CLASSIC IMMUNOTOXINS WITH PLANT OR MICROBIAL TOXINS - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 266

The third-generation immunotoxins have either a modified
toxin moiety to reduce immunogenicity and lysosomal pro-
tein degradation or a more complicated structure to increase
binding affinity for targeted antigens. For less immunogenic
and protease-resistant molecules, systemic studies on PE
immunotoxins have been performed [51,52]. In this study,
seven major B-cell epitopes have been indentified in PE38
and eliminated by introduction of eight point mutations
(R313A, Q332S, R432G, R467A, R490A, R513A, E548S,
and K590S). The new mutant, PE38-8X, is significantly
less immunogenic in three strains of mice, yet retains full
cytotoxic and antitumor activities [51]. In the other study, a
lysosomal protease-resistant PE immunotoxin has been devel-
oped by serial deletion of PE38 domain II. The immunotoxin
with PE25LR containing a small portion of domain II (resi-
dues 274-284) and full-length domain III, retains cytotoxic
activity and is less toxic to mice [52]. Combination of these
point mutations and major deletion of domain II would
generate a new PE immunotoxin with less immunogenicity,
less toxicity, and protease-resistance.
The molecules with complicated protein structure to
increase efficacy are designed by addition of an identical
ligand or a different ligand. The third-generation DT immu-
notoxins, A-dmDT390-bisFv(UCHT1) andDT2219ARL, are
being investigated in clinical trials. For molecules with two
identical ligands (normally sFv), there are two formats,
bivalent and fold-back formats. In the bivalent format
(DT-sFv-(G 4 S) 3 -sFv), the additional sFv (V L -(G 4 S) 3 -V H )is
fused to C-terminus of monovalent DT immunotoxin through
the (G 4 S) 3 linker. The bivalency provides much higher bind-
ing to targeted cells (20- to 30-fold increase) [69]. In the fold-
back format (DT-V L -G 4 S-V H -(G 4 S) 3 -V L -G 4 S-V H ), the G 4 S
linkers between V L and V H are shorter than the (G 4 S) 3 linker
between the first sFv and second sFv. The shorter linkers
prevent adjacent V L and V H domains from forming a sFv. The
(G 4 S) 3 linker is long enough so that the second V L and V H
domains can fold back to pair with the first V H and V L
respectively, forming two sFv antigen-binding sites. This
provides a more rigid structure compared to the bivalent,
resulting in increased potency by fivefold to sevenfold [70].
A good example of molecules with two different ligands
is DT2219ARL, which has the first 389 amino acid residues
of DT, and two different sFv moieties recognizing different
antigens (CD22 and CD19). The V L and V H domains in each
sFv moiety are connected by a flexible ARL linker derived
from human muscle aldolase (the first 20 amino acid resi-
dues). Two sFv molecules are linked by the (G 4 S) 3 linker.
This flexible ARL linker increased potency and product
yield in E. coli [71]. This bispecific molecule was a 1000-
fold more potent than DT22 alone, or an equal mixture of
DT22 and DT19. Three other bispecific DT immunotoxins
(DTEGF13, DTEpCAM23, and DTAT13) have been
reported [72-74]. More recently a deimmunized bispecific
PE immunotoxin (2219KDEL7mut) has shown in vivo

efficacy in a xenograft B-cell metastases model. This mole-
cule, an upgraded version of DT2219ARL, has two different
sFv moieties recognizing CD22 and CD19, and the less
immunogenic PE38-8X with KDEL [75]. Since this mole-
cule has much less immunogenicity, multiple drug treatment
may be given in future clinical trials.
For chemical conjugation, the conjugation reaction must
not severely alter the ligand affinity or impair the ability of
the toxin moiety to translocate to the cytosol and enzymati-
cally inactivate cell functions. Typically large amounts of
toxin and ligand molecules are needed to synthesize chemi-
cally conjugated immunotoxins because of low conjugation
yield. Chemical conjugations yield a heterogeneous product
because of the random reaction of derivatizing reagents
with multiple amino acid residues in the ligand and/or
toxin. Bifunctional conjugation reagents used include
N-succinimidyl-oxycarbonyl- a -methyl-(2-pyridyl-dithio)
toluene (SMPT), N-succinimidyl 3-(2-pyridyl-dithio)-propi-
onate (SPDP), m-maleimidobenzoyl-N-hydrosuccinimide
ester (MBS), and N-succinimidyl 4-(N-maleimidomethyl)
cyclohexane-1-carboxylate (SMCC). Amine-reactive
N-hydroxy-succinimide (NHS) ester that reacts with amine
group of lysine residues forms a stable amide bond.
The pyridyl disulfide group and the maleimide group of
bifunctional conjugation reagents react with thiol group of
cysteine residues to form a reversible disulfide bond and a
stable thioether bond, respectively. DTNB (5,5 0 -dithiobis
(2-nitobenzoic acid) is used as a thiol-protecting agent for
synthesis of Fab 0 and deglycosylated ricin A chain conju-
gate. Traut agent (2-iminothiolane) is often used to add a
thiol group to a ligand for conjugation. Synthesis method
and formulation of chemical conjugates tested in clinical
trials are summarized in Table 18.2. Typically, a whole IgG
or Fab2 0 fragment is derivatized with a bifunctional conju-
gation reagent. A free thiol group is introduced into a toxin if
it does not have the free thiol group. The derivatized ligand
and toxin are conjugated through a disulfide bond or thio-
ether bond. Then unconjugated molecules are removed from
conjugates by gel filtration, anion exchange, and/or affinity
chromatography.
Genetic fusions by recombinant DNA technology are
well defined and controlled, and generally have less effect
on toxin functions than chemical conjugation. E. coli is a
typical expression system for production of recombinant
immunotoxins. The trc and T7 promoters are popularly used
to express recombinant immunotoxins in E. coli. Since most
of recombinant immunotoxins are expressed in insoluble
inclusion bodies, an in vitro refolding step is required to
obtain functional immunotoxins. Immunotoxins in the inclu-
sion body preparation are washed extensively with detergent
to remove endotoxin, solubilized, denatured, and reduced.
The denatured and reduced recombinant immunotoxins are
then renatured in a refolding buffer containing L -arginine
and glutathione. The renatured proteins can be purified by