and n are integers such that 0<m<5 and 1 ≤n<3. In a preferred embodiment, the hapten is represented by the following radical:

Secondly, antibody producing cells that express catalytic antibody having a catalytic activity for converting cocaine to ecgonine methyl ester by hydrolysis are isolated from the immune responsive animal. Thirdly, the antibody producing cells are cloned and the catalytic monoclonal antibody are isolated therefrom.

Another aspect of the invention is directed to a process for converting cocaine to ecgonine methyl ester by hydrolysis. The process comprises the following steps. Firstly, an immune responsive animal is immunized with a quantity of an immunogen sufficient to elicit an immune response, the immunogen having a hapten represented by the following radical:

In the above structure, m and n are integers such that 0≤m≤5 and 1 ≤n≤3. In a preferred mode, the hapten is represented by the following radical:

Secondly, catalytic antibody is isolated from the immune responsive animal, the catalytic antibody having a catalytic activity for converting cocaine to ecgonine methyl ester by hydrolysis. Thirdly, the cocaine is contacted twith a concentration of the catalytic antibody sufficient to catalyze conversion of the cocaine by hydrolysis to ecgonine methyl ester.

In an alternative mode of this aspect of the invention, a process for converting cocaine to ecgonine methyl ester by hydrolysis comprises the following steps. Firstly, an immune responsive animal is immunized with an immunogen having a hapten represented by the following radical:

In the above structure, m and n are integers such that 0<m<5 and 1 ≤n≤3. In a preferred mode of this aspect of the invention, the hapten is represented by the following radical:

Secondly, antibody producing cells are isolated from the immune responsive animal that express catalytic antibody having a catalytic activity for converting cocaine to ecgonine methyl ester by hydrolysis. Thirdly, the antibody producing cells are cloned and catalytic monoclonal antibody is isolated therefrom. Fourthly, cocaine is contacted with a concentration of the catalytic monoclonal antibody sufficient to catalyze a conversion of the cocaine to ecgonine methyl ester by hydrolysis.

Brief Description of Drawings:

Figure 1 illustrates the antibody-catalyzed hydrolysis of cocaine.

Figure 2 illustrates the principle of TS stabilization and haptens based on this concept. Figure 3 illustrates a scheme for the synthesis of hapten 9 GNL.

Figure 4 is a scheme illustrating the synthetic steps used in the synthesis of hapten 13 GNK.

Figure 5 is a scheme illustrating the synthetic steps used in the synthesis of hapten 17 GNJ. Figure 6 is a table illustrating the mAbs screened for the hydrolysis of cocaine by following the release of benzoic acid using HPLC. Measurements were determined in 100 mM phosphate buffer, pH 7.4, 21 °C. Detailed Description:

Having tested a number of other modified phosphonate TS structures related to 3 without success, the present investigators decided to make a simple change in the linker of the hapten. A β-alanyl unit was appended at the linker terminus that afforded the GNL hapten 9, according to the synthetic method disclosed in Figure 3.

Mice were immunized with a GNL-KLH conjugate (immunogen) and the resultant mAbs were screened for the hydrolysis of cocaine by following the release of benzoic acid using HPLC. Remarkably, catalysis was detected in -25% of the total mAbs tested (>3-fold over background in the initial rate; 20 μM mAb, 500 μM cocaine), of which several were considered to have good activity (Figure 6). Significantly, as facilitated by the low K_m value, the most efficient mAb, GNL3A6, was able to completely degrade all offered cocaine (20 μM mAb, 900 μM cocaine). Notably, the K_m 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 k_cJK_m, the apparent second-order rate constant for the reaction of antibody and cocaine, that dictates mAb catalytic power.

The mAbs disclosed herein are similar in activity to all mAbs reported by Landry, except one, in which / _cat was ~60-fold better than GNL23A6 and k_cat/K_m ~19-fold better than GNL3A6 (Yang, G. X.-Q.; et al. J. Am. Chem. Soc. 1996, 118, 5881 ). However, the conditions for the Landry mAbs were optimized, which required an increase to pH 8. Cashman et al. reported the most efficient mAb (k K_m ~ 10³ M^"1 s^"1) (Cashman, J. R.; et al. J. Pharmacol. Exp. Ther. 2000, 293, 952), however this was at pH 8.4 and 37 °C, so the value under the conditions herein would likely be reduced ~10-fold. What the results demonstrate is that, despite efforts by three laboratories involving numerous mAbs and methods, efficient 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 effect incurred through a subtle change in the linker was of great interest. Based on the experience of the present investigators with hapten designs for a variety of hydrolytic reactions, the linker lengths in 3 and 4 should be adequate to allow recognition of the cocaine framework, and certainly the phosphonate moiety. However, since the β-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 efficacy 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 (Figure 4).

Only one mAb from a panel of 19 clones derived from GNK-KLH was found with a significant rate above background (Figure 6). Even though the activity was low, the one clone and its catalysis was more than previously observed for GNP mAbs. Hence, the longer linker length possibly promotes some elicitation of catalytic activity. However, the internal amide bond seems principally responsible for the linker-directed effects 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 β-alanyl linker was introduced at the nitrogen atom as in 4 to give the GNJ hapten 17 (Figure 5).

With GNJ-KLH three catalysts were found out of 24 tested (12.5%), fewer than with GNL-KLH. In addition, the best mAb, GNJ14G12, was less efficient than the GNL mAbs (Figure 6). 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.

It is disclosed herein that an amide linkage allows for more favorable hapten-peptide fragment presentation by MHC II and/or recognition by the T-cell receptor. Perhaps more tangible, the hydrogen bonding of the linker amide bond at the antibody binding site of B-cell surface immunoglobulin seems to elicit amino acid residues for chemical catalysis akin to the principle of "bait and switch" (Lavey, B. J.; Janda, K. D. In Antibody Expression and Engineering; Wang, H. Y.; Imanaka, T., Eds.; ACS Symposium Series 604, 1995; Chapter 10). 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 (Yang, G. X.-Q.; et al. J. Am. Chem. Soc. 1996, 118, 5881 ).

Both spontaneous (Garrett, E. R.; Seyda, K. J. Pharm. Sci. 1983, 72, 258; Stewart, D. J.; et al. Clin. Pharmacol. Therapeut. 1979, 25, 464; Cunningham, K. A.; Lakoski, J. M. Neuropsychopharmacology 1990, 3, 41 ; Li, P.; et al. Helv. Chim. Ada 1999, 82, 85) and esterase-catalyzed (Stewart, D. J.; et al. Clin. Pharmacol. Therapeut. 1979, 25, 464; Brzezinski, M. R.; et al. Biochem. Pharmacol. 1994, 48, 1747; Boyer, C. S.; Petersen, D. R. J. Pharmacol. Exp. Then 1992, 260, 939; Matsubara, K.; et al. Forensic Sci. Intl. 1984, 26, 169) hydrolysis of cocaine contribute to the short in vivo half-life of -30 minutes in human blood, comparable to that determined in the laboratory of the present investigators in rats (Carrera, M.; et al. Nature 1995, 378, 727). Yet, for an enzyme or catalytic antibody therapy to be effective, extensive clearance of cocaine must take place within seconds. The administration of purified human plasma cholinesterase reduced cocaine toxicity in mice (Hoffman, R. S.; et al. Clin. Toxicol. 1996, 34, 259). However, the catalytic power of this enzyme is not sufficient to achieve cocaine clearance in the human condition at clinically manageable concentrations of enzyme (Xie, W.; et al. Mol. Pharmacol. 1999, 55, 83). Landry et al. also reported some positive results in animal models using their best catalytic mAb (Mets, B.; et al. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 10176). However, high catalytic power is required to meet the demands of hydrolyzing cocaine rapidly enough to alter its pharmacokinetic profile and psychoactive effects in the human condition.

An estimate can be made as to the requirements of an anti-cocaine catalytic mAb during a period of rehabilitation from cocaine abuse. It is disclosed herein that an administered catalytic mAb, "humanized" or even "fully human" to minimize an immune response (James, K. In Handbook of Experimental Pharmacology : The Pharmacology of Monoclonal Antibodies; Rosenberg, M.; Moore, G. P., Eds.; Springer-Verlag: New York, 1994; Vol. 113, pp 3-19; Burton, D. R.; Barbas, C. F. III. Adv. Immunol. 1994, 57, 191), that is circulating at a practical, long-term clinical level of -1 mg/mL (-15 μM in active sites for whole IgG) must have a minimum k_cJK_m - 10⁴ M^"1 s^"1. A mAb operating with this rate constant affords sufficient clearance of a typical single dose of circulating cocaine (-10 μM) 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.

Detailed Description of Figures:

Figure 1 shows the antibody-catalyzed hydrolysis of cocaine. The products of this reaction are benzoic acid and the non-psychoactive methyl ecgonine ester 2.

Figure 2 shows the principle of TS stabilization and haptens based on this concept.

Figure 3 shows the scheme used for the synthesis of hapten 9 GNL. Reagents and conditions: (a) 1.25 M HCI, reflux; (b) 2-trimethylsilylethyl- 6-bromohexanoate, NaOH, pyridine, 80 °C; (c) (i) LDA, (ii) 11 ; (d) (i) TFA, (ii) β-alanine benzyl ester, EDC, HOBt; (e) H₂, Pd/C; (f) benzyl alcohol, NEt₃; (g) PCI₅, CHCl₃, 40 °C.

Figure 4 is a scheme showing the synthetic steps used in the synthesis of hapten 13 GNK. Reagents and conditions: (a) benzyl 10-bromodecanoate, Bu₄NOH, Bu₄NI, DMF; (b) (i) LDA, (ii) 11; (c) H₂, Pd/C.

Figure 5 is a scheme showing the synthetic steps used in the synthesis of hapten 17 GNJ. Reagents and conditions: (a) HCI, MeOH; (b) (i) LDA, (ii) 11; (c) (i) Troc-CI, NEt₃, (ii) Zn, formic acid; (d) f-butyl 6-bromohexanoate, NEt₃, CH₃CN; (e) (i) TFA, (ii) β-alanine benzyl ester, EDC, HOBt; (iii) H₂, Pd/C.

Figure 6 is a table showing the mAbs screened for the hydrolysis of cocaine by following the release of benzoic acid using HPLC. Remarkably, catalysis was detected in -25% of the total mAbs tested (>3-fold over background in the initial rate; 20 μM mAb, 500 μM cocaine), of which several were considered to have good activity (Table 1). Significantly, as facilitated by the low K_m value, the most efficient mAb, GNL3A6, was able to completely degrade all offered cocaine (20 μM mAb, 900 μM cocaine). Notably, the K_m 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 k_c K_m, the apparent second-order rate constant for the reaction of antibody and cocaine, that dictates mAb catalytic power.

Claims

What is claimed is:

1. A cocaine hapten represented by the following structure:

wherein m and n are integers such that 0<m≤5 and 1 ≤n≤3.

2. A cocaine hapten according to claim 1 represented by the following structure:

3. A process for eliciting a catalytic antibody from an immune responsive animal, the catalytic antibody having a catalytic activity for converting cocaine to ecgonine methyl ester by hydrolysis, the process comprising the step of immunizing the immune responsive animal with a sufficient quantity of an immunogen for eliciting an immune response, the immunogen having a hapten represented by the following radical:

wherein m and n are integers such that 0<m≤5 and 1 ≤n<3.

4. A process according to claim 3 wherein the hapten is represented by the following radical:

5. A catalytic antibody isolated from an immune responsive animal that has been immunized with a quantity of an immunogen sufficient to elicit an immune response, the catalytic antibody having a catalytic activity for converting cocaine to ecgonine methyl ester by hydrolysis, the immunogen having a hapten represented by the following radical:

I0 wherein m and n are integers such that 0≤m≤5 and 1 ≤n≤3.

6. A catalytic antibody according to claim 5 wherein the hapten is represented by the following radical:

7. A process for obtaining a catalytic monoclonal antibody having a catalytic 10 activity for converting cocaine to ecgonine methyl ester by hydrolysis, the process comprising the following steps:

immunizing an immune responsive animal with an immunogen having a hapten represented by the following radical:

wherein m and n are integers such that 0<m<5 and 1 <n≤3; then

30 isolating antibody producing cells from the immune responsive animal that express catalytic antibody having a catalytic activity for converting cocaine to ecgonine methyl ester by hydrolysis; and then

cloning the antibody producing cells and isolating the catalytic monoclonal antibody therefrom.

8. A process according to claim 7 wherein the hapten is represented by the following radical:

9. A catalytic monoclonal antibody having a catalytic activity for converting cocaine to ecgonine methyl ester by hydrolysis, the catalytic monoclonal antibody being isolated from an antibody producing cell, the antibody producing cell being obtained by the following process:

wherein m and n are integers such that 0≤m≤5 and 1 <n<3; then

isolating antibody producing cells from the immune responsive animal that express catalytic antibody having a catalytic activity for converting cocaine to ecgonine methyl ester by hydrolysis; and then

10. A catalytic monoclonal antibody according to claim 9 wherein the hapten is represented by the following radical:

11. A process for converting cocaine to ecgonine methyl ester by hydrolysis, the process comprising the following steps: immunizing an immune responsive animal with a quantity of an immunogen sufficient to elicit an immune response, the immunogen having a hapten represented by the following radical:

wherein m and n are integers such that 0<m<5 and 1 ≤n≤3; then

isolating a catalytic antibody from the immune responsive animal, the catalytic >0 antibody having a catalytic activity for converting cocaine to ecgonine methyl ester by hydrolysis; and then

contacting the cocaine with a concentration of the catalytic antibody sufficient to catalyze conversion of the cocaine by hydrolysis to ecgonine methyl 15 ester.

12. A process according to claim 11 wherein the hapten is represented by the following radical:

13. A process for converting cocaine to ecgonine methyl ester by hydrolysis, the process comprising the following steps:

0 wherein m and n are integers such that 0<m<5 and 1 ≤n<3; then

isolating antibody producing cells from the immune responsive animal that express catalytic antibody having a catalytic activity for converting cocaine to ecgonine methyl ester by hydrolysis; then 15 cloning the antibody producing cells and isolating catalytic monoclonal antibody therefrom; and then

contacting cocaine with a concentration of the catalytic monoclonal antibody \0 sufficient to catalyze a conversion of the cocaine to ecgonine methyl ester by hydrolysis.

14. A process according to claim 13 wherein the hapten is represented by the following radical: