Cytotoxicity of antiosteosarcoma recombinant immunotoxins composed of TP-3 Fv fragments and a truncated Pseudomonas exotoxin A.
Regrowth of drug-resistant tumor cells is responsible for approximately half of an unselected osteosarcoma population still dying of the disease despite aggressive combination therapy. Two monoclonal antibodies, TP-1 (immunoglobulin 2a) and TP-3 (immunoglobulin 2b) are available, which specifically recognize an antigen on osteosarcoma cells. In this work, we have fused the variable (V) genes of TP-3 to a truncated fragment of Pseudomonas exotoxin A, referred to as PE38. Two immunotoxins were made that differed in the Fv portion: TP-3(scFv)-PE38, which contains a peptide linker, and TP-3(dsFv)-PE38, which contains a disulfide bond for stabilization of the association between the V domains. Recombinant TP-3 immunotoxins were expressed in Escherichia coli and purified from inclusion bodies. We describe the design and expression of these immunotoxins, and their properties with regard to antigen binding, stability, and cytotoxicity. Toxicity studies were done in mice. We found that the immunotoxins exhibited very similar in vitro properties, whereas in vivo TP-3(dsFv)-PE38 was much better tolerated than TP-3(scFv)-PE38.
Onda
M
M
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-4255, USA.
Olafsen
T
T
Tsutsumi
Y
Y
Bruland
O S
OS
Pastan
I
I
D036002:ADP Ribose Transferases; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000378:metabolism; D000918:Antibody Specificity; D000951:Antigens, Neoplasm / Q000276:immunology; D001427:Bacterial Toxins; D016923:Cell Death; D004355:Drug Stability; D005098:Exotoxins / Q000235:genetics* / Q000627:therapeutic use; D005260:Female; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D000922:Immunotoxins / Q000235:genetics* / Q000494:pharmacology / Q000633:toxicity; D051379:Mice; D012516:Osteosarcoma / Q000276:immunology* / Q000473:pathology; D011993:Recombinant Fusion Proteins / Q000627:therapeutic use; D014407:Tumor Cells, Cultured; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D001427:Bacterial Toxins; D005098:Exotoxins; D007074:Immunoglobulin G; D007135:Immunoglobulin Variable Region; D000922:Immunotoxins; D011993:Recombinant Fusion Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A