Target-specific cytotoxic activity of recombinant immunotoxin scFv(MUC1)-ETA on breast carcinoma cells and primary breast tumors.
MUC1 is a mucin family protein, overexpressed in more than 90% of breast cancers in an underglycosylated form, exposing the core peptides of the extracellular domain that act as a potential target for antibody-mediated therapy. We have developed an anti-MUC1 scFv antibody from a phage library of mice immunized with synthetic peptide MUC1-variable number of tandem repeats. MUC1 binding phages were affinity selected through biopanning using a biotin-streptavidin pull-down method. The selected phage clones showed target-specific binding to MUC1-expressing cells. Fusion of truncated Pseudomonas aeruginosa exotoxin A (ETA) to a high binder, phage-derived scFv clone and bacterial expression and purification of recombinant scFv(MUC1)-ETA immunotoxin were done with good yield and purity. In vitro target-specific cytotoxic activity and target-specific binding of immunotoxin were shown on MUC1-expressing cells and primary breast tumor samples. A truncated ETA fusion protein expressed from the same vector but lacking scFv did not show cytotoxic effects, confirming target specificity. Our results suggest that the scFv(MUC1)-ETA immunotoxin has therapeutic potential and deserves further development and characterization for MUC1-specific breast cancers treatment.
Singh
Ravibhushan
R
Protein Engineering and Biotherapy, Advanced Centre for Treatment Research and Education in Cancer, Tata Memorial Centre, Navi Mumbai-410 208, India.
Samant
Urmila
U
Hyland
Stephen
S
Chaudhari
Pradip R
PR
Wels
Winfried S
WS
Bandyopadhyay
Dilip
D
D036002:ADP Ribose Transferases / Q000276:immunology / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000494:pharmacology; D001427:Bacterial Toxins / Q000276:immunology / Q000494:pharmacology*; D001943:Breast Neoplasms / Q000276:immunology* / Q000628:therapy*; D049109:Cell Proliferation / Q000187:drug effects; D005098:Exotoxins / Q000276:immunology / Q000494:pharmacology*; D005260:Female; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000276:immunology*; D000922:Immunotoxins / Q000276:immunology / Q000494:pharmacology*; D018396:Mucin-1 / Q000737:chemistry / Q000276:immunology*; D019151:Peptide Library; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D037521:Virulence Factors / Q000276:immunology / Q000494:pharmacology*; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D000970:Antineoplastic Agents; D001427:Bacterial Toxins; D005098:Exotoxins; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D000922:Immunotoxins; D018396:Mucin-1; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A