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

Abstract

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.

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

“…As discussed in past reports from this laboratory (14,36), the variable region sequences were organized in a VH-linker-VL order to convert the two heavy and light chain sequences of mAb7F9 to scFv7F9Cys. Sequence elements for a 6-histidine tag and a free cysteine were engineered at the carboxy terminus for purification and site-specific conjugation, as described (14,36). cDNA encoding scFv7F9Cys was synthesized by GenScript (Piscataway, NJ) and cloned into a pUC57 vector (14,36,37).…”

“…Sequence elements for a 6-histidine tag and a free cysteine were engineered at the carboxy terminus for purification and site-specific conjugation, as described (14,36). cDNA encoding scFv7F9Cys was synthesized by GenScript (Piscataway, NJ) and cloned into a pUC57 vector (14,36,37). For maintenance the plasmids were transformed into E. coli strain DH5α (Invitrogen, Carlsbad, CA).…”

Affinity improvement of a therapeutic antibody to methamphetamine and amphetamine through structure-based antibody engineering

“…Saturation binding assay was performed as described in Nanaware-Kharade et al25. The scFv concentration used in all saturation binding experiments was optimized to maximize the signal to noise ratio as well as minimize errors resulting from possible ligand depletion.…”

Polymer-antibody fragment conjugates for biomedical applications

“…Numerous research groups have introduced C-terminal cysteine residuesfordirected conjugation of scFvs via sulfhydryl groupsdistal to the antigen-binding site [62,79], though functional yields of the modified scFv can be greatly compromized by the accumulation of large aggregates of disulfide crosslinked polypeptides in the expression host. This cysteine addition approach has been used to PEGylate scFvs via a flexible maleimide linker, resulting in an increase inin vivo half-life by up to 100-fold [80].C-terminal cysteines have alsorecently been utilizedto conjugateantimethamphetamine scFvs to polyamidoamine dendrimer nanoparticlesvia a heterobifunctional PEG cross-linker, in an approach the authors suggest may be of broad use in preclinical characterization and treatment of drug abuse [81]. An alternative approach has seen the production of a glycosylated scFv in E. coli followed by oxidation of the sugar moiety for covalent, site-specific conjugation to amine groups [63].…”