Dean Pettit

Engineering the isoelectric point of a renal cell carcinoma targeting antibody greatly enhances scFv solubility

BACKGROUND The murine A6H monoclonal antibody targets a cell surface antigen associated with renal cell carcinoma with high specificity and excellent biodistribution properties. Tumor to blood ratios of > 40:1 have been achieved in clinical studies. OBJECTIVES In order to generate an antibody engineering system that would allow the construction of improved derivatives for diagnostics and therapeutics, a single-chain Fv antibody (scFv) derived from A6H was constructed. The initial single-chain Fv, constructed with a cysteine residue and hexa-histidine sequence at the C-terminus, displayed a limited solubility of 100 microg/ml at pH 7.4. The low solubility and refolding yield of the original single-chain Fv required that a more soluble variant be designed and constructed. STUDY DESIGN We hypothesized that lowering the pI of the scFv antibody away from the physiological range would yield a more soluble antibody. A derivative was thus subsequently engineered with five glutamic acid residues followed by the cysteine and hexa-histidine residues. The cysteine was included to provide a conjugation site for future radiolabeling studies. RESULTS The redesigned A6H single-chain Fv has a predicted pI of 6.1, relative to 7.5 for the native scFv. The redesigned A6H scFv displayed a greatly enhanced solubility of > 15 mg/ml at pH 7.4. Both the original scFv and the redesigned single-chain Fv exhibited a strong tendency to form dimers and soluble high molecular weight aggregates. The monomer and disulfide bonded dimer were separated from the aggregates and complete cell binding isotherms were obtained, demonstrating that the purified A6H scFv retains much of the activity of the parent monoclonal. CONCLUSION The addition of glutamic acid to the C-terminus of poorly soluble scFv antibodies could provide a straightforward avenue for improving their solubility properties. The increased solubility of the A6H scFv allowed the purification of the monomeric and dimeric species from the soluble aggregated species.

Release of PEGylated granulocyte-macrophage colony-stimulating factor from chitosan/glycerol films.

We have prepared a new formulation for mucosal delivery of GM-CSF or PEGylated GM-CSF based on a chitosan carrier plus added glycerol to control the rate of release of the protein. Thin dry films comprised of various weight ratios of chitosan to glycerol and containing either granulocyte-macrophage colony-stimulating factor (GM-CSF) or PEGylated GM-CSF, PEG-(GM-CSF), were prepared. The amount of GM-CSF or PEG-(GM-CSF) released from the chitosan/glycerol films was determined using size exclusion high performance liquid chromatography (HPLC-SEC). The amount of PEG-(GM-CSF) released from the films decreased with an increase in the amount of glycerol present in the film. In parallel with this, films with higher glycerol content exhibited a lower degree of equilibrium swelling when immersed in release media. pH measurements of the release media and analysis of the dried films by Fourier-transform infrared spectroscopy (FTIR) suggested that the amount of residual acetic acid in the dry films decreased as the glycerol content increased. This indicates that glycerol may act by displacing and releasing bound acetic acid from the chitosan molecules, resulting in chitosan--glycerol hydrogen bond formation as the film dries. Further, it was found that the release rate and the amount of PEG-(GM-CSF) released decreased with increasing molecular weight of the conjugated PEG. This effect was not observed with films containing physical mixtures of PEG and GM-CSF. The decrease in the fraction of PEG-(GM-CSF) released with increasing PEG molecular weight is believed to be due to the increased steric hindrance of the PEGylated protein molecule during its diffusion out of the swollen chitosan/glycerol film.