Redesigned anti-human placental alkaline phosphatase single-chain Fv: soluble expression, characterization and in vivo tumour targeting.
Although much progress has been made in the production of recombinant antibodies and their fusions, there are still problems with solubility and folding. Useful antibodies produced from cloned hybridomas do not always result in scFvs behaving favourably. We report here further work on an scFv (H17E2) against the oncofetal antigen human placental alkaline phosphatase. The overall expression was greatly improved and the H17E2 scFv was redesigned by manipulation of the interdomain linker, resulting in much higher expression levels of the soluble scFv in its active conformation at 0.2-0.5 mg/l of bacterial culture. We show that the new soluble version of this scFv has similar characteristics to the refolded version in terms of antigen and tumour cell binding, stability and in vivo pharmacokinetics. The final tumour uptake behaviour of these scFvs is superior to that of the parental whole antibody with respect to tumour:organ ratios, but still requires further development before considering it as a suitable molecule for clinical use in ovarian or testicular cancer.
Deonarain
M P
MP
Tumour Targeting Laboratory, Royal Postgraduate Medical School, Hammersmith Hospital, London, UK.
Rowlinson-Busza
G
G
George
A J
AJ
Epenetos
A A
AA
D000469:Alkaline Phosphatase / Q000276:immunology*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000097:blood / Q000235:genetics* / Q000276:immunology*; D000912:Antibodies, Neoplasm / Q000737:chemistry* / Q000235:genetics*; D000951:Antigens, Neoplasm / Q000276:immunology; D014408:Biomarkers, Tumor; D003001:Cloning, Molecular / Q000379:methods; D015870:Gene Expression / Q000235:genetics; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000235:genetics*; D007074:Immunoglobulin G / Q000097:blood / Q000235:genetics* / Q000276:immunology; D007624:KB Cells; D051379:Mice; D010920:Placenta / Q000201:enzymology*; D011485:Protein Binding / Q000235:genetics / Q000276:immunology; D014018:Tissue Distribution / Q000502:physiology
D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; C106201:H17E2 monoclonal antibody; D007128:Immunoglobulin Fragments; D007074:Immunoglobulin G; C031192:oncofetal antigens; D000469:Alkaline Phosphatase