Treatments specific to the medical problems caused by (+)-methamphetamine (METH) abuse are greatly needed. Toward this goal, we have developed new multivalent anti-METH antibody fragment-nanoparticle conjugates with customizable pharmacokinetic (PCKN) properties. For this, we first designed novel anti-METH single chain antibody fragments (scFvs) with an engineered terminal cysteine (proto-type scFv6H4Cys and scFv7F9Cys).
Major Aim 1 focused on generating and characterizing the dendrimer nanoparticle-antibody fragment conjugates. Generation 3 (G3) polyamidoamine (PAMAM) dendrimer nanoparticles were chosen for conjugation due to their monodispersive properties and multiple primary amine functional groups. Further, a heterobifunctional PEG was used as a crosslink the free amine of the dendrimer to the thiol group of cysteine on the antibody fragment. PEG crosslinker was reacted with dendrimers in a stoichiometric ratio of 11:1 to synthesize PEG modified dendrimers, which were further reacted with 3-fold molar excess of anti-METH scFv6H4Cys. This generated G3-PEG-scFv6H4Cys conjugates (dendribodies). The dendribodies were separated from the unreacted PEG modified dendrimers and scFv6H4Cys using affinity chromatography. This reaction resulted in a heterogeneous mix of G3-PEG-scFv6H4Cys conjugates with three to six scFv6H4Cys conjugated to each dendrimer.
A detailed optimization of the conjugation reaction along with in vitro characterization of size, purity, and METH binding function) of the PEG modified dendrimers and the dendribodies was performed using SDS-PAGE, UV-Vis spectroscopy, size exclusion chromatography (SEC) and saturation equilibrium dialysis. The dendribodies showed affinity for METH, identical to that of the unconjugated scFv6H4Cys, whereas the PEG modified dendrimers had no affinity for METH. The PEG modified dendrimers and the scFv6H4Cys based dendribodies were then analyzed for in vitro cytotoxicity using hemolysis assay. We found that G3 PAMAM dendrimers exhibited toxicity towards erythrocytes, whereas PEG modified dendrimers, scFv6H4Cys, and dendribodies showed no toxicity. These data suggest that an anti-METH scFvCys can be conjugated to a PEG modified dendrimer nanoparticle without affecting the METH binding properties. The Major Aim 1 studies were a critical step toward preclinical characterization and development of a novel nanomedicine for the treatment of METH abuse.
In Major Aim 2, we compared scFv6H4Cys and scFv7F9Cys nanoparticle conjugation products for: 1) reaction efficiency, 2) size of the antibody-dendrimer conjugates, 3) multivalency, 4) METH-binding function, and 5) potential for efficient scale up. We found that scFv7F9Cys was a more efficient protein for dendrimer nanoparticle conjugation compared to scFv6H4Cys. Further, considering that the chimeric anti-METH monoclonal antibody Ch-mAb7F9 has recently completed Phase 1a clinical trials, we pursued subsequent Major Aims 2 and 3 studies with scFv7F9Cys based dendribodies. Using size exclusion chromatography (SEC) analysis, the formulation was enriched with dendribodies, and well separated from the unreacted scFv7F9Cys, PEG modified dendrimers and scFv7F9Cys. The dissociation constants (KDs) of the scFv7F9Cys and dendribodies for [ 3H]-METH were 6.2 and 3.2 nM, respectively. Similarly, the IC 50 values of scFv7F9Cys and dendribodies for AMP were 8 and 4 μM, respectively.
Densitometry was used to estimate the conjugation and purification efficiency of the dendribody reaction. On average, for small scale conjugation reactions, we conjugated 46 ± 4% of scFv7F9Cys to PEG modified dendrimers and removed 50 ± 2% of the unreacted scFv7F9Cys from the dendribody reaction for a final yield of ∼23%. However, during the medium level scale up of the dendribody reaction process, the total purified dendribody yield was ∼14%. Using standard protein quantitation assays, we found that the BCA assay consistently overestimated the concentration of protein in the presence of dendrimers and PEG24. In Bradford assay, only the presence of dendrimers affected protein quantitation. Next, a formulation and storage buffer containing 50 mM sodium phosphate, 150 mM NaCl, 2 mM EDTA and protein was used to stabilize excipients with 5% w/v sucrose and 0.02% v/v Tween 80; pH 6.4 was used. Before preclinical evaluation, a preliminary stability study of the dendribody formulation was performed to ensure retention of activity under storage conditions. Results from our preliminary stability studies suggested that our formulations were stable at -80°C despite repeated freeze/thaw induced stress.
In Major Aim 3, we tested our central hypothesis that the efficacy and duration of action of an anti-METH antibody fragment can be customized and improved by conjugating it to a dendrimer delivery system. Thus, the PCKN parameters of the dendribodies and their ability to favorably alter METH disposition in a rat model of chronic METH dosing were investigated. The average serum scFv7F9Cys and dendribody concentration-time curves were best fit with a two-compartment PCKN model. The clearance and volume of distribution of scFv7F9Cys upon conjugation to dendrimers were decreased 45 and 1.6 fold, respectively, whereas the terminal elimination half-life (t1/2λz) increased 20 fold. In biodistribution studies, major organs, blood, and urine distribution data of scFv7F9Cys and dendribody (determined by percent injected [3H]-tracer dose) agreed well with the PCKN data. Renal clearance appeared to be the major route of elimination for both treatments. Further, both scFv7F9Cys and dendribodies redistributed METH to a much smaller Vd, which caused an immediate 61 and 63 fold increase in the METH serum concentrations, respectively. The calculated molar stoichiometry of METH to scFv7F9Cys and dendribodies was nearly one-to-two. Finally, the dendribodies maintained higher serum METH concentrations for up to 96 hr compared to 8 hr for the scFv7F9Cys control.
In conclusion, this re-design of scFv7F9Cys resulted in considerably improved t1/2λz of the scFv7F9Cys from 80.4 (± 8.6) to 1560 (± 34.8) min. Taken together, these data suggest that an anti-METH scFv can be successfully conjugated to functionalized dendrimer nanoparticles, creating a new potential nanomedicine platform for treating METH abuse. This approach of combining high affinity antibody fragments with nanotechnology is the first to investigate the design features and molecular principles needed to create innovative nanomedicines to treat drug abuse. Overall, our data suggest that the dendribody design could be used as a novel platform to generate multivalent antibody fragment conjugates with customizable PCKN profiles.
|Advisor:||Peterson, Eric C.|
|Commitee:||Crooks, Peter A., DeSilva, Binodh, Owens, Michael, Rusch, Nancy J.|
|School:||University of Arkansas for Medical Sciences|
|School Location:||United States -- Arkansas|
|Source:||DAI-B 76/01(E), Dissertation Abstracts International|
|Subjects:||Pharmacology, Education, Pharmacy sciences|
|Keywords:||Antibody, Bioconjugation, Methamphetamine, Multimerization, Nanoparticles, Pharmacokinetics|
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