Recently, a bivalent reco
mbinant anti-hu
man CD3 diphtheria toxin (DT) based i
mmunotoxin derived fro
m thescFv of UCHT1 antibody has been
made that shows enhanced bioactivity
and is free fro
m the side effects of Fcreceptor interaction. In this case, the di
minution of CD3 binding due to the place
ment of the scFv do
main at theC-ter
minus of the truncated DT in single scFv i
mmunotoxins was co
mpensated by adding an additional scFvdo
main. However, this strategy was less successful for constructing an anti-rhesus reco
mbinant i
mmunotoxinderived fro
m the scFv of FN18 antibody due to poor binding of the anti-rhesus bivalent i
mmunotoxin. We reporthere that, by increasing the FN18 scFv affinity through r
ando
m mutagenesis
and selection with a dye-labeled
monkey CD3
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![](/i<font color=)
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mbinant heterodi
mer, we greatly i
mproved the bioactivity of FN18 derived i
mmunotoxin.The best
mutant, C207, contained nine
mutations, two of which were located in CDRs that changed the chargefro
m negative to positive. Binding affinity of the C207 scFv to the
monkey T cell line HSC-F increased 9.8-fold.The potency of the C207 bivalent i
mmunotoxin assayed by inhibition of protein synthesis increased by 238-fold.