175 Structure-based engineering to generate high-affinity immunotherapy for the drug of abuse

Methamphetamine (METH) abuse is a major threat in the USA and worldwide without any FDA approved medications. Anti-METH antibody antagonists block or slow the rate of METH entry into the brain and have shown efficacy in preclinical studies (Peterson, Laurenzana, Atchley, Hendrickson, & Owens, 2008 ). A key determinant of the antibody's efficacy is its affinity for METH and we attempted to enhance the efficacy by designing mutations to alter the shape or the electrostatic character of the binding pocket. Towards this goal, we developed a single chain anti-METH antibody fragment (scFv6H4) from a parent IgG (1). The crystal structure of scFv-6H4 in complex with METH was determined (Celikel, Peterson, Owens, & Varughese, 2009 ). Based on its elucidated binding interactions, we designed point mutations in the binding pocket to improve its affinity for METH and amphetamine (AMP), the active metabolite of METH. The mutants, scFv-S93T,-I37 M and -Y34 M were cloned, expressed in yeast and tested for affinity against METH and AMP. Two mutants showed enhanced binding affinity for METH: scFv-I37 M by 1.3-fold and scFv-S93T by 2.6-fold. Additionally, all the mutants showed increase in affinity for AMP: scFv-I37 M by 56-fold, scFv-S93T by 17-fold and scFvY34 M by 5-fold. Crystal structure for one of the high-affinity mutant, scFv-S93T, in complex with METH was determined (Figure 1). Binding pocket of the mutant is more hydrophobic in comparison with the wild type. ScFv-6H4 binds METH in a deep pocket containing two water molecules. The substitution of a serine residue by a threonine leads to the expulsion of a water molecule (Figure 2), relieving some unfavorable contacts between the hydrocarbon atoms of METH and the water molecule and increasing the affinity to sub-nanomolar range. Therefore, the present study shows that efficacy could be enhanced by altering the hydrophobicity or the shape of the binding pocket

Therapeutic Anti-Methamphetamine Antibody Fragment-Nanoparticle Conjugates: Synthesis and In Vitro Characterization

Treatments specific to the medical problems caused by methamphetamine (METH) abuse are greatly needed. Towards this goal, we are developing new multivalent anti-METH antibody fragment-nanoparticle conjugates with customizable pharmacokinetic properties. We have designed a novel anti-METH single chain antibody fragment with an engineered terminal cysteine (scFv6H4Cys). Generation 3 (G3) polyamidoamine dendrimer nanoparticles were chosen for conjugation due to their monodisperse properties and multiple amine functional groups. ScFv6H4Cys was conjugated to G3 dendrimers via a heterobifunctional PEG crosslinker that is reactive to a free amine on one end and a thiol group on the other. PEG modified dendrimers were synthesized by reacting PEG crosslinker with dendrimers in a stoichiometric ratio of 11:1, which were further reacted with three-fold molar excess of anti-METH scFv6H4Cys. This reaction resulted in a heterogeneous mix of G3-PEG-scFv6H4Cys conjugates (dendribodies) with three to six scFv6H4Cys conjugated to each dendrimer. The dendribodies were separated from the unreacted PEG modified dendrimers and scFv6H4Cys using affinity chromatography. A detailed in vitro characterization of the PEG modified dendrimers and the dendribodies was performed to determine size, purity, and METH-binding function. The dendribodies were found to have identical affinity for METH as the unconjugated scFv6H4Cys in saturation binding assays, whereas the PEG modified dendrimers had no affinity for METH. These data suggest that an anti-METH scFv can be successfully conjugated to a PEG modified dendrimer nanoparticle with no adverse effects on METH binding properties. This study is a critical step towards preclinical characterization and development of a novel nanomedicine for the treatment of METH abuse

Development and testing of AAV-delivered single-chain variable fragments for the treatment of methamphetamine abuse

Methamphetamine (METH) substance abuse disorders have major impact on society, yet no medications have proven successful at preventing METH relapse or cravings. Anti-METH monoclonal antibodies can reduce METH brain concentrations; however, this therapy has limitations, including the need for repeated dosing throughout the course of addiction recovery. An adeno-associated viral (AAV)-delivered DNA sequence for a single-chain variable fragment could offer long-term, continuous expression of anti-METH antibody fragments. For these studies, we injected mice via tail vein with 1 x 10(12) vector genomes of two AAV8 scFv constructs and measured long-term expression of the antibody fragments. Mice expressed each scFv for at least 212 days, achieving micromolar scFv concentrations in serum. In separate experiments 21 days and 50 days after injecting mice with AAV-scFvs mice were challenged with METH in vivo. The circulating scFvs were capable of decreasing brain METH concentrations by up to 60% and sequestering METH in serum for 2 to 3 hrs. These results suggest that AAV-delivered scFv could be a promising therapy to treat methamphetamine abuse

Development and testing of AAV-delivered single-chain variable fragments for the treatment of methamphetamine abuse

<div><p>Methamphetamine (METH) substance abuse disorders have major impact on society, yet no medications have proven successful at preventing METH relapse or cravings. Anti-METH monoclonal antibodies can reduce METH brain concentrations; however, this therapy has limitations, including the need for repeated dosing throughout the course of addiction recovery. An adeno-associated viral (AAV)-delivered DNA sequence for a single-chain variable fragment could offer long-term, continuous expression of anti-METH antibody fragments. For these studies, we injected mice via tail vein with 1 x 10¹² vector genomes of two AAV8 scFv constructs and measured long-term expression of the antibody fragments. Mice expressed each scFv for at least 212 days, achieving micromolar scFv concentrations in serum. In separate experiments 21 days and 50 days after injecting mice with AAV-scFvs mice were challenged with METH <i>in vivo</i>. The circulating scFvs were capable of decreasing brain METH concentrations by up to 60% and sequestering METH in serum for 2 to 3 hrs. These results suggest that AAV-delivered scFv could be a promising therapy to treat methamphetamine abuse.</p></div

A comparison of METH or AMP brain and serum concentrations over time, after a 0.56 mg/kg <i>ip</i> injection of METH, between AAV-scFv6H4, AAV-scFv7F9, and a saline control at day 21 post AAV8 administration.

<p>Mice treated with either AAV-scFv6H4 or AAV-scFv7F9 showed significantly lower brain METH concentrations (a) and significantly higher serum concentrations of METH (b) than the saline-treated mice (*, p < 0.05; #, p < 0.001). There was also a significant decrease in AMP brain concentrations (c) in the AAV-scFv treated groups compared to control mice but no difference in serum AMP concentrations (d). Points are shown as mean ± SEM (n = 3–4 per group).</p

PEGylation of a High-Affinity Anti-(+)Methamphetamine Single Chain Antibody Fragment Extends Functional Half-Life by Reducing Clearance

PURPOSE: Methamphetamine (METH) abuse is a worldwide drug problem, yet no FDA-approved pharmacological treatments are available for METH abuse. Therefore, we produced an anti- METH single chain antibody fragment (scFv7F9Cys) as a pharmacological treatment for METH abuse. ScFv’s have a short half-life due to their small size, limiting their clinical use. Thus, we examined the pharmacokinetic effects of conjugating poly(ethylene) glycol (-PEG) to scFv7F9Cys to extend its functional half-life. METHODS: The affinity of scFv7F9Cys and PEG conjugates to METH was determined in vitro via equilibrium dialysis saturation binding. Pharmacokinetic and parameters of scFv7F9Cys and scFv7F9Cys-PEG20K (30 mg/kg i.v. each) and their ability to bind METH in vivo were determined in male Sprague-Dawley rats receiving a subcutaneous infusion of METH (3.2 mg/kg/day). RESULTS: Of three PEGylated conjugates, scFv7F9Cys-PEG20K was determined the most viable therapeutic candidate. PEGylation of scFv7F9Cys did not alter METH binding functionality in vitro, and produced a 27-fold increase in the in vivo half-life of the antibody fragment. Furthermore, total METH serum concentrations increased following scFv7F9Cys or scFv7F9Cys-PEG20K administration, with scFv7F9Cys-PEG20K producing significantly longer changes in METH distribution than scFv7F9Cys. CONCLUSIONS: PEGylation of scFv7F9Cys significantly increase the functional half-life of scFv7F9Cys, suggesting it may be a long-lasting pharmacological treatment option for METH abuse

Schematic of the prototype scFv design.

<p>V_H, variable heavy region; V_L, variable light region; Linker, 15 amino acid linker; His6, 6-histidine tag for purification and identification; FLAG, FLAG tag for identification; HMM38, a secretory signal sequence. The HMM38 at the 5’ end of the sequences is cleaved during secretion at the site indicated (triangle).</p