These studies were designed to determine the mechanism by which some anti-methamphetamine (anti-METH) monoclonal antibodies (mAbs) rapidly lose METH-binding function and efficacy after administration to the rat. Anti-METH mAb6H4 was chosen as the prototype for this short-acting group of mAbs due to its high affinity for METH and rapid loss of in vivo METH-binding function. The central hypothesis was that mAb6H4 is modified in vivo leading to decreased METH affinity (higher KD), decreased METH binding capacity (reduced Bmax), or both.
In the first set of experiments, the effect of increased mAb clearance and METH metabolic processing were determined in studies comparing a short-acting (mAb6H4) and long-acting (mAb4G9) anti-METH mAb. Pharmacokinetic results revealed that increased mAb clearance did not significantly alter the relative METH-binding function of the mAbs, and neither METH nor METH metabolites covalently bound to mAb6H4. Importantly, the in vivo percent occupancy of METH-binding sites on mAb6H4 rapidly decreased over the first several hrs after administration, suggesting the antibody lost substantial METH binding capacity after exposure to the in vivo environment.
The second series of studies aimed to develop a novel mAb probe for discovering modification to antibodies after in vivo administration. The probe, termed mAb6H4-6His, was engineered with a polyhistidine tag to facilitate purification from serum. Characterization of mAb6H4-6His using size exclusion chromatography, isoelectric focusing electrophoresis, and radioimmunoassay showed that the probe retained essential immunochemical properties of native mAb6H4, including high affinity for METH (KD 4.39 nM). A method for purifying mAb6H4-6His from rat serum was also developed using Protein G and immobilized metal ion affinity chromatography.
In the final series of experiments, in vivo testing and ex vivo immunochemical analysis of mAb6H4-6His following 24 hrs of in vivo exposure was conducted. The results showed that METH affinity (KD) was unchanged, but binding capacity (Bmax) was substantially reduced. In conclusion, these studies report the discovery of a novel mechanism leading to significantly reduced in vivo function of an anti-METH mAb. This mechanism, which substantially shortens the duration of mAb efficacy, could occur to other therapeutic antibodies and, therefore, has broad clinical implications for improving protein-based medications.
|Advisor:||Owens, S. Michael|
|Commitee:||Henry, Ralph L., Peterson, Eric C., Raney, Kevin D., Wessinger, William D.|
|School:||University of Arkansas for Medical Sciences|
|Department:||Interdisciplinary Biomedical Sciences|
|School Location:||United States -- Arkansas|
|Source:||DAI-B 74/09(E), Dissertation Abstracts International|
|Keywords:||Immunotherapy, Methamphetamine, Monoclonal antibodies, Pre-clinical|
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