Cancer Immune Therapy Edited by G. Stuhler and P. Walden ...

368 17 Immunotoxins and Recombinant Immunotoxins in Cancer Therapy
inhibitors that will prevent the binding of the toxin or the targeting moiety to endothelial
cells.
Toxicity can also be reduced by modifications in the scFv targeting moiety. For example,
reduction of the non-specific animal toxicity of recombinant Fv±immunotoxin
anti-Tac(Fv)±PE38 (which targets the IL-2 receptor) was achieved by introducing mutations
in the framework regions of the Fv which lower the isoelectric point (pI)
[189]. The dose escalation with this recombinant Fv±immunotoxin (that has produced
eight responses, including a durable clinical complete remission in a recently
completed phase I trial of leukemias and lymphomas) was limited by liver toxicity. It
was noted that the Fv of anti-Tac has a pI of 10.2, which brought about the hypothesis
that the overall positive charge on the Fv portion of anti-Tac(Fv)±PE38 contributes to
non-specific binding to liver cells and results in dose-limiting liver toxicity. A mouse
model was used to investigate the basis of this toxicity, and it was found that lowering
the pI of the Fv of anti-Tac from 10.2 to 6. 82 by selective mutation of surface residues
causes a 3-fold decrease in animal toxicity and hepatic necrosis. This change in
pI did not significantly alter the CD25 binding affinity, the cytotoxic activity toward
target cells, or antitumor activity, resulting in a 3-fold improvement in the therapeutic
index. If this decreased toxicity occurs in humans, it should greatly increase the
clinical utility of this immunotoxin.
Another strategy to overcome the problems of non-specific toxicity and antigenicity
is by the chemical modification of the recombinant Fv±immunotoxins. An example
for this was also demonstrated recently in which site-specific chemical modification
with polyethylene glycol (PEGylation) of anti-Tac(Fv)±PE38 (LMB-2) improved its
antitumor activity, and reduced animal toxicity and immunogenicity. PEGylation can
increase plasma half-lives, stability and therapeutic potency. To produce a PEGylated
recombinant immunotoxin with improved therapeutic properties, a mutant form of
anti-Tac(Fv)±PE38 (LMB-2) in which one cysteine residue was introduced into the
peptide connector (ASGCGPE) between the Fv and the toxin was constructed [190].
This mutant LMB-2 (Cys1-LMB-2), which retained full cytotoxic activity, was then
site-specifically conjugated with 5 or 20 kDa of polyethylene glycol±maleimide.
When compared with unmodified LMB-2, both PEGylated immunotoxins showed similar
cytotoxic activities in vitro, but superior stability at 37 8C in mouse serum, a 5to
8-fold increase in plasma half-lives in mice and a 3- to 4-fold increase in antitumor
activity. This was accompanied by a substantial decrease in animal toxicity and immunogenicity.
Site-specific PEGylation of recombinant immunotoxins may thus increase
their therapeutic potency in humans.
17.8.2
Specificity Dictated by the Targeting Moiety
Specificity of the recombinant immunotoxin is determined by the distribution of the
target antigens; several target antigens are relatively cancer specific, but are present
on some normal cells in small amounts. For example, erbB2, although over-expressed
on tumor cells, is also expressed on a limited number of normal cells. This
reactivity with normal cells may cause side effects during immunotoxin therapy. It