Lowering the isoelectric point of the Fv portion of recombinant immunotoxins leads to decreased nonspecific animal toxicity without affecting antitumor activity.
Recombinant immunotoxins are genetically engineered proteins in which the Fv portion of an antibody is fused to a toxin. Our laboratory uses a 38-kDa form of Pseudomonas exotoxin A termed PE38 for this purpose. Clinical studies with immunotoxins targeting CD25 and CD22 have shown that dose-limiting side effects are attributable to liver damage and other inflammatory toxicities. We recently showed that mutating exposed surface neutral residues to acidic residues in the framework region of the Fv portion of an immunotoxin targeting CD25 [anti-Tac(scFv)-PE38] lowered its isoelectric point (pI) and decreased its toxicity in mice without impairing its cytotoxic or antitumor activities. We have now extended these studies and made mutations that change basic residues to neutral or acidic residues. Initially the pI of the mutant Fv (M1) of anti-Tac(scFv)-PE38 was decreased further. Subsequently, mutations were made in two other immunotoxins, SS1(dsFv)-PE38 targeting ovarian cancer and B3(dsFv)-PE38 targeting colon and breast cancers. We have found that all these mutant molecules fully retained specific target cell cytotoxicity and antitumor activity but were considerably less toxic to mice. Therefore, lowering the pI of the Fv may be a general approach to diminish the nonspecific toxicity of recombinant immunotoxins and other Fv fusion proteins without losing antitumor activity.
Onda
M
M
Laboratory of Molecular Biology, Division of Basic Sciences, National Cancer Institute, NIH, Bethesda, Maryland 20892-4255, USA.
Nagata
S
S
Tsutsumi
Y
Y
Vincent
J J
JJ
Wang
Q
Q
Kreitman
R J
RJ
Lee
B
B
Pastan
I
I
D036002:ADP Ribose Transferases; D000410:Alanine Transaminase / Q000097:blood; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001427:Bacterial Toxins; D004220:Disulfides / Q000737:chemistry / Q000633:toxicity; D005098:Exotoxins / Q000737:chemistry / Q000493:pharmacokinetics / Q000633:toxicity*; D005260:Female; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000235:genetics / Q000633:toxicity*; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics / Q000633:toxicity; D000922:Immunotoxins / Q000737:chemistry / Q000235:genetics / Q000493:pharmacokinetics / Q000633:toxicity*; D007526:Isoelectric Point; D008099:Liver / Q000187:drug effects; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009374:Neoplasms, Experimental / Q000276:immunology / Q000628:therapy; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000633:toxicity; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D000410:Alanine Transaminase; D000906:Antibodies; D001427:Bacterial Toxins; D004220:Disulfides; D005098:Exotoxins; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D000922:Immunotoxins; D011994:Recombinant Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C407312:antitac(FV)-PE38 recombinant immunotoxin; C055966:immunoglobulin Fv