title: Bivalent disulfide-stabilized fragment variable immunotoxin directed against mesotheliomas and ovarian cancer.
abstract: We have used protein engineering to generate a stable bivalent fragment variable (Fv) molecule from the antimesothelin antibody SS, in which the VH and VL domains of the Fv are linked to each other by a disulfide bond, and the two Fvs are connected by a flexible 15-amino acid (Gly4-Ser)3 linker. The SS (dsFv)2 molecule is fused to a M(r) 38,000 truncated form of Pseudomonas exotoxin to generate a bivalent, disulfide stabilized, (dsFv)2 immunotoxin. The immunotoxin was expressed in Escherichia coli, refolded in vitro, and purified to approximately 95% purity with a high yield of > 10%. Binding studies demonstrated that the (dsFv)2 molecule has 40 times higher apparent affinity for recombinant mesothelin than a monovalent dsFv molecule. The (dsFv)2 immunotoxin was 4-10-fold more cytotoxic to three mesothelin antigen-positive cell lines than the monovalent dsFv immunotoxin. However, when tested in mice bearing tumor cells expressing mesothelin, the antitumor activity of the bivalent immunotoxin is very similar to the activity of the lower affinity monovalent immunotoxin. Our data indicate that increasing affinity of an antibody fragment does not necessarily lead to higher antitumor activity of an immunotoxin in vivo.
authors.affiliation: Laboratory of Molecular Biology, National Cancer Institute, NIH, Bethesda, Maryland 20892-4255, USA.
authors.forename: T K
authors.initials: TK
authors.lastname: Bera
authors.forename: J
authors.initials: J
authors.lastname: Williams-Gould
authors.forename: R
authors.initials: R
authors.lastname: Beers
authors.forename: P
authors.initials: P
authors.lastname: Chowdhury
authors.forename: I
authors.initials: I
authors.lastname: Pastan
mesh_terms: D036002:ADP Ribose Transferases / Q000235:genetics / Q000493:pharmacokinetics / Q000494:pharmacology*; D000818:Animals; D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm / Q000378:metabolism; D001427:Bacterial Toxins / Q000235:genetics / Q000493:pharmacokinetics / Q000494:pharmacology*; D004220:Disulfides / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005098:Exotoxins / Q000235:genetics / Q000493:pharmacokinetics / Q000494:pharmacology*; D005260:Female; D058851:GPI-Linked Proteins; D015870:Gene Expression; D006801:Humans; D007128:Immunoglobulin Fragments / Q000276:immunology; D000922:Immunotoxins / Q000235:genetics / Q000493:pharmacokinetics / Q000494:pharmacology*; D008562:Membrane Glycoproteins / Q000378:metabolism; D000090204:Mesothelin; D008654:Mesothelioma / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009374:Neoplasms, Experimental / Q000188:drug therapy / Q000378:metabolism; D010051:Ovarian Neoplasms / Q000276:immunology*; D010957:Plasmids; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D037521:Virulence Factors / Q000235:genetics / Q000493:pharmacokinetics / Q000494:pharmacology*; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D001427:Bacterial Toxins; D004220:Disulfides; D005098:Exotoxins; D058851:GPI-Linked Proteins; D007128:Immunoglobulin Fragments; D000922:Immunotoxins; D008562:Membrane Glycoproteins; D000090204:Mesothelin; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C000715528:Msln protein, mouse; C055966:immunoglobulin Fv