Production and characterization of a recombinant anti-MUC1 scFv reactive with human carcinomas.
Recombinant single-chain fragments (scFv) of the murine anti-MUC1 monoclonal antibody C595 have been produced using the original hybridoma cells as a source of variable heavy (V(H))- and variable light (V(L))-chain-encoding antibody genes. The use of the polymerase chain reaction (PCR), bacteriophage (phage) display technology and gene expression systems in E. coli has led to the production of soluble C595 scFv. The scFv has been purified from the bacterial supernatant by peptide epitope affinity chromatography, leading to the recovery of immunoreactive C595 scFv, which was similar in activity to the C595 parent antibody. Analysis by DNA sequencing, SDS-PAGE and Western blotting has demonstrated the integrity of the scFv, while ELISA, FACScan analysis, fluorescence quenching, quantitative immunoreactivity experiments and immunohistochemistry confirm that the activity of the scFv compares favourably with that of the parent antibody. The retention of binding activity to MUC1 antigen on human bladder and breast carcinoma tissue specimens illustrates the potential application of this novel product as an immunodiagnostic and immunotherapeutic reagent.
Denton
G
G
Cancer Research Laboratories, Department of Pharmaceutical Sciences, University of Nottingham, University Park, UK.
Sekowski
M
M
Spencer
D I
DI
Hughes
O D
OD
Murray
A
A
Denley
H
H
Tendler
S J
SJ
Price
M R
MR
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D015153:Blotting, Western; D003001:Cloning, Molecular / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis; D004926:Escherichia coli; D005434:Flow Cytometry; D006801:Humans; D007128:Immunoglobulin Fragments / Q000096:biosynthesis / Q000302:isolation & purification; D007150:Immunohistochemistry; D051379:Mice; D008969:Molecular Sequence Data; D018396:Mucin-1 / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D009362:Neoplasm Metastasis; D009369:Neoplasms / Q000473:pathology*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007128:Immunoglobulin Fragments; D018396:Mucin-1; D011994:Recombinant Proteins