Stabilization of the Fv fragments in recombinant immunotoxins by disulfide bonds engineered into conserved framework regions.
Disulfide-stabilized Fv's (dsFv's) are recombinant Fv fragments of antibodies in which the unstable variable heavy (VH) and variable light (VL) heterodimers are stabilized by disulfide bonds engineered at specific sites that lie between structurally conserved framework positions of VH and VL. We have recently described one example of a recombinant immunotoxin, B3(dsFv)-PE38KDEL, that is composed of such a dsFv connected to a truncated form of Pseudomonas exotoxin [Brinkmann, U., Reiter, Y., Jung, S.-H., Lee, B., & Pastan, I. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 7538-7542]. This disulfide-stabilized immunotoxin has the same cytotoxic activity and specificity as its single-chain immunotoxin counterpart. To determine whether the stabilization of Fv's by disulfides at these positions is generally applicable, we made and analyzed two other dsFv-containing immunotoxins. One is made from the e23 antibody, which binds to the carcinoma-associated antigen erbB2; the other is made from the anti-Tac antibody, which binds to the p55 subunit of the IL-2 receptor. Comparison of the specificity and activity of these immunotoxins with those of their scFv counterparts revealed that e23(dsFv)-PE38KDEL was considerably more active than e23(Fv)-PE38KDEL, whereas anti-Tac(dsFv)-PE38KDEL was only somewhat more active than its single-chain counterpart. These results suggest that dsFv's have at least the same binding properties as scFv's, and in some cases they may have better binding. Thus, it should be feasible to use the positions we have identified in the conserved framework region to disulfide-stabilize many different Fv's.(ABSTRACT TRUNCATED AT 250 WORDS)
Reiter
Y
Y
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Brinkmann
U
U
Kreitman
R J
RJ
Jung
S H
SH
Lee
B
B
Pastan
I
I
D036002:ADP Ribose Transferases; D000595:Amino Acid Sequence; D001427:Bacterial Toxins; D001483:Base Sequence; D002460:Cell Line; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D066246:ErbB Receptors / Q000276:immunology; D005098:Exotoxins / Q000737:chemistry* / Q000235:genetics; D007128:Immunoglobulin Fragments / Q000737:chemistry*; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry; D007147:Immunoglobulin Light Chains / Q000737:chemistry; D000922:Immunotoxins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011518:Proto-Oncogene Proteins / Q000276:immunology; D011550:Pseudomonas aeruginosa; D018719:Erb-b2 Receptor Tyrosine Kinases; D015375:Receptors, Interleukin-2 / Q000276:immunology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D003545:Cysteine; D004220:Disulfides; D066246:ErbB Receptors; D005098:Exotoxins; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D000922:Immunotoxins; D009838:Oligodeoxyribonucleotides; D011518:Proto-Oncogene Proteins; D018719:Erb-b2 Receptor Tyrosine Kinases; D015375:Receptors, Interleukin-2; D011994:Recombinant Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A