title: Engineering the isoelectric point of a renal cell carcinoma targeting antibody greatly enhances scFv solubility.
abstract: 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.
  
  
  OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Bioengineering, University of Washington, Seattle 98195, USA.
authors.forename: P H
authors.initials: PH
authors.lastname: Tan
authors.forename: V
authors.initials: V
authors.lastname: Chu
authors.forename: J E
authors.initials: JE
authors.lastname: Stray
authors.forename: D K
authors.initials: DK
authors.lastname: Hamlin
authors.forename: D
authors.initials: D
authors.lastname: Pettit
authors.forename: D S
authors.initials: DS
authors.lastname: Wilbur
authors.forename: R L
authors.initials: RL
authors.lastname: Vessella
authors.forename: P S
authors.initials: PS
authors.lastname: Stayton
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology*; D000912:Antibodies, Neoplasm / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D002292:Carcinoma, Renal Cell / Q000276:immunology*; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D007526:Isoelectric Point; D007680:Kidney Neoplasms / Q000276:immunology*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D015202:Protein Engineering; D012995:Solubility
chemical_list: D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; D007135:Immunoglobulin Variable Region