Treatment of methamphetamine abuse: an antibody-based immunotherapy approach

Abstract

Methamphetamine is a highly addictive psychostimulant with tens of millions of abusers around the world, and currently there is no effective or approved medication for addiction to it. Monoclonal antibodies with a high affinity for methamphetamine have the potential to sequester the drug in the vascular compartment and reduce entry into the brain, acting as peripheral pharmacokinetic antagonists without inducing adverse effects on neurons. However, in order to maintain the antibodies at an effective level, repeated administration is required, which would be expensive and problematic for patient compliance. In this study, we intended to investigate whether using a recombinant adeno-associated virus-mediated gene transfer technique can be an effective approach to achieve long-term expression of anti-methamphetamine monoclonal antibodies in mouse models. We generated a recombinant adeno-associated virus vector encoding the heavy and light chains of an anti-methamphetamine monoclonal antibody, which were constructed in a single open reading frame and linked with a 2A self-processing sequence. In the context of virus-mediated gene transfer, expression of full-length and functional monoclonal antibodies was successfully demonstrated in vitro and in vivo. Further investigations on dose optimization, long-term expression, and protection from methamphetamine challenge in mouse models are ongoing.

1. Introduction

Methamphetamine (Meth) is an illicit, highly addictive stimulant of the central nervous system (CNS); it is estimated that there are over 35 million Meth abusers in the world [1]. Although governments throughout the world have attempted to conquer Meth abuse over the past two decades, it continues to be a major public health problem that needs to be tackled on a global scale. Meth abuse can cause a number of adverse biological effects on many organ systems, including acute toxic effects on the cardiovascular system and CNS, acute renal failure, altered behavioral and cognitive functions, and persistent neurological damages to the brain [2,3]. In addition, a growing body of evidence has indicated that Meth could suppress both innate and adoptive immunity, and increase susceptibility to viral pathogens, such as human immunodeficiency virus [4,5]. Currently, there is no approved medication for Meth abuse, and the broad mechanism of Meth action has made it difficult to find an effective small molecule for the development of pharmacotherapeutic intervention [6,7].

2. The principle of anti-methamphetamine immunotherapy

Alternatively, antibodies with a high affinity for Meth can sequester the drug molecules in the bloodstream and prevent their gaining access to their activation sites in the CNS or other vulnerable organs (Fig. 1), acting as peripheral pharmacokinetic antagonists to terminate the drug-induced reinforcing effects [8,9]. Because antibodies are too big to readily traverse the blood-brain barrier, the antibody-bound drug molecules cannot freely enter the brain, resulting in a drug concentration gradient allowing a free drug molecule to leave the brain at a relatively higher rate than it enters [8].

Figure 1.

Concept of antibody-based therapy for methamphetamine abuse. When methamphetamine is taken and enters the bloodstream, it can move across the blood-brain barrier (BBB) and freely access its activation sites in the brain, causing adverse psychological and craving effects (left side). In the presence of anti-methamphetamine antibodies in the bloodstream, methamphetamine molecules will be captured and confined in the circulation, resulting in a reduced entry of drugs into the central nervous system (right side). Red closed circle: methamphetamine molecule; Green Y shaped figure: anti-methamphetamine antibody.

3. Approaches for generating anti-methamphetamine antibodies

In general, drug-specific antibodies can be generated in a patient’s bloodstream by active and passive immunization approaches (Fig. 2). In active immunization, drug-like antigens are repeatedly administrated via traditional vaccination procedures to generate a patient’s own polyclonal antibodies against the target drugs [10]. The passive immunization approach is performed by intravenous administration of well-characterized and genetically engineered monoclonal anti-drug antibodies, which are derived from vaccinated animals or antibody libraries [11,12]. In fact, these immunization approaches have been investigated for the treatment of Meth abuse in several rodent models, with promising results [13,14].

Figure 2.

Approaches for generating anti-methamphetamine antibodies in the bloodstream for the treatment of methamphetamine abuse. Antibody-based therapy for methamphetamine (Meth) abuse encompasses three potential approaches: (I) individuals can receive drug-specific vaccines to generate their own anti-Meth polyclonal antibodies; (II) antibodies with high affinity to Meth can be isolated from antibody libraries expressed in various microorganism systems, or from antibody-producing hybridoma cells derived from vaccinated animals; those preselected antibodies can be genetically modified to form human compatible ones in suitable expression systems for passive transfer into the peripheral circulation; and (III) expression of anti-Meth antibodies can be achieved by recombinant adeno-associated virus (rAAV) mediated gene transfer into humans.

Although drug-specific immunization approaches could potentially avoid inducing unwanted neuromodulatory effects in the brain, several limitations should be carefully considered before treating drug abuse with this approach. The drug molecule itself cannot reactivate the vaccine-induced anti-drug immune response to regenerate anti-drug antibodies [15,16]; the preformed antibodies will be disabled by forming long-lived complexes with the captured drugs and be eliminated gradually after being infused into the circulation [10,17].

Therefore, in order to maintain an effective level of anti-drug antibodies, a well-scheduled and repeated administration course is required for both immunization approaches, which would be costly and become a major barrier to patient adherence. In addition, although the drug vaccine is designed to induce drug-specific antibodies, active immunization could potentially induce antibodies with unwanted reactivity to endogenous molecules or tissue antigens, which might reduce the therapeutic efficacy. Therefore, we proposed an alternative approach for a long-term expression of Meth-specific antibodies in vivo via recombinant adeno-associated virus (rAAV) mediated gene transfer.

4. Recombinant adeno-associated virus-mediated expression of anti-methamphetamine antibodies

The rAAV vector has been widely used in gene therapy applications, largely due to its low immunogenicity and non-pathogenicity for humans, and its capability to transduce a broad range of cell types and achieve a long-term gene expression [18,19]. In this study, we generated a rAAV serotype-8 vector carrying an antibody expression cassette (Fig. 3), in which the heavy- and light-chain sequences of a well-characterized anti-Meth monoclonal antibody (K_D = 11 nM) were separated by a furin-2A self-cleavage site [13,20].

Figure 3.

Schematic illustration of genetic construction and biosynthesis of anti-methamphetamine monoclonal antibody. The heavy- and light-chain genes of a preselected anti-methamphetamine antibody were constructed in a single open reading frame, which were linked together by a sequence coding for a combination of furin cleavage site (FCS) and 2A self-processing sequence (derived from foot-and-mouth-disease virus), driven by the cytomegalovirus (CMV) promoter. During protein biosynthesis, the encoded 2A sequence can disrupt peptide-bond formation at the C-terminus of 2A, without devastating synthesis of the downstream protein. The FCS allows enzymatic removal of 2A sequence from the C-terminus of translated heavy chain. Finally, the co-expressed heavy- and light-chain proteins self-assemble to form a functional antibody in cells receiving the expression cassette of anti-methamphetamine antibody. SP: signal peptide.

The anti-Meth antibodies expressed in HEK 293 cells infected with the rAAV8 vector were purified from the culture medium and shown to capture Meth molecules in a colorimetric assay (Fig. 4). Thus, anti-Meth antibodies expressed from the furin-2A construct are produced in the secreted form and retain the binding activity to Meth molecules. To evaluate rAAV8-mediated expression of anti-Meth antibodies in vivo, adult mice were intraperitoneally injected with the rAAV8 vector (at a dose of 10⁹ genome copies/mouse), and the serum levels of anti-Meth antibodies were evaluated over time. The anti-Meth antibody was detected at 14 days post-administration of the rAAV8 vector, and a 12-fold higher serum level (p < 0.001) was achieved at 47 days post-administration (Fig. 5A); long-term evaluation is still ongoing. We further investigated whether anti-Meth antibodies generated by rAAV8-mediated gene transfer can attenuate Meth-induced behavioral changes. Mice were intraperitoneally injected with Meth (1 mg/kg) at 50 days post-administration of the rAAV8 vector, and the post-challenge locomotor activity was recorded for 90 min as a total distance traveled. Compared with the mock-infected group (n = 4), the Meth-induced locomoter activity was reduced by 30% (p = 0.084) in the group (n = 3) receiving the rAAV8 vector (Fig. 5B).

Figure 4.

A colorimetric assay for quantitative determination of anti-methamphetamine antibodies. Antibodies can be captured by protein-G-conjugated sepharose beads. After washing off the unbound methamphetamine (Meth) conjugated with horseradish peroxidase (HRP), if the added HRP substrates are converted to blue color substances, it indicates the presence of anti-Meth antibodies; conversely, if color changes do not occur, it is inferred that the captured antibody has no binding activity to Meth. The color intensity produced is proportional to the amount of anti-Meth antibodies captured by the beads. Anti-V5: a mouse monoclonal antibody specific to a 14-amino acid V5 epitope derived from simian parainfluenza virus type 5; Green Y shaped figure: antibody.

Figure 5.

Expression of anti-methamphetamine monoclonal antibody in vivo by rAAV8 mediated gene transfer. Male ICR strain mice (8-week age) were intraperitoneally injected with PBS (n = 4) or rAAV8 (n = 3) carrying the expression cassette of an anti-methamphetamine (Meth) antibody. Serum samples were collected at indicated time points (0, 14, 47 days post-administration) for measuring the expression levels of anti-Meth antibodies by colorimetric assay (A). Animals were challenged with Meth at a dose of 1 mg/kg through intraperitoneal injection on day 50 post-administration of rAAV8, and the induced locomotor activity was recorded as total distance traveled (cm) in 90 min (B). Horizontal lines: mean of distance traveled. Statistical differences were determined by using two-sample t-test. All animal studies were conducted under protocols reviewed and approved by the National Health Research Institutional Animal Care and Use Committee.

Although the difference did not reach statistical significance, these data demonstrated that a single administration of a low dose of rAAV8 is able to achieve peripheral expression of functional anti-Meth antibodies to attenuate Meth-induced behavioral changes in mice. It should be noted that this study only presented preliminary results, and the animal numbers were not enough to satisfy statistical criteria; in addition, the virus vector was administrated at a low dosage. In future work, there should be numerous chances and many ways for us to improve protection from a Meth-challenge following rAAV8-mediated gene transfer.

5. Conclusions

Currently, we are conducting further investigations focusing on increasing virus dosages to achieve higher serum levels of anti-Meth antibodies in a sample size with sufficient statistical power; furthermore, an antibody highly specific to amphetamine, a pharmacologically active metabolite of Meth, is also applied in the rAAV8-mediated gene transfer approach. Although the safety and efficacy of rAAV-mediated expression of anti-Meth antibodies in humans remains to be addressed, we envision that this gene transfer approach used along or in combination with appropriate counseling would greatly increase the likelihood of successful treatment of Meth dependence. Furthermore, this gene transfer approach could potentially be applied to treatment for other drug abuse.